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Many experiments show that vitrification significantly reduces the fertilization capacity of mammalian oocytes, restricting the application of vitrified oocytes. It has been proven that the JUNO protein plays a vital role in mammalian oocytes fertilization. However, little information is available about the effects of vitrification on the JUNO protein and the procedure to protect it in bovine oocytes. Here, the present study was designed to investigate the effect of vitrification on the JUNO protein level in bovine oocytes. In this study, MII oocytes were treated with cholesterol‐loaded methyl‐β‐cyclodextrin (CLC; 0, 10, 15, 20 mM) for 45 min before vitrification and methyl‐β‐cyclodextrin (MβCD; 0, 2.25, 4.25, 6.25 mM) for 45 min after thawing (38–39°C). Then, the expression level and function of JUNO protein, cholesterol level in the membrane, the externalization of phosphatidylserine, sperm binding capacity and the developmental ability of vitrified bovine oocytes were examined. Our results showed that vitrification significantly decreased the JUNO protein level, cholesterol level, sperm binding capacity, development ability, and increased the promoter methylation level of the JUNO gene and apoptosis level of bovine oocytes. Furthermore, 15 mM CLC + 4.25 mM MβCD treatment significantly improved the cholesterol level and increased sperm binding and development ability of vitrified bovine oocytes. In conclusion, the combination treatment of cholesterol‐loaded methyl‐β‐cyclodextrin and methyl‐β‐cyclodextrin significantly improves the fertilization capacity of vitrified bovine oocytes by protecting fertilization protein JUNO. [ABSTRACT FROM AUTHOR]

Folate receptor β (FR-β), a marker expressed on macrophages, is a promising target for imaging of inflammation. Here, we report the radiosynthesis and preclinical evaluation of [68Ga]Ga-NOTA-folate (68Ga-FOL). After determining the affinity of 68Ga-FOL using cells expressing FR-β, we studied atherosclerotic mice with 68Ga-FOL and 18F-FDG PET/CT. In addition, we studied tracer distribution and co-localization with macrophages in aorta cryosections using autoradiography, histology, and immunostaining. The specificity of 68Ga-FOL was assessed in a blocking study with folate glucosamine. As a final step, human radiation doses were extrapolated from rat PET data. We were able to produce 68Ga-FOL with high radiochemical purity and moderate molar activity. Cell binding studies revealed that 68Ga-FOL had 5.1 nM affinity for FR-β. Myocardial uptake of 68Ga-FOL was 20-fold lower than that of 18F-FDG. Autoradiography and immunohistochemistry of the aorta revealed that 68Ga-FOL radioactivity co-localized with Mac-3–positive macrophage-rich atherosclerotic plaques. The plaque-to-healthy vessel wall ratio of 68Ga-FOL was significantly higher than that of 18F-FDG. Blocking studies verified that 68Ga-FOL was specific for FR. Based on estimations from rat data, the human effective dose was 0.0105 mSv/MBq. Together, these findings show that 68Ga-FOL represents a promising new FR-β–targeted tracer for imaging macrophage-associated inflammation. [ABSTRACT FROM AUTHOR]

Abstract Metal-based coordination compounds, including the well-known cytostatic drug cisplatin, are widely used in the anticancer therapy. Generally, they exhibit high cytotoxicity not only towards malignant cells, but also towards non-malignant cells, which represents main problem of their clinical use. Herein, we describe the synthesis, characterization and biological testing of three trinuclear nickel(II) coordination compounds. Central nickel atoms are bridged by trithiocyanurate anion and coordinated by triamine and bis-benzimidazoles, respectively. To delineate a potential usage in anticancer therapy, we encapsulated the most cytotoxic complex into biomacromolecular protein cage apoferritin (FRT), forming FRTNi. FRT encapsulation markedly decreased the hemotoxicity of free Ni compounds. Despite FRTNi can be internalized through passive targeting by enhanced permeability and retention effect, we further introduced active targeting utilizing folate receptor (FR) via folic acid (FA)-modified FRT (FRTNiFA). Using breast cancer cell lines T-47D (FR+), MCF-7 (FR-) and non-malignant mammary gland derived cell line HBL-100 (FR-), we show pronounced FR-dependent internalization of FRTNiFA. Overall, we demonstrate that the FRT macromolecular nanocarrier provides a very low off-target toxicity, which could enable the use of highly toxic Ni compounds in cancer nanomedicine. [ABSTRACT FROM AUTHOR]

Abstract Introduction Nonalcoholic steatohepatitis (NASH) afflicts 20–36% of individuals with nonalcoholic fatty liver disease (NAFLD). A lipotoxic hepatic environment, altered innate immune signaling and inflammation are defining features of progression to NASH. Activated resident liver macrophages express folate receptor beta (FR-β) which may be an indicator of progression from steatosis to NASH. The goals of this study were to characterize FR-β protein expression in human NAFLD and rodent models of NASH, and demonstrate liver targeting of an FR-β imaging agent to the liver of a rodent NASH model using FR-β. Methods Rat liver lysates from methionine choline deficient (MCD) fed rats, high fat diet (HFD) and methionine choline sufficient (MC+) rat controls were analyzed for hepatic FR-β protein. The FR-β-targeted agent, Etarfolatide was injected into MCD and MC + -fed C57BL/6 mice for efficient FastSPECT hepatic imaging. Additionally, FR-β expression across the stages of human NAFLD from normal to NASH was assessed. Results FastSPECT images show targeting of Etarfolatide to the liver of mice fed 8 weeks of MCD diet but not control-fed mice. The MCD rat model exhibited significantly increased protein expression of hepatic FR-β in contrast to HFD or normal samples. Similarly human liver samples categorized as NASH Fatty or NASH Not Fatty showed elevated FR-β protein when compared to normal liver. FR-β transcript expression levels were elevated across both NASH Fatty and NASH Not Fatty samples. Conclusion The findings in this study indicate that FR-β expression in NASH may be harnessed to target agents directly to the liver. Highlights • Inflammation in NASH is associated with macrophage activation. • Etarfolatide, a folate-linked imaging agent is targeted to activated macrophages. • Targeting of Etarfolatide to the liver occurs in mice with MCD diet-induced NASH. • Etarfolatide targeting to the liver does not occur in control mice fed an MC+ diet. • FR-β protein and mRNA expression are increased in human NASH. [ABSTRACT FROM AUTHOR]

A highly sensitive homogeneous electrochemiluminescence (ECL) biosensor for folate receptor (FR) has been developed based on hyperbranched rolling circle amplification (HRCA) and terminal protection. FR was combined to folic acid modified single stranded DNA (FA‐ssDNA) through the strong affinity between FA and FR, which prevented ssDNA from enzymolysis of exonuclease I (Exo I). The protected ssDNA was then applied as a template of padlock looping to trigger the HRCA proceeding. The products of HRCA contains large amount of double strand DNA (dsDNA), which provided excellent carries for Ru(phen)32+ intercalation. The extra free Ru(phen)32+ was removed by ultrafiltration and the Ru(phen)32+ embedded into dsDNA can act as a high‐efficiency ECL indicator to quantify the FR concentration. The enhanced ECL intensity had a linear relationship with logarithm of FR concentration in the range of 4 fM to 120 pM with a correlation coefficient of 0.998. Furthermore, the proposed ECL biosensor has been applied to detect FR in Hela cells and showed a linear relationship with the logarithm of cell concentration ranging from 100–5000 cell/mL. Compared to other FR biosensors, the proposed ECL biosensor presents the merits of the simplicity, high sensitivity and specificity, which shows the great potential application in the field of early cancers diagnosis. Let there be light: an electrochemiluminescence (ECL) biosensor for detection of the folate receptor is fabricated in a homogeneous system. The combination of an effective ECL indicator (Ru(phen)32+) and the hyperbranched rolling circle amplification (HRCA) technique promotes the high sensitivity of the fabricated biosensor. [ABSTRACT FROM AUTHOR]

