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The use of FR + CTC to distinguish lung cancer from benign lung disease has been well studied. However, the effective method to differentiate precursor glandular lesions from benign/malignant pulmonary diseases is rare.380 patients with indeterminate pulmonary nodules were prospectively recruited. Peripheral blood samples were collected from all participants before surgery for analyzing FR + CTC levels. The performance of FR + CTC to identify lung precursor lesions were analyzed by receiver operating characteristic (ROC) curve.FR + CTC can effectively differentiate precursor from benign pulmonary diseases in all included patients (cutoff: 9.22 FU/3 ml, AUC = 0.807, (p 0.0001, sensitivity: 69.17%, specificity: 82.46%) and patients with single pulmonary lesion (cutoff: 9.03 FU/3 ml, AUC = 0.842, p = 0.0001, sensitivity: 75.20%, specificity: 83.00%). However, FR + CTC cannot differentiate precursor from benign pulmonary diseases in multiple lesions patients (p = 0.110). FR + CTC neither differentiate precursor from malignant pulmonary lesions in all included patients (p = 0.715), single nor multiple lesions patients (p = 0.867, p = 0.692, respectively). Total number of pulmonary nodules, MTD, location (lower vs upper) were independent risk factors for malignancy (AOR, 95% CI: 3.104 (1.525, 6.316), 3.148 (1.722, 5.754), 2.098 (1.132, 3.888), respectively.Preoperative FR + CTC can be identified in precursor glandular lesions and utilized to differentiate from benign pulmonary diseases. Total number of pulmonary nodules, MTD, location (lower vs upper) were independent risk factors for malignancy.

Background The objective of this study was to conduct a bibliometric analysis of the entire field of folate receptor research. Folate receptor is expressed on a wide variety of cancers and certain immune cells. Methods A Web of Science search was performed on folate receptor or folate binding protein (1969-to June 28, 2019). The following information was examined: publications per year, overall citations, top 10 authors, top 10 institutions, top 10 cited articles, top 10 countries, co-author collaborations and key areas of research. Results In total, 3248 documents for folate receptor or folate binding protein were retrieved for the study years outlined in the methods section search query. The range was 1 per year in 1969 to 264 for the last full year studied (2018). A total of 123,720 citations for the 3248 documents retrieved represented a mean citation rate per article of 38.09 and range of 1667 citations (range 0 to 1667). Researchers in 71 countries authored publications analyzed in this study. The US was the leader in publications and had the highest ranking institution. The top 10 articles have been cited 7270 times during the time frame of this study. The top cited article had an average citation rate of 110 citations per year. Network maps revealed considerable co-authorship among several of the top 10 authors. Conclusion Our study presents several important insights into the features and impact of folate receptor research. To our knowledge, this is the first bibliometric analysis of folate receptor.

Retinoblastoma (RB) is a malignant intraocular neoplasm that occurs in children. Diagnosis and therapy are frequently delayed, often leading to metastasis, which necessitates effective imaging and treatment. In recent years, the use of nanoplatforms allowing both imaging and targeted treatment has attracted much attention. Herein, we report a novel nanoplatform folate-receptor (FR) targeted laser-activatable liposome termed FA-DOX-ICG-PFP@Lip, which is loaded with doxorubicin (DOX)/indocyanine green (ICG) and liquid perfluoropentane (PFP) for photoacoustic/ultrasound (PA/US) dual-modal imaging-guided chemo/photothermal RB therapy. The dual-modal imaging capability, photothermal conversion under laser irradiation, biocompatibility, and antitumor ability of these liposomes were appraised. The multifunctional liposome showed a good tumor targeting ability and was efficacious as a dual-modality contrast agent both