Study Question: Is JUNO protein present at the surface membrane of human oocytes and involved in the fertilisation process?Summary Answer: JUNO protein is expressed on the plasma membrane of human oocytes and its inhibition by a monoclonal antibody completely blocks gamete fusion.What Is Known Already: Fusion of gamete membranes is the culminating event of the fertilisation process, but its molecular mechanisms are poorly understood. Until now, three molecules have been shown to be essential: CD9 tetraspanin in the oocyte, Izumo1 protein on the sperm and Juno, its corresponding receptor on the oocyte. Oocyte CD9 and sperm IZUMO1 have been identified in human gametes and their interaction is also well-conserved among several mammalian species. The presence of JUNO on human oocytes, however, has not yet been reported, nor has its role in fertilisation been investigated.Study Design, Size, Duration: We selected an anti-human JUNO antibody in order to investigate the presence of JUNO on the oocyte membrane surface and studied its potential involvement in gamete membrane interaction during fertilisation.Participants/materials, Setting, Methods: Monoclonal antibodies against human JUNO (anti-hJUNO mAb) were produced by immunisation of mice with HEK cells transfected with the putative human JUNO sequence (HEK-hJUNO). These antibodies were used for immunostaining experiments and in vitro fertilisation assays with human gametes (GERMETHEQUE Biobank).Main Results and the Role Of Chance: Three hybridoma supernatants, verified by immunostaining, revealed specifically HEK-hJUNO cells. The three purified monoclonal antibodies, FJ2E4 (IgG1), FJ8E8 (IgG1) and FJ4F5 (IgG2a), recognised the soluble recombinant human JUNO protein and, in a western blot of HEK-hJUNO extracts, a protein with an expected MW of 25 kDa. In addition, soluble recombinant human IZUMO protein inhibited the binding of anti-hJUNO mAbs to cells expressing hJUNO. Using these anti-hJUNO mAbs in immunostaining, we identified the presence of JUNO protein at the plasma membrane of human oocytes. Furthermore, we revealed a progressive expression of JUNO according to oocyte maturity. Finally, we showed that human zona-free oocytes, inseminated in the presence of anti-hJUNO mAb, were not fertilised by human sperm. These results suggest that, as seen in the mouse, JUNO is indeed involved in human gamete membrane fusion during fertilisation.Large-scale Data: N/A.Limitations, Reasons For Caution: In accordance with French bioethics laws, functional tests were performed using zona-free oocytes, which of course does not fully encompass all normal in vivo physiological conditions. However, these in vitro tests do provide direct information regarding sperm-oocyte membrane interactions.Wider Implications Of the Findings: Mechanisms of gamete fusion appear to be homologous between mice and humans. However, some differences do exist and analysing the human mechanisms is essential. In fact, this is the first report describing the presence of JUNO on human oocytes and its involvement in human fertilisation. This discovery allows further examination of the understanding of molecular mechanisms that drive gamete fusion: a crucial challenge at a time when infertility affects 16% of reproductively active couples.Study Funding/competing Interests: This work was supported by the Agence Nationale pour la Recherche, Grant no. ANR-13-BVS5-0004, and by Association Institut du Cancer et d'Immunogénétique (ICIG). There are no competing interests. [ABSTRACT FROM AUTHOR]

Pancreatic cancer has a poor prognosis; hence, novel prognostic markers and effective therapeutic targets should be identified. We aimed to evaluate folate receptor alpha (FR-α) expression in pancreatic cancer and examine its association with clinicopathological features. We utilized tissue samples from 100 primary pancreatic cancer patients who underwent surgery. FR-α was expressed in 37 of 100 cases (37%). The FR-α-positive group (median, 18.8 months) had a significantly poorer prognosis than the FR-α-negative group [median 21.3 months; HR 1.89 (1.12-3.12); P = 0.017]. These groups were not significantly different regarding progression-free survival (P = 0.196). Furthermore, other serum tumor markers including CA19-9 (mean, 186 vs. 822 U/ml; P = 0.001), Dupan-2 (286 vs. 1133 U/ml; P = 0.000), and Span-1 (69.7 vs. 171.9 U/ml; P = 0.006) were significantly downregulated in the FR-α-positive group. CA19-9 was another prognostic factor, in addition to FR-α, and patient prognosis showed clear stratification curves with the expression of these two molecules. Along with CA19-9, FR-α expression was an independent prognostic factor for the overall survival. FR-α and CA19-9 helped predict patient prognosis based on stratification curves. [ABSTRACT FROM AUTHOR]

Objective: The objective of this study is to investigate the clinical significance of folate-receptor-positive circulating tumor cells (FR+CTC) for the diagnosis of lung cancer, especially in early-stage patients.Materials and Methods: A total of 72 lung cancer patients, including 31 with stage I diseases and two with stage 0 diseases, were enrolled in this study. Twenty-four patients with benign lung diseases and two healthy volunteers served as the control group. Three milliliters of peripheral blood were collected from each participant for FR+CTC analysis on enrollment. FR+CTC enumeration was performed using immunomagnetic leukocyte depletion and ligand-targeted polymerase chain reaction techniques.Results: The study results revealed that using a cutoff value of 8.7 CTC Units/3 mL, the sensitivity, and specificity of FR+CTC for diagnosis of lung cancer were 81.94% and 73.08%, respectively. Such high sensitivity (74.19%) and specificity (73.08%) persisted even if only stage I lung cancer patients were retained in the analysis. In receiver operating characteristic analysis, the performance of FR+CTC (area under the curve = 0.8153) was superior to other clinical biomarkers such as carcinoembryonic antigen, neuron-specific enolase, and cytokeratin 19 fragments. In a subgroup analysis, patients with nodule size of >3 cm showed an improved sensitivity (88.46%); although, the specificity appeared to decrease (40%). All five patients with benign diseases in this subgroup had inflammatory diseases, indicating that large inflammatory nodules may also release FR -expressing cells into the circulatory system.Conclusion: FR+CTC is a reliable biomarker for the early diagnosis of small-sized lung cancer. Further study with larger sample size is required to assess the diagnostic efficiency of FR+CTC in patients with large nodule sizes. [ABSTRACT FROM AUTHOR]

Folate receptor (FR) is overexpressed in various cancer cells while its expression in normal cells is restricted. The present study provides a new folic acid/folate (FA) functionalized nanomaterials to sense and the differentiation of the cancer cells from normal ones. The reported nanoprobe is based on the mesoporous materials that are functionalized with FA to specify the FR overexpressed cancerous cells. MCF 7 cell lines were used as a model to show the ability of the developed probe for cancer cell detection. The selective binding of FA to FR-positive cells causes the endocytosis of the mesoporous materials into the cells where it can be observed by fluorescence microscopy images. The specific nature of the binding of the FA functionalized mesoporous silica prevents the false detection of normal cells from cancerous cells even in the presence of each other. The cytotoxicity of the n-Pr-NH 2 -MCM 41-FA on the MCF 7 cells was investigated using MTT assay. The reported method can detect the MCF 7 cells from 100 to 1000 cells/mL. This method provides a selective and nontoxic approach towards detection of breast cancer cell lines while it can be developed as a point of care (POC) device for early detection of cancer. Finally, the MCF 7 cancer cells were treated with doxorubicin anti-cancer drug and our device detect the trace amount of MCF 7 based on their electrochemical activity. [ABSTRACT FROM AUTHOR]

Methotrexate (MTX), as a disease modifying antirheumatic drug (DMARD), was first line drug to treat rheumatoid arthritis. However, the severe side effect during long term and high dosage usage limit its application. The aim of this study was to develop dual-functional lipid polymeric hybrid pH-responsive nanoparticles to deliver MTX to inflamed joints selectively. The designed MTX loaded stearic acid-octa-arginine and folic acid decorated poly lactic-co-glycolic acid (PLGA) -PK3-based lipid polymeric hybrid nanoparticles (Sta-R8-FA-PPLPNs/MTX) were composed of PK3, Folate-PEG-PLGA, egg PC, and Sta-R8. The nanoparticles exhibited smooth spherical morphology and particle size of 100–150 nm. The in vitro release study indicated that MTX was released faster in phosphate buffered solution (PBS) of pH 5.0 than that in PBS of pH 7.4 from Sta-R8-FA-PPLPNs/MTX. The cellular uptake study revealed that Sta-R8-FA-PPLPNs/MTX were internalized through folate receptor mediated endocytosis into activated macrophages. Therapeutic effects on adjuvant-induced arthritis (AIA) rats further confirm that Sta-R8-FA-PPLPNs/MTX could be promising against rheumatoid arthritis. [ABSTRACT FROM AUTHOR]

Folate deficiency in pregnancy is associated with neural tube defects, restricted fetal growth and fetal programming of diseases later in life. Fetal folate availability is dependent on maternal folate levels and placental folate transport capacity, mediated by two key transporters, Folate Receptor-α and Reduced Folate Carrier (RFC). We tested the hypothesis that intrauterine growth restriction (IUGR) is associated with decreased folate transporter expression and activity in isolated syncytiotrophoblast microvillous plasma membranes (MVM). Women with pregnancies complicated by IUGR (birth weight <3rd percentile, mean birth weight 1804±110 g, gestational age 35.7±0.61 weeks, n=25) and women delivering an appropriately-for gestational age infant (control group, birth weight 25th-75th centile, mean birth weight 2493±216 g, gestational age 33.9±0.95 weeks, n=19) were recruited and placentas were collected at delivery. MVM was isolated and folate transporter protein expression was measured using Western blot and transporter activity was determined using radiolabelled methyltetrahydrofolic acid and rapid filtration. Whereas the expression of FR-α was unaffected, MVM RFC protein expression was significantly decreased in the IUGR group (-34%, P<.05). IUGR MVM had a significantly lower folate uptake compared to the control group (-38%, P<.05). In conclusion, placental folate transport capacity is decreased in IUGR, which may contribute to the restricted fetal growth and intrauterine programming of childhood and adult disease. These findings suggest that continuation of folate supplementation in the second and third trimester is of particular importance in pregnancies complicated by IUGR. [ABSTRACT FROM AUTHOR]

The folate receptor (FR) is a valued target that is highly expressed in various cancers, which will expedite the development of ligand-receptor binding based cancer therapeutics. In the present investigation, through tissue microarray analysis, we report higher levels of folate receptor expression in prostate cancer (PCa) tissue derived from patients, which were minimal in normal tissue. For folate-receptor based targeted therapy of PCa, we generated novel planetary ball milled (PBM) nanoparticles (NPs) encapsulated with resveratrol (RES), and in combination with docetaxel (DTX) and conjugated with folic acid (FA) on the surface. The cytotoxic effect of FA-conjugated DTX-nanoparticles was found effectual that reduced the concentration of free drug (DTX) to 28 times. Flow cytometry analysis showed a significant increase in the number of apoptotic cells by 30.92% and 65.9% in the FA-conjugated RES and in combination with DTX nanoparticle formulation respectively. However, only 8.9% apoptotic cells were found with control (empty NP). The expressions of NF-kB p65, COX-2, pro (BAX, BAK) and anti-apoptotic (BCL-2, BCL-XL) genes were significantly reduced after treatment with FA-RES + DTX-NP. In addition, the FA-conjugated DTX formulation exhibited additional cytotoxic effects with the down-regulation of survivin and an increased expression of Cleaved Caspase-3 in PCa cells. Further, we observed that treating DTX resistant PCa cells with FA-RES + DTX-NP exhibited a reversal of the ABC-transporter markers thereby limiting the multidrug resistance phenotype of the cancer cells. Our results strongly suggested that FA conjugated nanoparticle drugs acted as effective inhibitors of drug efflux that effectually enhances the intracellular concentration of the drug to exhibit their cytotoxic effect. [ABSTRACT FROM AUTHOR]

Gastric cancer is a malignancy that has a high mortality rate. Although progress has been made in the treatment of gastric cancer, many patients experience cancer recurrence and metastasis. Folate receptor 1 (FOLR1) is overexpressed on the cell surface in over one-third of gastric cancer patients, but rarely is expressed in normal tissue. This makes FOLR1 a potential target for chimeric antigen receptor (CAR) T cell immunotherapy, although the function of FOLR1 has not been elucidated. CAR are engineered fusion receptor composed of an antigen recognition region and signaling domains. T cells expressing CAR have specific activation and cytotoxic effects against cancer cells containing the target antigen. In this study, we generated a CAR that targets FOLR1 composed of a single-chain variable fragment (scFv) of FOLR1 antibody and signaling domains consisting of CD28 and CD3ζ. Both FOLR1-CAR KHYG-1, a natural killer cell line, and FOLR1-CAR T cells recognized FOLR1-positive gastric cancer cells in a MHC-independent manner and induced secretion of various cytokines and caused cell death. Conclusively, this is the first study to demonstrate that CAR KHYG-1/T cells targeting FOLR1 are effective against FOLR1-positive gastric cancer cells. [ABSTRACT FROM AUTHOR]

In this work, a microfluidic paper-based fluorescent sensor device was constructed for quantitative detection of microRNA (miRNA) and determination of the expression of folate receptor (FR) on the cell surface. It is worth pointing out that simultaneous detection of two biomarkers in cancer was achieved through dynamic self-assembly of T-shaped duplex structure. T-shaped duplex structure can respond to specific triggers and convert to an output due to the conformation changes, it is meaningful to fabricate T-shaped duplex structure into powerful biosensors that can monitor chemical stimuli. Moreover, lamellar MnO 2 nanosheets were synthesized, which exhibited excellent quenching ability via adsorbed fluorophore-labeled ssDNA, but the adsorption of the dsDNA was significantly lower than ssDNA because of the shielding of nucleobase pairs by the phosphate groups. This newly designed biosensor provided a sensitive detection of miR-21 from 0.01 to 5.0 fM with a relatively low detection limit of 0.0033 fM. Besides, the present sensing system has a detection of FR in the range from 2.0 to 30.0 ng/mL with the detection limit of 0.667 ng/mL. As biological markers for disease diagnosis, the detection of FR and miRNA will provide a new approach and therapeutic tool for early clinical diagnosis. [ABSTRACT FROM AUTHOR]

Cluster of differentiation (CD)68 may be used as a pan-macrophage or M1 marker, whereas CD163 may be used as an M2 marker. Furthermore, folate receptor (FR)β exhibits an M2-like functional profile. In the present study, CD68 and CD163 were used to evaluate and classify tumor-associated macrophages (TAMs). The expression of CD68, CD163 and FRβ by TAMs in hepatocellular carcinoma (HCC) Tissues was investigated. Samples from 105 patients with HCC were evaluated using immunohistochemistry. The results revealed that CD68 and CD163 overexpression was associated with a worse prognosis. The number of CD68 positive cells observed was significantly higher in patients with stage IV cancer. Furthermore, an increase in CD68 positive cells was observed in patients with median tumor size ≥3.5 cm and in patients with poorly differentiated HCC. The number of CD163 positive cells was also significantly increased in patients with median tumor size ≥3.5 cm and in those with poorly differentiated HCC. A low CD163/68 ratio was correlated with a worse outcome. The ratio was significantly lower in patients with stage IV cancer, patients with des-gamma-carboxy prothrombin abnormalities, patients with blood vessel infiltration and patients with intrahepatic metastasis. The number of FRβ positive cells was not correlated with clinicopathological features. The results of the present study indicate that overexpression of CD68 and CD163 may be associated with a worse patient outcome. The evaluation of CD68 and CD163 positive cells in a cancer microenvironment is controversial. TAMs are not simply cells with single markers or restricted M1 or M2 phenotypes; they are more diverse and heterogeneous. Further studies are required to determine the cross-interaction between diverse TAMs and the tumor microenvironment. [ABSTRACT FROM AUTHOR]

Activated macrophages play a key role in the development and maintenance of inflammatory diseases such as atherosclerosis, lupus, psoriasis, rheumatoid arthritis, ulcerative colitis, and many others. These activated macrophages, but not resting or quiescent macrophages highly up-regulate folate receptor beta (FR-β). This differential expression of FR-β provides a mechanism to selectively deliver imaging and therapeutic agents utilizing folate as a targeting molecule. In an effort to determine whether inflammatory diseases can be targeted utilizing a folate-linked nanosize carrier, a PEG-coated liposome was prepared that incorporated a folate conjugated PEG that also could transport imaging or therapeutic cargo. We demonstrate that these folate-liposomes specifically bind to folate receptor positive cells and accumulate at sites of inflammation in mouse models of colitis and atherosclerosis. These two animal models show that folate-targeted liposomes could be successfully utilized to deliver fluorescent molecules and an anti-inflammatory drug (betamethasone) for diagnostic and therapeutic applications. [ABSTRACT FROM AUTHOR]

Within the last decade, several folate-based radiopharmaceuticals for Single Photon Emission Computed Tomography (SPECT) and Positron Emission Tomography (PET) have been evaluated; however, there is still a lack of suitable 18F-folates for clinical PET imaging. Herein, we report the synthesis and evaluation of two novel 18F-folates employing strain-promoted and copper-catalyzed click chemistry. Furthermore, the influence of both click-methods on lipophilicity and pharmacokinetics of the 18F-folates was investigated. 18F-Ala-folate and 18F-DBCO-folate were both stable in human serum albumin. In vitro studies proved their high affinity to the folate receptor (FR). The lipophilic character of the strain-promoted clicked 18F-DBCO-folate (logD = 0.6) contributed to a higher non-specific binding in cell internalization studies. In the following in vivo PET imaging studies, FR-positive tumors could not be visualized in a maximum intensity projection images. Compared with 18F-DBCO-folate, 18F-Ala-folate (logD = -1.4), synthesized by the copper-catalyzed click reaction, exhibited reduced lipophilicity, and as a result an improved in vivo performance and a clear-cut visualization of FR-positive tumors. In view of high radiochemical yield, radiochemical purity and favorable pharmacokinetics, 18F-Ala-folate is expected to be a promising candidate for FR-PET imaging. [ABSTRACT FROM AUTHOR]

One type of biocompatible nanoparticles functionalized with folate and 99mTc was successfully synthesized. Maleimide‐folic acid (Mal‐FA) was selected to covalently conjugate with ‐SH of the nanoparticles (NPs) to prepare NPs‐FA for targeting. 99mTc was selected to conjugate with ‐NH2 and ‐SH groups of cysteine residues on the surface of NPs to prepare NPs‐FA‐99mTc for radioactive counting. The ability to target folate receptors of NPs‐FA‐99mTc was assessed in uptake studies with folate‐receptor–positive human HepG2 cells. The results showed that the as‐prepared NPs can selectively uptake by folate receptor‐overexpressing HepG2 tumor cells in vitro. The oligomeric hybrid NPs radiolabeled with 99mTc may develop to be SPECT/CT imaging biomaterials with high selectivity. [ABSTRACT FROM AUTHOR]