Summary: Background: Due to the critical role of folates in neurodevelopment, it is important to understand potential interactions between anti-HIV drugs used during pregnancy, and folate delivery pathways in the placenta. This study investigates the effect of dolutegravir (DTG) exposure on the functional expression of the reduced folate carrier (RFC), proton-coupled folate transporter (PCFT), and folate receptor-α (FRα) in the placenta. Methods: Human placental cell lines, human placental explants, and a pregnant mouse model treated with clinically relevant concentrations of DTG were used. Gene and protein expression were assessed by qPCR, immunoblot and immunohistochemical assays. Folate transport function was measured by applying radioisotope-based transport assays. Findings: In placental cells, clinically relevant DTG exposure for 3h or 6h was associated with a modest but significant reduction in the expression of RFC and PCFT both at the mRNA and protein levels, as well as decreased uptake of RFC and PCFT substrates [3H]-methotrexate and [3H]-folic acid, respectively. In pregnant mice, DTG administration was associated with an increase in both placental RFC and PCFT mRNA expression, accompanied by a decrease in placental FRα mRNA under folate-deficient dietary conditions. Interpretation: These findings demonstrate a potential interaction between DTG and folate transport pathways in the placenta, particularly in vivo, under folate deficient conditions, potentially impacting folate delivery to the foetus in the context of DTG-based ART during pregnancy. Funding: Funded by Ontario HIV Treatment Network, grant #506657; and Eunice Kennedy Shriver National Institute of Child Health & Human Development of the National Institutes of Health, award #R01HD104553.

Dual-targeted drug delivery system has established their reputation as potent vehicles for cancer chemotherapies. Herein, gemcitabine (Gem) was conjugated to human serum albumin (HSA) via dithiodipropionic anhydride to fabricate Gem-HSA nanoparticles. It was hypothesized that this system can enhance the low stability of Gem and can improve its intracellular delivery. Furthermore, folate was applied as targeting agent on HSA nanoparticles for increasing the tumor selectivity of Gem. To evaluate the structural properties of synthesized products, 1 H NMR and FT-IR were performed. Moreover, HPLC was implemented for confirming the conjugation between HSA and Gem. Nanoparticles have shown spherical shape with negative charge. The release rate of Gem was dependent to the concentration of glutathione and pH. Folate-targeted HSA nanoparticles have shown higher cytotoxicity, cellular uptake, and apoptosis induction on folate receptor overexpressing MDA-MB-231 cells in comparison to non-targeted nanoparticles. Finally, it is considered that the developed dual-targeted nanoparticles would be potent in improving the stability and efficacy of intracellular delivery of Gem and its selective delivery to cancer cells.

DNA synthesis and methylations are crucial during pre-implantation embryonic development, and are mediated by one-carbon metabolism of folates. Folates, transported into the cells via folate receptors (FOLR1 and FOLR2) and carriers (SLC19A1), are metabolized by various enzymes involved in folate-methionine cycle. However, the variations in temporal expression of folate transporters and folate-methionine cycle enzymes during pre-implantation embryo development is obscure. Thus, the present study aimed to investigate the differential expression of the genes for folate transporters and folate-methionine cycle enzymes. We also examined the expression of folate transport proteins in different pre-implantation development stages. Immature buffalo oocytes were matured in maturation medium followed by in vitro fertilization and culture at standard culture conditions. The temporal pattern of gene expression in buffalo, when compared to previous studies, indicated an inter-specific variation. The transcripts of some enzymes and folate transporters were significantly upregulated after zygotic genome activation. The transcripts as well as proteins for FOLR1, FOLR2 and SLC19A1 were present in oocytes and all the pre-implantation embryo stages. FOLR1 was present in the nuclei of different stages of developing embryos but not in the metaphase (MII) oocytes. As a result, the present study advocates the existence of active folate transport in buffalo oocytes and pre-implantation embryos. The data provided by the analysis of differential gene expression of folate transporters and metabolic enzymes would likely contribute to a better understanding of the role of folates in embryo development as well as advancements in assisted reproductive technologies.