Clustered regularly interspaced short palindromic repeats (CRISPR)-caspase 9 (Cas9) genome editing technology holds great promise for the field of human gene therapy. However, a lack of safe and effective delivery systems restricts its biomedical application. Here, a folate receptor-targeted liposome (F-LP) was used to deliver CRISPR plasmid DNA co-expressing Cas9 and single-guide RNA targeting the ovarian cancer-related DNA methyltransferase 1 ( DNMT1) gene (gDNMT1). F-LP efficiently bound the gDNMT1 plasmid and formed a stable complex (F-LP/gDNMT1) that was safe for injection. F-LP/gDNMT1 effectively mutated endogenous DNMT1 in vitro, and then expressed the Cas9 endonuclease and downregulated DNMT1 in vivo. The tumor growth of both paclitaxel-sensitive and -resistant ovarian cancers were inhibited by F-LP/gDNMT1, which shows fewer adverse effects than paclitaxel injection. Therefore, CRISPR-Cas9-targeted DNMT1 manipulation may be a potential therapeutic regimen for ovarian cancer, and lipid-mediated delivery systems represent promising delivery vectors of CRISPR-Cas9 technology for precise genome editing therapeutics. [ABSTRACT FROM AUTHOR]

Pulmonary metastasectomy for osteosarcoma provides a select group of patients an opportunity for long-term survival and possible cure. Unfortunately, a complete metastasectomy is challenging due an inability to accurately identify lesions that lay below the threshold of preoperative imaging or intraoperative visual and tactile inspection. Growing evidence suggests that osteosarcomas express a number of unique molecular markers, including the folate receptor alpha. In this case report, we describe the application of a folate receptor-targeted, near-infrared optical contrast agent (OTL38) to improve osteosarcoma localization during minimally invasive pulmonary resection. In addition to localizing preoperatively identified lesions, this technology helped identify additional disease that was undetected on preoperative imaging or with traditional intraoperative techniques. This report marks the first successful utilization of a molecular imaging probe useful for osteosarcomas. This technology may provide a unique approach to improve pulmonary metastasectomy of osteosarcomas. [ABSTRACT FROM AUTHOR]

The 68Ga is a positron‐emitting radionuclide that can be combined with bifunctional chelating agents and bioactive substances for use as positron‐emission tomography (PET) diagnostic agents. The HBED‐CC is an acyclic chelating agent that is rapidly labeled with 68Ga under mild conditions. To target cancer cells, bioactive substances can be conjugated to the carboxyl terminus of HBED‐CC. Because folic acid strongly binds to folate receptors that are overexpressed on the surfaces of many types of cancer cells, it was coupled with HBED‐CC through a small polyethylene glycol‐based linker (EDBE) to generate an active, receptor‐selective targeting system. The HBED‐CC‐EDBE‐folate (HCEF) precursor was readily labeled with 68Ga in 5 minutes at room temperature (98% radiochemical yield; 99% radiochemical purity after isolation). In cellular uptake tests, higher uptakes of 68Ga‐HCEF were observed for the CT26 and KB cell lines (which express folate receptors) than for the A549 cell line (which does not). Finally, in vivo micro‐PET measurements over 2 hours of binding in BALB/c mice into which CT26 tumors had been transplanted showed the selective accumulation of 68Ga‐HCEF in the folate receptor‐expressing CT26 tumors. These results confirmed the potential of 68Ga‐HCEF as a PET diagnostic agent for tumors that express folate receptors. [ABSTRACT FROM AUTHOR]

Purpose To characterize folate receptor alpha (FRα) expression in archival and fresh biopsy tumor samples from relapsed ovarian cancer patients. Methods Patients with ovarian tumors amenable to biopsy were eligible to enroll. Eligibility included a minimum requirement of FRα positivity in archival tumor samples (≥ 25% of cells with ≥ 2 + staining intensity). Patients received mirvetuximab soravtansine at 6 mg/kg once every 3 weeks. Core needle biopsies were collected before and after treatment and FRα levels assessed by immunohistochemistry. Descriptive statistics were used to summarize the association between receptor expression and response. Results Twenty-seven heavily pre-treated patients were enrolled. Six individuals (22%) did not have evaluable pre-treatment biopsies due to insufficient tumor cells. The concordance of FRα expression in archival and biopsy tissues was 71%, and no major shifts in receptor expression were seen in matched pre- and post-treatment biopsy samples. Adverse events were generally mild (≤ grade 2) with keratopathy (48%), fatigue (44%), diarrhea, and blurred vision (each 37%) being the most common treatment-related toxicities. The confirmed objective response rate (ORR) was 22%, including two complete responses and four partial responses. Superior efficacy measures were observed in the subset of patients with the highest FRα levels (ORR, 31%; progression-free survival, 5.4 months). Conclusion Concordance of FRα expression in biopsy versus archival tumor samples suggests that archival tissue can reliably identify patients with receptor-positive tumors and is appropriate for patient selection in mirvetuximab soravtansine clinical trials. Regardless of the tissue source analyzed, higher FRα expression was associated with greater antitumor activity. [ABSTRACT FROM AUTHOR]

Folate receptor (FR)-mediated drug delivery is a promising approach for active targeting of drugs to the FR-positive tumor cells. Bleomycin (BLM) is an antitumor antibiotic with poor therapeutic activity as a result of its limited diffusion into tumor cells. The aim of this study was to investigate whether FR-targeted PEGylated nanoliposomes (FPNL) can effectively deliver BLM to tumor cells and enhance its in vitro and in vivo efficacy. FPNL and PNL (non-targeted) were prepared by thin film hydration method, and their physiochemical properties, cellular uptake, tissue distribution and tumor inhibitory effects were investigated. In Lewis lung cancer (LLC1) cells, FPNL containing BLM showed 2.38-fold and 3.26-fold higher cytotoxicity compared to PNL-BLM and free BLM, respectively. Moreover, the uptake of FPNL by these cells was increased as compared to the PNL. Furthermore, FPNL showed significantly higher tumor distribution of BLM in the LLC1 cells and more tumor inhibition efficacy compared to free BLM and PNL. Both formulations of nanoliposomes had longer plasma half-life than that of free BLM. Therefore, FPNL may be suitable carriers for targeted drug delivery to FR-positive tumor cells. [ABSTRACT FROM AUTHOR]

The fundamentals of folic acid and folate receptors functioning in the body, changes in the expression level of folate receptors in carcinogenesis, as well as use of folic acid and its derivatives for targeted delivery of photosensitizers to tumors have been reviewed. The ways of increasing the efficacy of photodynamic therapy by creating multifunctional nanoplatforms that ensure both passive targeting and receptor-mediated internalization of photosensitizers in tumor cells have been discussed. [ABSTRACT FROM AUTHOR]

Diagnosis and therapy exploiting overexpressed receptors on the cell surface is one important strategy in medicine. Determination of the over expression level of a particular receptor is prerequisite for it to be of clinical use. Differentiation between FR-positive (FR=folate receptor) and -negative cells via fluorescence microscopy using a substrate mimetic fluorophore is presented in this work. The strategy adopted here is not the classical FA-conjugated (FA=folic acid) fluorescent probe but a small and environment-sensitive pterin-based (pterin is part of folate, i.e., vitamin B9) fluorescent probe. Electronically diverse pterin-based fluorescent probes have been designed and synthesized to understand the effect of the binding environment on the receptor-substrate interactions. By utilizing steady-state UV/Vis and fluorescence along with time-resolved fluorescence spectroscopy, the effects on the electronic and acid-base properties of the substrate were investigated. Evidently, one synthesized probe showed FA-mimicking behavior with strong binding interaction with FR. [ABSTRACT FROM AUTHOR]

Folate-receptor-positive activated macrophages are critical for the development and maintenance of many chronic inflammatory and autoimmune diseases. Previously, small-molecule folate-targeted conjugates were found to specifically bind to these activated macrophages in vitro and selectively accumulate at sites of inflammation in vivo. While these small-molecule conjugates have shown promise, the use of a folate-targeted, higher cargo capacity nanovehicle may prove superior in delivering imaging or therapeutic agents in vivo. This nanoparticle strategy has been demonstrated in oncology, where targeted dendrimers have shown superior delivery capabilities; however, little research has been pursued in the area of folate-targeted dendrimers for inflammation and autoimmune diseases. Therefore, we endeavored to create a folate-decorated dendrimer to explore its uptake in mouse models of ulcerative colitis and atherosclerosis. We demonstrate that our final poly(ethylene glycol)-coated, acetic-anhydride-capped, folate-targeted poly(amidoamine) dendrimer exhibits no discernible cytotoxicity in vitro, specifically binds to a folate-receptor-expressing macrophage cell line in vitro, and selectively accumulates in areas of inflammation in vivo. [ABSTRACT FROM AUTHOR]

Bufalin (BF), a traditional Chinese medicine, exhibited inhibitory activities against a broad spectrum of tumor cells. The present study elaborates that bufalin was successfully encapsulated into the cavity of β-cyclodextrin (β-CD), which was determined by Fourier transform infrared spectroscopy (FT-IR), proton nuclear magnetic resonance spectroscopy ( 1 H NMR), differential scanning calorimetry (DSC) and scanning electron microscopy (SEM). The best reaction mole ratio of BF/β-CD was 1:5. The solubilities of bufalin in water and phosphate buffer solution (pH = 7.4) were increased up to 24 and 34 times after encapsulated into the cavity of β-CD respectively. The inclusion efficiency (IE) and drug loading (DL) of bufalin in the inclusion complex were (94.22 ± 0.85)% and (14.11 ± 0.20)%, respectively. Then β-CD conjugated with folic acid (FA) were further prepared and employed to improve the anti-tumor efficacy of inclusion complex. The in vitro dissolution and solubility study showed better values of inclusion complex and FA targeted inclusion complex than that of pure BF. Cytotoxicity experiments by using HCT116 cell line revealed that the antitumor efficiency of bufalin were enhanced more than two folds in the presence of β-CD and folate conjugated β-CD (FA-PEI-β-CD), which demonstrated the potential application of β-CD (FA-PEI-β-CD) as delivery vehicles of bufalin for antitumor therapy. [ABSTRACT FROM AUTHOR]

The current study utilizes folic acid conjugated poly(styrene-co-maleic anhydride) block copolymer (FA-SMA) to enhance the solubility of a hydrophobic but very potent synthetic curcumin-difluorinated (CDF) analog and its targeted delivery to folate receptor-alpha overexpressing cancers. The nanomicelles showed high aqueous solubility. Importantly, the encapsulation of CDF in nanomicelles resulted in high photo-stability of the otherwise photo-labile drug. When the nanomicelles were tested in folate-receptor overexpressing ovarian and cervical cancer cells they exhibited high anticancer activity causing significant cell population to undergo apoptosis due to upregulation of tumor suppressor phosphatase and tensin homolog (PTEN) and inhibition of nuclear factor kappa-B (NFκB), which further confirmed the targeting ability and anticancer potentials of folate-targeted formulations. [ABSTRACT FROM AUTHOR]

Background: Mirvetuximab soravtansine (IMGN853) is an antibody-drug conjugate that selectively targets folate receptor α (FRα). In this phase 1 dose-escalation study, the authors investigated IMGN853 in patients with FRα-positive solid tumors.Methods: Patients received IMGN853 on day 1 of a 21-day cycle (once every 3 weeks dosing), with cycles repeated until patients experienced dose-limiting toxicity or progression. Dose escalation commenced in single-patient cohorts for the first 4 planned dose levels and then followed a standard 3 + 3 scheme. The primary objectives were to determine the maximum tolerated dose and the recommended phase 2 dose. Secondary objectives were to determine safety and tolerability, to characterize the pharmacokinetic profile, and to describe preliminary clinical activity.Results: In total, 44 patients received treatment at doses escalating from 0.15 to 7.0 mg/kg. No meaningful drug accumulation was observed with the dosing regimen of once every 3 weeks. The most common treatment-related adverse events were fatigue, blurred vision, and diarrhea, the majority of which were grade 1 or 2. The dose-limiting toxicities observed were grade 3 hypophosphatemia (5.0 mg/kg) and grade 3 punctate keratitis (7.0 mg/kg). Two patients, both of whom were individuals with epithelial ovarian cancer, achieved confirmed tumor responses according to Response Evaluation Criteria in Solid Tumors 1.1, and each was a partial response.Conclusions: IMGN853 demonstrated a manageable safety profile and encouraging preliminary clinical activity, particularly in patients with ovarian cancer. The results establish a recommended phase 2 dosing of 6.0 mg/kg (based on adjusted ideal body weight) once every 3 weeks. Cancer 2017. © 2017 American Cancer Society. Cancer 2017;123:3080-7. © 2017 American Cancer Society. [ABSTRACT FROM AUTHOR]

Combination chemotherapy is a promising method of improving cancer treatment, but the distinct pharmacokinetics of combined drugs and non-specific drug distribution slow down the development in the clinic. In this study, folate (FA) receptor-targeted polymersomes with apparent bilayered lamellar structure were successfully developed to co-encapsulate a hydrophobic-hydrophilic chemotherapeutic drug pair (PTX and DOX) in a single vesicle for enhancing the combination chemotherapeutic effect. Hydrophobic PTX was loaded into the thick hydrophobic lamellar membrane by the self-assembly of triblock copolymer PCL 8000 -PEG 8000 -PCL 8000 , while hydrophilic DOX was encapsulated into the hydrophilic reservoir using a trans-membrane ammonium sulfate gradient method. In vitro release study indicated that the drugs were released from the polymersomes in a controlled and sustained manner. Cellular uptake study indicated that FA-targeted Co-PS had higher internalization efficiency in FA receptor-overexpressing BEL-7404 cells than non-targeted Co-PS. In vitro cytotoxicity assay demonstrated that FA-targeted Co-PS exhibited less cytotoxic effect than free drug cocktail, but suppressed the growth of tumor cells more efficiently than non-targeted Co-PS. Ex vivo imaging biodistribution studies revealed that FA-targeted Co-PS led to highly efficient targeting and accumulation in the BEL-7404 xenograft tumor. Furthermore, the in vivo antitumor study showed that the combination chemotherapy of polymersomes to BEL-7404 tumor via intravenous injection was superior to free drug cocktail treatment, and the FA-targeted Co-PS exhibited significantly higher tumor growth inhibition than non-targeted Co-PS group. Therefore, the newly developed FA-targeted co-delivery polymersomes hold great promise for simultaneous delivery of multiple chemotherapeutics and would have great potential in tumor-targeting and combination chemotherapy. Statement of Significance Combination chemotherapy is a promising method of improving cancer treatment, but the distinct pharmacokinetics of combined drugs and non-specific drug distribution slow down the development in the clinic. In our study, novel folate-targeted co-delivery polymersomes (Co-PS) were successfully developed to encapsulate a hydrophobic-hydrophilic chemotherapeutic drug pair (paclitaxel and doxorubicin) into the different compartments of the vesicle. In vivo studies revealed that the combination chemotherapy of polymersomes to BEL-7404 xenograft tumor via intravenous injection was superior to free drug cocktail treatment, and the FA-targeted Co-PS exhibited significantly higher tumor growth inhibition than non-targeted Co-PS group. Therefore, the newly developed FA-targeted co-delivery polymersomes hold great promise for simultaneous delivery of multiple chemotherapeutics and would have great potential in tumor-targeting and combination chemotherapy. [ABSTRACT FROM AUTHOR]

A rapid, simple, and novel method for folate receptor α (FRα) determination is reported here. A probe of gold nanoparticles (Au NPs) modified with anti-FRα antibody was synthesized under the optimized conditions first. The antibody-modified Au NPs would aggregate when FRα was added to the probe for the specific interaction between antibody and antigen, resulting in the enhancement of resonance Rayleigh scattering (RRS) intensity. There is a linear relationship between the change of RRS intensity (ΔIRRS) and the concentration of FRα, with the detecting range of 0.50–37.50 ng·mL−1 and the limit of determination of 0.05 ng·mL−1. The determination of FRα in serum samples was realized with the advantages of high selectivity, high sensitivity, and easy operation. [ABSTRACT FROM AUTHOR]

Folate receptor (FR) is overexpressed in many epithelial cancers and tumor-associated macrophages (TAMs), which enable it to function as an appropriate target for cancer treatment. We have successfully synthesized multifunctional folate-targeted and oxygen/paclitaxel loaded microbubbles (TOPLMBs) for ultrasound (US) mediated delivery for combination therapy in an intraperitoneal ovarian cancer xenograft model. The TOPLMBs target both ovarian cancer cells and TAMs and provide a promising drug delivery strategy for the combination treatment of ovarian cancer and tumor microenvironment. Microscopic imaging and flow cytometric analysis showed that TOPLMBs significantly penetrated into ovarian cancer cells and tumor-associated macrophages (TAMs) within tumor ascites fluid and the tumor nodules. Immunohistochemical analyses of dissected tumor tissue confirmed the increased tumor apoptosis, the reduced expression of vascular endothelial growth factor (VEGF) and microvascular density (MVD), and the reduced expression of CD68 after treatment (P < 0.05). Our experiment results suggest that intraperitoneal injection of dual-targeting TOPLMBs followed by US mediation provide a promising drug delivery strategy for combination treatment of ovarian cancer and tumor microenvironment with the therapeutic outcome superior to that of conventional therapeutic options. [ABSTRACT FROM AUTHOR]