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

Objectives The purpose of this study was to assess the expression of the 3 major folate transporters—folate receptors (FRs), reduced folate carrier (RFC), and proton-coupled folate transporter (PCFT)—in oral squamous cell carcinoma (OSCC). We hypothesized that patterns of expression of folate transporters would be different in OSCC compared with normal oral epithelium. Study Design We used immunohistochemistry to examine the expression of FR, RFC, and PCFT in 15 primary specimens collected from patients with OSCC, 2 human cadaveric samples of OSCC, and 12 normal human cadaveric oral tissues from a medical gross anatomy laboratory. Possible correlations between the expression of each folate transporter and patients’ clinical data were determined. Results All 3 folate transporters were highly expressed in normal oral epithelium. In contrast, OSCC samples generally demonstrated low expression of FR, RFC, and PCFT, with wide distribution in the invading cancer cells. There were no differences in folate transporter expression between OSCC samples collected from patients and from human cadavers. The lowest expression of FR and PCFT characterized less-differentiated tumors, and the lowest expression of RFC correlated with higher lymph node involvement. Conclusions Human oral cancer samples expressed decreased amounts of all 3 major folate transport proteins compared with controls from normal cadaveric oral tissues.

The aim of the study was to evaluate the performance of a folate receptor-mediated tumor detection (FRD) assay for detection of cervical high-grade lesions.A total of 1504 patients with abnormal cytology and/or positive human papillomavirus (HPV) testing during primary screening from November 2014 to August 2015 were enrolled. The patients were recruited from the Peking University People's Hospital and 12 other hospitals. Folate receptor-mediated tumor detection was applied in all the patients before colposcopy to compare the detection rate, sensitivity, specificity, positive predictive value, negative predictive value, and coincidence rate with HPV and cytology tests according to the pathologic diagnosis.In the total of 1504 patients, 503 patients were negative for intraepithelial lesion or malignancy, 440 patients were cervical intraepithelial neoplasia (CIN) 1, 254 patients were CIN 2, 257 patients were CIN 3, 46 patients were squamous cell carcinoma, and 4 patients were adenocarcinoma in situ. The sensitivity of FRD was 77.72%, which was less than cytology (80.39%) and HPV testing (95.54%). The specificity of FRD was 60.02%, which was greater than cytology (30.12%) and HPV testing (14.95%). The coincidence rate of FRD to the pathologic diagnosis (66.62%) was also significantly greater than atypical squamous cells of undetermined significance cytology and above (48.87%) and HPV testing (45.01%, p.0001). The detection rate of FRD for all grades of lesions increased with the severity of lesions.Folate receptor-mediated tumor detection has a slightly lower sensitivity and a higher specificity than cytology and HPV testing for detection of CIN 2+. Simplicity of FRD requires less professional skill. Folate receptor-mediated tumor detection could be a candidate test for cervical cancer screening especially in low- and middle-income countries. However, FRD still needs more clinical trial data to demonstrate its ability in general screening population.

Folic acid (FA), also termed folate, is an essential vitamin for health at all ages since it participates in the biosynthesis of nucleotides, amino acids, neurotransmitters, and certain vitamins. It is therefore crucial for rapidly growing tissues such as those of the fetus. It is becoming clear that FA deficiency and impaired folate pathways are implicated in many diseases of both early life and old age. FA can be transported into the cell by the folate receptor, the reduced folate transporter, and proton-coupled folate transporter. Folate transport proteins are present in certain eye tissues, which explains why FA plays an important role in eye development. The purpose of this literature review is to investigate the evidence relating FA deficiency to eye diseases.