Microglia, as cellular mediators of neuroinflammation, are implicated in the pathogenesis of a wide range of neurodegenerative diseases. Positron emission tomography (PET) imaging of microglia has matured over the last 20 years, through the development of radiopharmaceuticals targeting several molecular biomarkers of microglial activation and, among these, mainly the translocator protein-18 kDa (TSPO). Nevertheless, current limitations of TSPO as a PET microglial biomarker exist, such as low brain density, even in a neurodegenerative setting, expression by other cells than the microglia (astrocytes, peripheral macrophages in the case of blood brain barrier breakdown), genetic polymorphism, inducing a variation for most of TSPO PET radiopharmaceuticals’ binding affinity, or similar expression in activated microglia regardless of its polarization (pro- or anti-inflammatory state), and these limitations narrow its potential interest. We overview alternative molecular targets, for which dedicated radiopharmaceuticals have been proposed, including receptors (purinergic receptors P2X7, cannabinoid receptors, α7 and α4 β2 nicotinic acetylcholine receptors, adenosine 2A receptor, folate receptor β) and enzymes (cyclooxygenase, nitric oxide synthase, matrix metalloproteinase, β-glucuronidase, and enzymes of the kynurenine pathway), with a particular focus on their respective contribution for the understanding of microglial involvement in neurodegenerative diseases. We discuss opportunities for these potential molecular targets for PET imaging regarding their selectivity for microglia expression and polarization, in relation to the mechanisms by which microglia actively participate in both toxic and neuroprotective actions in brain diseases, and then take into account current clinicians’ expectations. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The purpose of this study was to develop a novel drug delivery system for a sustained and targeted delivery of honokiol (HK) to the nasopharyngeal carcinoma (NPC) HNE-1 cell lines, since the folate receptor (FR) is over-expressed on their surface. Emulsion solvent evaporation was used to develop the active targeting nanoparticles-loaded HK (ATNH) using copolymerpoly (ɛ-caprolactone)-poly (ethyleneglycol)-poly (ɛ-caprolactone) (PCEC), which was modified with folate (FA) by introducing Polythylenimine (PEI). ATNH characterization, including particle size distribution, morphology, drug loading, encapsulation efficiency and drug release, was performed. Transmission electron microscopy (TEM) and Fourier transform infrared spectroscopy (FTIR) were employed to evaluate the shape and construction, respectively. MTT assay, cell uptake study and apoptosis test were assayed to detect the antitumor properties and targeting uptake by HNE-1 cellsin vitro. Cell-cycle redistribution,18 F-FDG PET/CT and immunohistochemistry were performedin vivo. The ATNH we developed were successfully synthesized and showed a suitable size distribution, high encapsulation efficiency, gradual release, and targeting uptake by the cellsin vitro. Moreover, ATNH significantly inhibited tumor growth, metabolism, proliferation, micro-vessel generation, and caused cell-cycle arrest at G1phase. Thus, these nanoparticles we developed might represent a novel formulation for HK delivery and a promising potential therapy in the treatment of cancer. [ABSTRACT FROM AUTHOR]

New folic acid (FA) coupled three layered micelles (3LM) were designed to encapsulate DNA, and their application as delivery system that specifically targets activated macrophages was investigated for new treatment options in rheumatoid arthritis (RA). FA coupled poly( l -lactide)- b -poly(ethylene glycol) (FA-PEG-PLLA) was synthesized via the NHS-ester activated/amine coupling method. Fluorescein labeled folic acid was used for flow cytometric detection of the expression of functional folic receptor β in LPS-activated and resting macrophages. FA coupled 3LM were formulated in a two-step procedure and characterized regarding hydrodynamic diameters and zeta potentials. The presence of the targeting ligand was shown not to increase the size of the 3LM compared to their non-targeted counterparts. Targeted and non-targeted 3LM were used in vitro to optimize uptake conditions in the RAW 264.7 macrophage cell line. The amount of FA coupled polymer in the final formulation was found to be optimal at 75% FA-PEG-PLLA and 25% PLLA-PEG-PLLA. Subsequently, transgene expression in vitro in RAW 264.7 cells and ex vivo in primary activated and resting mouse macrophages was determined as a function of FR-mediated internalization of 3LM encapsulating GFP expressing plasmid. FR-overexpressing activated cells, as successfully identified by internalization of FA-fluorescein, showed significantly higher GFP expression in vitro and ex vivo than resting macrophages with only a basal level of FR expression. Lastly, injectable hydrogels as depot formulation were formed by stereocomplexation, and their degradation, DNA release profiles, and dissociation into intact 3LM were found to be beneficial for potential in vivo application. Our findings confirm that FA-3LM are taken up by activated macrophages via folate receptor mediated endocytosis and that their hydrogels release intact 3LM for efficient transfection of primary macrophages. Therefore, FA-3LM could become a promising delivery system for receptor-mediated drug or gene delivery and novel therapy for rheumatoid arthritis in an in situ forming gel formulation. [ABSTRACT FROM AUTHOR]

Small interfering RNA (siRNA) promises high efficacy and excellent specificity to silence the target gene expression, which shows potential for cancer treatment. However, systemic delivery of siRNA with selectivity to the tumor site and into the cytosol of tumor cells remains a major limitation. To achieve this, we generated oligoaminoamide-based sequence-defined polycationic oligomers by solid-phase assisted synthesis, which can form polyplexes with anionic siRNA by electrostatic interaction to serve as siRNA carrier. Targeting for folate receptor (FR)-overexpressing tumors, we optimized the physicochemical properties of polyplexes by combinatorial optimization of PEGylated folate-conjugated oligomer (for FR targeting and shielding of surface charges) and 3-arm oligomer (for size modification and particle stability). For uni-directional fast coupling between the two groups of oligomers, we activated the cysteine thiol groups of one of the oligomers with 5,5′-dithio-bis(2-nitrobenzoic acid) to achieve a fast chemical linkage through disulfide formation with the free thiol groups of the other oligomer. These targeted combinatorial polyplexes (TCPs) are homogeneous spherical particles with favorable size and surface charge, which showed strong siRNA binding activity. TCPs were internalized into cells by FR-mediated endocytosis, triggered significant eGFP-luciferase marker gene silencing, and transfection with antitumoral EG5 siRNA suppressed cell proliferation in FR-expressing tumor cells. Moreover, the most promising formulation TCP1 after i.v. administration in tumor-bearing mice exhibited siRNA delivery into the tumor, resulting in EG5 gene silencing at mRNA level. Therefore, by covalent combination of two sequence-defined functional oligomers, we developed a siRNA carrier system with optimized size and surface charge for efficient tumor cell-directed gene silencing and cytotoxicity in vitro and in vivo . [ABSTRACT FROM AUTHOR]

In this work, we report a facile one-pot strategy for protein detection based on sterically and allosterically tunable hybridization chain reaction (HCR). In our strategy, DNA hairpins H1 and H2 are dual-labeled with pyrene moieties through a six-carbon-atom spacer at each end; and a single-stranded DNA primer is designed to contain two small molecules near each end. In the absence of target protein, the primer can trigger HCR events between alternating H1 and H2 hairpins to form a nicked double-helix. As a result, the pyrene excimers are formed to emit at approximately 485 nm. On the contrary, upon binding of the specific target protein onto the primer through the protein-small molecule interaction, the HCR will be inhibited due to the steric and allosteric effect. The changes of the fluorescent signals of pyrene excimers are in response to the concentration of target protein, so that the detection of protein can be realized. We have demonstrated the feasibility of this strategy by using streptavidin (SA) and folate receptor (FR) as model targets. Results show that both of them can be well detected with a detection limit of 1.07 nM and 2.7 nM, respectively. The developed method for protein assay is flexible, so we infer that the one-pot strategy holds great potential for the detection of other proteins. [ABSTRACT FROM AUTHOR]

The determination of folate receptor (FR) that over expressed in vast quantity of cancerous cells frequently is significant for the clinical diagnosis and treatment of cancers. Many DNA-based electrochemical biosensors have been developed for FR detection with high selectivity and sensitivity, but most of them need complicated immobilization of DNA on the electrode surface firstly, which is tedious and therefore results in the poor reproducibility. In this study, a simple, sensitive, and selective electrochemical FR biosensor in cancer cells has been proposed, which combines the advantages of the convenient immobilization-free homogeneous indium tin oxide (ITO)-based electrochemical detection strategy and the high selectivity of the terminal protection of small molecule linked DNA. The small molecule of folic acid (FA) and an electroactive molecule of ferrocence (Fc) were tethered to 3′- and 5′-end of an arbitrary single-stranded DNA (ssDNA), respectively, forming the FA-ssDNA-Fc complex. In the absence of the target FR, the FA-ssDNA-Fc was degraded by exonuclease I (Exo I) from 3′-end and produced a free Fc, diffusing freely to the ITO electrode surface and resulting in strong electrochemical signal. When the target FR was present, the FA-ssDNA-Fc was bound to FR through specific interaction with FA anchored at the 3′-end, effectively protecting the ssDNA strand from hydrolysis by Exo I. The FR-FA-ssDNA-Fc could not diffuse easily to the negatively charged ITO electrode surface due to the electrostatic repulsion between the DNA strand and the negatively charged ITO electrode, so electrochemical signal reduced. The decreased electrochemical signal has a linear relationship with the logarithm of FR concentration in range of 10 fM to 10 nM with a detection limit of 3.8 fM (S/N=3). The proposed biosensor has been applied to detect FR in HeLa cancer cells, and the decreased electrochemical signal has a linear relationship with the logarithm of cell concentration ranging from 100–10000 cell/mL. Compared with the traditional heterogeneous electrochemical FR biosensors, the proposed biosensor owns the merits of the simplicity and high specificity, presenting the great potential application in the area of early diagnosis of cancers. [ABSTRACT FROM AUTHOR]