Acute or chronic kidney disease can cause micronutrient deficiency. Patients with end-stage renal disease, kidney transplantation or on dialysis have reduced circulating levels of folate, an essential B vitamin. However, the molecular mechanism is not well understood. Reabsorption of folate in renal proximal tubules through folate transporters is an important process to prevent urinary loss of folate. The present study investigated the impact of acute kidney injury (AKI) on folate transporter expression and the underlying mechanism. AKI was induced in Sprague-Dawley rats that were subjected to kidney ischemia (45 min)-reperfusion (24 h). Both male and female rats displayed kidney injury and low plasma folate levels compared with sham-operated rats. The plasma folate levels were inversely correlated to plasma creatinine levels. There was a significant increase in neutrophil gelatinase-associated lipocalin (NGAL) and IL-6 mRNA expression in the kidneys of rats with ischemia-reperfusion, indicating kidney injury and increased inflammatory cytokine expression. Ischemia-reperfusion decreased mRNA and protein expression of folate transporters including folate receptor 1 (FOLR1) and reduced folate carrier (RFC); and inhibited transcription factor Sp1/DNA binding activity in the kidneys. Simulated ischemia-reperfusion through hypoxia-reoxygenation or Sp1 siRNA transfection in human proximal tubular cells inhibited folate transporter expression and reduced intracellular folate levels. These results suggest that ischemia-reperfusion injury downregulates renal folate transporter expression and decreases folate uptake by tubular cells, which may contribute to low folate status in AKI. In conclusion, ischemia-reperfusion injury can downregulate Sp1 mediated-folate transporter expression in tubular cells, which may reduce folate reabsorption and lead to low folate status.

Folate and related derivatives are essential small molecules required for survival. Of significant interest is the biological role and necessity of folate in the crosstalk between commensal organisms and their respective hosts, including the tremendously complex human distal gut microbiome. Here, we designed a folate-based probe consisting of a photo-crosslinker to detect and quantitate folate-binding proteins from proteomic samples. We demonstrate the selectivity of our probe for the well-established human folate-binding protein dihydrofolate reductase and show no promiscuous labeling occurs with human caspase-3 or bovine serum albumin, which served as negative controls. Affinity-based enrichment of folate-binding proteins from an E. coli lysate in combination with mass spectrometry proteomics verified the ability of our probe to isolate low-abundance folate-dependent proteins. We envision that our probe will serve as a tool to elucidate the roles of commensal microbial folate-binding proteins in health and microbiome-related diseases.

In order to improve the therapeutic efficiency of the chemotherapeutic drug paclitaxel in tumors, a folate-based Paclitaxel nanoemulsion (FNEs) was developed for tumor targeted treatment.In this study, we designed a folate-targeted nanoemulsion (folate/PEG-DSPE/nanoemulsion, FNEs) based on the traditional nanoemulsion using the principle of long-circulation targeting receptor mediated. The nanoemulsion (folate/PEG-DSPE/nanoemulsion, FNEs) was fabricated using high-pressure homogenization with a microfluidizer.The nanoemulsion (folate/PEG-DSPE/nanoemulsion, FNEs) can improve the delivery efficiency of nanocarriers at the tumor site by virtue of the high expression of folate receptors on the tumor surface. Malvern Nanoseries device and transmission electron microscopy (TEM) analyses showed that the nanoemulsions were spherical with an average diameter of 140 nm. The nanoemulsions can effectively carry paclitaxel (PTX) with an encapsulation rate of about 95%. And in vitro experiments have shown that it can efficiently increase the uptake of PTX in 4 T1 breast cancer cells and FNEs had a targeting capability hundredfold higher than that of PTX-loaded nanoemulsions (PTX-NEs) without folate. In vivo experiments have shown that the pharmacokinetic parameters of FNEs were better than those of other PTX groups and FNEs can significantly enhance circulation time in the body of the subcutaneously implanted 4 T1 breast cancer in mice, increase the accumulation of chemotherapy drugs at tumor sites and effectively inhibit tumor growth with lower system toxicity.This study can effectively improve the therapeutic efficiency of chemotherapy drugs for tumors, and provide an useful reference for solving the problem of low efficacy of chemotherapy drugs in clinical treatment of tumors. Graphical Abstract Schematic representation of Folic acid/PEG-DSPE/nano-emulsion (FNEs) specifically target tumor cells and enhanced anti-tumor effects.