Breast cancer is a diverse and complex disease that remains one of the leading causes of death among women. Novel, outside-of-the-box imaging and treatment methods are needed to supplement currently available technologies. In this study, we present evidence for the intracellular delivery and ultrasound-stimulated activation of folate receptor (FR)-targeted phase-change contrast agents (PCCAs) in MDA-MB-231 and MCF-7 breast cancer cells in vitro . PCCAs are lipid-coated, perfluorocarbon-filled particles formulated as nanoscale liquid droplets capable of vaporization into gaseous microbubbles for imaging or therapy. Cells were incubated with 1:1 decafluorobutane (DFB)/octafluoropropane (OFP) PCCAs for 1 h, imaged via confocal microscopy, exposed to ultrasound (9 MHz, MI = 1.0 or 1.5), and imaged again after insonation. FR-targeted PCCAs were observed intracellularly in both cell lines, but uptake was significantly greater (p < 0.001) in MDA-MB-231 cells (93.0% internalization at MI = 1.0, 79.5% at MI = 1.5) than MCF-7 cells (42.4% internalization at MI = 1.0, 35.7% at MI = 1.5). Folate incorporation increased the frequency of intracellular PCCA detection 45-fold for MDA-MB-231 cells and 7-fold for MCF-7 cells, relative to untargeted PCCAs. Intracellularly activated PCCAs ranged from 500 nm to 6 μm (IQR = 800 nm–1.5 μm) with a mean diameter of 1.15 ± 0.59 (SD) microns. The work presented herein demonstrates the feasibility of PCCA intracellular delivery and activation using breast cancer cells, illuminating a new platform toward intracellular imaging or therapeutic delivery with ultrasound. [ABSTRACT FROM AUTHOR]

Supplemental digital content is available in the text. Objective: This exploratory analysis evaluated the incidence of adverse events (AEs) by folate receptor (FR) status in the randomized, multicenter, open-label PRECEDENT study in women with platinum-resistant ovarian cancer receiving pegylated liposomal doxorubicin (PLD) ± the small-molecule drug conjugate vintafolide. Methods: Women 18 years or older with platinum-resistant ovarian cancer were randomized 2:1 to vintafolide (2.5 mg intravenously, 3 times per week, weeks 1 and 3, every 28 days) + PLD (50 mg/m2 intravenously, day 1, every 28 days) or PLD alone (same dose/schedule). The expression of functionally active FR was evaluated by single-photon emission computed tomography with etarfolatide. Patients were categorized according to FR positivity: patients with all target lesions positive for FR expression (FR 100%), patients with 1 or more but not all target lesions positive for FR expression (FR 10%–90%), and patients with all lesions negative for FR expression (FR 0%). Results: Data on FR status were available for 94 patients: 38 were FR 100%, 36 were FR 10% to 90%, and 20 were FR 0%. Across all FR subgroups, the duration of treatment was longer, and the number of cycles was higher in combination-therapy arms than PLD-alone arms. Although the frequency of AEs was relatively consistent across subgroups, the FR 100% subgroup had a higher incidence of patients with at least 1 AE for combination therapy versus PLD alone. No surprising safety signals were shown according to FR status. The incidence of grade 3 or 4 treatment-emergent drug-related AEs was generally low across all FR subgroups and treatment arms. Conclusions: This exploratory analysis suggests that FR status does not influence the AE profile of vintafolide + PLD combination therapy or PLD alone in patients with platinum-resistant ovarian cancer. Future a priori analyses in larger populations are needed to confirm these findings. [ABSTRACT FROM AUTHOR]

We report the development and characterization of a novel nanometric system for specific delivery of therapeutic siRNA for cancer treatment. This vector is based on a binary mixture of the cationic surfactant dioctadecyldimethylammonium chloride (DODAC) and thehelperlipid monoolein (MO). These liposomes were previously validated by our research group as promising non-viral vectors for nucleic acid delivery. In this work, the DODAC:MO vesicles were for the first time functionalized with polyethylene glycol and PEG-folate conjugates to achieve both maximal stability in biological fluids and increase selectivity toward folate receptor α expressing cells. The produced DODAC:MO:PEG liposomes were highly effective in RNA complexation (close to 100%), and the resulting lipoplexes also demonstrated high stability in conditions simulating their administration by intravenous injection (physiological pH, high NaCl, heparin and fetal bovine serum concentrations). In addition, cell uptake of the PEG-folate-coated lipoplexes was significantly greater in folate receptor α positive breast cancer cells (39% for 25 µg/mL of lipid and 31% for 40 µg/mL) when compared with folate receptor α negative cells (31% for 25 µg/mL of lipid and 23% for 40 µg/mL) and to systems without PEG-folate (≈13% to 16% for all tested conditions), supporting their selectivity towards the receptor. Overall, the results support these systems as appealing vectors for selective delivery of siRNA to cancer cells by folate receptor α-mediated internalization, aiming at future therapeutic applications of interest. [ABSTRACT FROM PUBLISHER]

To assess targeting of an epothilone folate conjugate (BMS-753493) to the folate receptor (FR)-overexpressed tumor in mice bearing both FR+ and FR– tumors, a series of experiments were conducted by quantitative whole-body autoradiography (QWBA) and LC–MS/MS following i.v. administration of BMS-753493 or its active moiety, BMS-748285 in mice bearing FR+ (98M109) and FR– (M109) tumors. QWBA showed [ 3 H]BMS-753493–derived radioactivity was extensively distributed to various tissues. The FR over-expressing 98M109 tumors showed consistently higher level of radioactivity than FR-negative tumors ( i.e ., M109 tumors) up to 48 h post dose of [ 3 H]BMS-753493, despite the magnitude of difference between the tumors is relatively small (generally 3~5-fold). The radioactivity level in 98M109 tumors was 2~12-fold of normal tissues except intestine/content at 48 h post dose. No selective radioactivity uptake into 98M109 tumors over M109 or normal tissues was observed after i.v. administration of the active epothilone, [ 3 H]BMS-748285. LC–MS/MS measurements demonstrated that the concentrations of BMS-748285, presumably from hydrolysis of the folate conjugate, in 98M109 tumors were greater than those in M109 tumors after i.v. administration of BMS-753493 (2–3-fold) whereas no differential uptake in the tumors following BMS-748285 administration. Those data were consistent with radioactivity determinations. Those results demonstrated that the folate conjugation in BMS-753493 enabled moderately preferential distribution of the active epothilone to FR over-expressing 98M109 tumors, thereby supporting targeted delivery of cytotoxics through the folate receptor. [ABSTRACT FROM AUTHOR]

Background: In this retrospective study we evaluated the respective correlations and clinical relevance of FOLR1 mRNA expression, FOLR1 promoter specific methylation and global DNA hypomethylation in type I and type II ovarian cancer.Methods: Two hundred fifty four ovarian cancers, 13 borderline tumours and 60 samples of healthy fallopian epithelium and normal ovarian epithelium were retrospectively analysed for FOLR1 expression with RT-PCR. FOLR1 DNA promoter methylation and global DNA hypomethylation (measured by means of LINE1 DNA hypomethylation) were evaluated with MethyLight technique.Results: No correlation between FOLR1 mRNA expression and its specific promoter DNA methylation was found neither in type I nor in type II cancers, however, high FOLR1 mRNA expression was found to be correlated with global DNA hypomethylation in type II cancers (p = 0.033). Strong FOLR1 mRNA expression was revealed for Grades 2-3, FIGO stages III-IV, residual disease > 0, and serous histotype. High FOLR1 expression was found to predict increased platinum sensitivity in type I cancers (odds ratio = 3.288; 1.256-10.75; p = 0.020). One-year survival analysis showed in type I cancers an independent better outcome for strong expression of FOLR1 in FIGO stage III and IV. For the entire follow up period no significant independent outcome for FOLR1 expression was revealed. In type I cancers LINE 1 DNA hypomethylation was found to exhibit a worse PFS and OS which were confirmed to be independent in multivariate COX regression model for both PFS (p = 0.026) and OS (p = 0.012).Conclusion: No correlations were found between FOLR1 expression and its specific promoter methylation, however, high FOLR1 mRNA expression was associated with DNA hypomethylation in type II cancers. FOLR1 mRNA expression did not prove to predict clinical outcome in type II cancers, although strong FOLR1 expression generally denotes ovarian cancers with highly aggressive phenotype. In type I cancers, however, strong FOLR1 expression has been found to be a reliable indicator of improved platinum responsiveness reflecting a transient better one-year follow up outcome in highly FOLR1 expressing type I cancers. An independent prognostic role of global DNA hypomethylation was demonstrated in type I tumours. [ABSTRACT FROM AUTHOR]