In this study, the sulfated polysaccharide (SP) of Codium fragile was conjugated to folic acid (SP-FA). FT-IR and 1H NMR techniques revealed the occurrence of esterification reaction between the hydroxyl group of SP and the γ-carboxyl group of FA that confirming the SP-FA conjugation. SP and SP-FA did not show any direct toxicity on NK cells and HeLa cells. However, the treatment of SP and SP-FA enhance the NK cells cytotoxicity against HeLa cells by the upregulation of IFN-γ, TNF-α, perforin, and Granzyme-B. Moreover, NK cells activation was stimulated through NF-кB and MAPK pathways. The binding capacity studies exposed the targeting ability of HeLa cells by folate receptor (FR) which was assessed by a confocal quantitative image cytometer analysis. These results indicate that SP-FA could be used as selective drug delivery systems for targeting FR-overexpressed cancer cells with less toxicity.

The complex physiological barriers impose extremely conflicting demands on systemic drug delivery, so both particle size and surface charge of the nanoplatforms become vital factors. As a carbon-based nanomaterial with excellent optical properties, carbon dots are not suitable for direct systemic transport in vivo, which limits their application in the field of biomedical imaging, especially in the areas of diagnosis and cancer treatment. Liposomes have been developed as universal nanocarriers for various drugs. In this study, we aimed to build a highly precise and penetrative drug delivery system (DDS) using carbon dots encapsulated by liposomes. Carbon dots (CDs) were synthesized by the hydrothermal method using citric acid and ethylenediamine. Furthermore, simian virus 40 large T-antigen derived the nuclear targeting sequence (NLS) was bonded on the surface of CDs to obtain CDs-NLS. The antitumor drug doxorubicin was loaded onto the CDs-NLS through an acid-labile hydrazine bond to obtain DOX@CDs. Finally, DOX@CDs were encapsulated in aqueous centers of folate-coated and pH-sensitive liposomes, named pHSL-FA. In this paper, a nucleus-targeted nanocomposite (DOX@CDs), which bonds with the nuclear targeting sequence (NLS) and the anticancer drug doxorubicin (DOX), has physicochemical properties of particle size of about 3.8 nm, zeta potential of +31.8 mV and high quantum yield of 64.53%. The negatively charged folate-coated and pH-sensitive liposomes (pHSL-FA) are used as a carrier to reverse the surface charge of DOX@CDs. Compared to free DOX@CDs, pHSL-FA show higher tumor accumulation in 4 T1 tumor-bearing mice and further improve cytotoxicity to tumor cells. This work proposes a unique nanomedical approach that enables the precise delivery of chemotherapy drugs and significantly reduces side effects, which is promising for clinical translation.

One of the main challenges in clinical translation of polymeric micelles is retention of the drug in the nanocarrier system upon its systemic administration. Core crosslinking and coupling of the drug to the micellar backbone are common strategies to overcome these issues. In the present study, polymeric micelles were prepared for tumor cell targeting of the kinase inhibitor dactolisib which inhibits both the mammalian Target of Rapamycin (mTOR) kinase and phosphatidylinositol-3-kinase (PI3K). We employed platinum(II)-based linker chemistry to couple dactolisib to the core of poly(ethylene glycol)-b-poly(acrylic acid) (PEG-b-PAA) polymeric micelles. The formed dactolisib-PEG-PAA unimers are amphiphilic and self-assemble in an aqueous milieu into core–shell polymeric micelles. Folate was conjugated onto the surface of the micelles to yield folate-decorated polymeric micelles which can target folate receptor over-expressing tumor cells. Fluorescently labeled polymeric micelles were prepared using a lissamine-platinum complex linked in a similar manner as dactolisib. Dactolisib polymeric micelles showed good colloidal stability in water and released the coupled drug in buffers containing chloride or glutathione. Folate decorated micelles were avidly internalized by folate-receptor-positive KB cells and displayed targeted cellular cytotoxicity at 50–75 nM IC50. In conclusion, we have prepared a novel type of folate-receptor targeted polymeric micelles in which platinum(II) linker chemistry modulates drug retention and sustained release of the coupled inhibitor dactolisib.