Ribosome-inactivating proteins (RIPs) are enzymes (3.2.2.22) that possess N-glycosilase activity that irreversibly inhibits protein synthesis. RIPs have been found in plants, fungi, algae, and bacteria; their biological role is still under investigation, even if it has been recognized their role in plant defence against predators and viruses. Nevertheless, several studies on these toxins have been performed to evaluate their applicability in the biomedical field making RIPs selectively toxic towards target cells. Indeed, these molecules are extensively used to produce chimeric biomolecules, such as immunotoxins or protein/peptides conjugates. However, to date, clinical use of most of these bioconiujates has been limited by toxicity and immunogenicity. More recently, material sciences have provided a wide range of nanomaterials to be used as excellent vehicles for toxin-delivery, since they are characterized by improved stability, solubility, and in vivo pharmacokinetics. This review discusses progresses in the development of RIPs bioconjugates, with particular attention to the recent use of nanomaterials, whose appropriate design opens up a broad range of different possibilities to the use of RIPs in novel therapeutic approaches in human diseases. [ABSTRACT FROM AUTHOR]

Background: The poor prognosis and the limited efficacy of targeted therapy in patients with triple-negative breast cancer (TNBC) have raised the need for alternative therapies. Recent studies have demonstrated that folate receptor-alpha (FRα) may represent an ideal tumor-associated marker for immunotherapy for TNBC. Methods: The aim of the present study was to apply a chimeric antigen receptor (CAR) approach for the targeting of FRα expressed on TNBC cells and evaluate the antitumor activity of CAR-engineered T cells in vitro and in vivo. Results: We found that human T cells expressing a FRα-specific CAR were potent and specific killers of TNBC cells that express moderate levels of FRα in vitro and significantly inhibited tumor outgrowth following infusion into immunodeficient mice bearing an MDA-MB-231 tumor xenograft. However, the antitumor activity of the FRα CAR T cells was modest when compared to the same CAR T cells applied in an ovarian tumor xenograft model where FRα expression is more abundant. Notably, FRα CAR T cells induced superior tumor regression in vivo against MDA-MB-231 that was engineered for overexpression of FRα. Conclusions: Taken together, our results show that FRα CAR T cells can mediate antitumor activity against established TNBC tumor, particularly when FRα is expressed at higher levels. These results have significant implications for the pre-selection of patients with high antigen expression levels when utilizing CAR-based adoptive T cell therapies of cancer in future clinical trials. [ABSTRACT FROM AUTHOR]

Background: A folate-receptor-targeted poly (lactide-co-Glycolide) (PLGA)-Polyethylene glycol (PEG) nanoparticle is developed for encapsulation and delivery of disulfiram into breast cancer cells. After a comprehensive characterization of nanoparticles, cell cytotoxicity, apoptosis induction, cellular uptake and intracellular level of reactive oxygen species are analyzed. In vivo acute and chronic toxicity of nanoparticles and their efficacy on inhibition of breast cancer tumor growth is studied. Results: The folate-receptor-targeted nanoparticles are internalized into the cells, induce reactive oxygen species formation, induce apoptosis and inhibit cell proliferation more efficiently compared to the untargeted nanoparticles. The acute and toxicity test show the maximum dose of disulfiram equivalent of nanoparticles for intra-venous injection is 6 mg/kg while show significant decrease in the breast cancer tumor growth rate. Conclusion: It is believed that the developed formulation could be used as a potential vehicle for successful delivery of disulfiram, an old and inexpensive drug, into breast cancer cells and other solid tumors. [ABSTRACT FROM AUTHOR]

Folate receptor (FR) α expression in normal tissues is restricted to a subpopulation of epithelial cells. In contrast, FRa is overexpressed in epithelial ovarian cancer (EOC) and non-small-cell lung carcinoma. Therefore, FRα is considered a promising therapeutic target for EOC and non-small-cell lung carcinoma. Farletuzumab (MORAb-003) is a humanized monoclonal antibody of immunoglobulin G subtype 1 kappa, targeting human FRα. To date, Phase I/II clinical trials have clearly demonstrated the feasibility and safety of farletuzumab as a treatment option against solid tumors. However, in Phase III clinical trial that was conducted to verify the combined effect of paclitaxel-carboplatin combination therapy and farletuzumab for patients with recurrent EOC, improvement in progression-free survival was not statistically significant. This result might be owing to the fact that the eligibility criteria for these studies did not include FRα expression. The significance of FRα as a predictive/prognostic biomarker remains unclear. In addition, there is currently no established biomarker to predict the response and toxicities among patients receiving farletuzumab therapy. Furthermore, the primary mechanism of action of farletuzumab has not yet been identified. Therefore, further research to identify the mechanism of farletuzumab in tumor suppression is necessary to clarify the full potential of this chemotherapeutic agent. [ABSTRACT FROM AUTHOR]

Folic acid-conjugated d -α-tocopheryl polyethylene glycol 1000 succinate (TPGS–FOL) decorated methotrexate (MTX)-conjugated nanoparticles were developed for targeted delivery of MTX to folate receptor-expressed tumor cells. The synthesis of TPGS–FOL followed 3-step process. Firstly, the terminal hydroxyl group of TPGS was converted to sulfonyl chloride using mesyl chloride in comparison with nosyl and tosyl chlorides. The highest conversion efficiency and yield were obtained by mesyl chloride due to the formation of higher reactive intermediate in a presence of triethylamine. Secondly, the substitution of sulfonyl group by sodium azide produced considerably high yield with conversion efficiency of over 90%. Lastly, the coupling reaction of azido-substituted TPGS and propargyl folamide by click reaction resulted in 96% conjugation efficiency without polymer degradation. To fabricate the folate receptor-targeted nanoparticles, 10 and 20%mol MTX-conjugated PEGylated poly(ϵ-caprolactone) nanoparticles were decorated with TPGS–FOL. The size and size distribution of MTX-conjugated nanoparticles relatively increased with %MTX. The MTX release from the nanoparticles was accelerated in acidic medium with an increase of %MTX but retarded in physiological pH medium. The decoration of TPGS–FOL onto the nanoparticles slightly enlarged the size and size distribution of the nanoparticles; however, it did not affect the surface charge. The cytotoxicity and cellular uptake of MCF-7 cells demonstrated that 10% MTX-conjugated nanoparticles and FOL-decorated nanoparticles possessed higher toxicity and uptake efficiency than 20% MTX-conjugated nanoparticles and undecorated nanoparticles, respectively. The results indicated that FOL-10% MTX-conjugated nanoparticles exhibited potential targeted delivery of MTX to folate receptor-expressed cancer cells. [ABSTRACT FROM AUTHOR]

Introduction Application of therapeutic folate radioconjugates is a promising option for the treatment of folate receptor (FR)-positive tumors, although high uptake of radiofolates in the kidneys remains a critical issue. Recently, it was shown that enhancing the blood circulation of radiofolates results in increased tumor uptake and reduced retention of radioactivity in the kidneys. In this study, we investigated and compared the anti-tumor effects and potential long-term damage to the kidneys after application of an albumin-binding ( 177 Lu-cm09), and a conventional ( 177 Lu-EC0800) folate radioconjugate. Methods In vivo studies were performed with KB tumor-bearing nude mice. 177 Lu-EC0800 and 177 Lu-cm09 were applied at variable quantities (10–30 MBq/mouse), and the tumor growth was monitored over time. Mice without tumors were injected with the same radiofolates and investigated over eight months by determination of creatinine and blood urea nitrogen plasma levels and by measuring renal uptake of 99m Tc-DMSA using SPECT. At the study end, the morphological changes were examined on renal tissue sections using variable staining methods. Results Compared to untreated controls, dose-dependent tumor growth inhibition and prolonged survival was observed in all treated mice. In line with the resulting absorbed dose, the treatment was more effective with 177 Lu-cm09 than with 177 Lu-EC0800, enabling complete tumor remission after application of ≥ 20 MBq (≥ 28 Gy). Application of radiofolates with an absorbed renal dose ≥ 23 Gy showed increased levels of renal plasma parameters and reduced renal uptake of 99m Tc-DSMA. Morphological changes observed on tissue sections confirmed radionephropathy of variable stages. Conclusions 177 Lu-cm09 showed more favorable anti-tumor effects and significantly less damage to the kidneys compared to 177 Lu-EC0800 as was expected based on improved tumor-to-kidney ratios. It was demonstrated that enhancing the blood circulation time of radiofolates was favorable regarding the risk–benefit profile of a therapeutic application. These results hold promise for future translation of the albumin-binder concept to the clinics, potentially enabling FR-targeted radionuclide therapy in patients. [ABSTRACT FROM AUTHOR]