The folate receptor (FR) is a promising cell membrane-associated target for molecular imaging and radionuclide therapy of cancer (FR-α) and potentially also inflammatory diseases (FR-β) through use of folic acid-based radioconjugate. FR is often overexpressed by cells of epithelial tumors, including tumors of ovary, cervix, endometrium, lungs, kidneys, etc. In healthy tissues, FR can be found in small numbers by the epithelial cells, mainly in the kidneys. Extremely high undesired accumulation of the folate radioconjugates in the renal tissue is a main drawback of FR-targeting concept. In the course of this work, we aimed to reduce the undesirable accumulation of folate radioconjugates in the kidneys by introducing a histidine/glutamic acid tag into their structure. Two folic acid based compounds were synthesized: NODAGA-1,4-butanediamine-folic acid (FA-I, as control) and NODAGA-[Lys-(HE)2]-folic acid (FA-II) which contains a (His-Glu)2 fragment. In vitro studies with FR (+) cells (KB and others) showed that both compounds have specificity for FR. Introduction of (HE)2-tag does not affect FR binding ability of the conjugates. In vivo biodistribution studies with normal laboratory animals, as well as with KB tumor bearing animals, were carried out. The results showed that introduction of the (HE)2 tag into the structure of folate radioconjugates can significantly reduce the accumulation of these compounds in non-target tissues and important organs (the accumulation in the kidneys is reduced 2–4 times), leaving the accumulation in tumor at least at the same level, and even increasing it.

Malaria parasites are fast replicating unicellular organisms and require substantial amounts of folate for DNA synthesis. Despite the central role of this critical co-factor for parasite survival, only little is known about intraparasitic folate trafficking in Plasmodium. Here, we report on the expression, subcellular localisation and function of the parasite's folate transporter 2 (FT2) during life cycle progression in the murine malaria parasite Plasmodium berghei. Using live fluorescence microscopy of genetically engineered parasites, we demonstrate that FT2 localises to the apicoplast. In invasive P. berghei stages, a fraction of FT2 is also observed at the apical end. Upon genetic disruption of FT2, blood and liver infection, gametocyte production and mosquito colonisation remain unaltered. But in the Anopheles vector, FT2-deficient parasites develop inflated oocysts with unusual pulp formation consisting of numerous single-membrane vesicles, which ultimately fuse to form large cavities. Ultrastructural analysis suggests that this defect reflects aberrant sporoblast formation caused by abnormal vesicular traffic. Complete sporogony in FT2-deficient oocysts is very rare, and mutant sporozoites fail to establish hepatocyte infection, resulting in a complete block of parasite transmission. Our findings reveal a previously unrecognised organellar folate transporter that exerts critical roles for pathogen maturation in the arthropod vector.

Folate is an essential micronutrient for biological function. The liver, a primary organ for folate metabolism and storage, plays an important role in folate homeostasis. Proton-coupled folate transporter (PCFT) and reduced folate carrier (RFC) are the major folate transporters responsible for folate uptake at basolateral membrane of hepatocytes. Low serum folate levels are frequently associated with obesity. We investigated the mechanism that regulated folate status in a mouse model with diet-induced obesity. Mice (C57BL/6J) were fed a high-fat diet (60% kcal fat) for 8 weeks. Mice displayed increased hepatic lipid accumulation and decreased folate levels in the liver and serum compared to mice fed a normal chow diet (10% kcal fat). High-fat diet-fed mice had low expression of PCFT and RFC and decreased nuclear respiratory factor-1 (NRF-1)/DNA-binding activity. Treatment with NRF-1 siRNA or palmitic acid reduced folate transporter expression in hepatocytes. Inhibition of NRF-1 mediated folate transporter expression significantly reduced intracellular folate levels. These results suggest that chronic consumption of high-fat diets impairs folate transporter expression via NRF-1-dependent mechanism, leading to reduced hepatic folate storage. Understanding the regulation of folate homeostasis in obesity may have an important implication in current guideline of folate intake. KEY MESSAGES: Serum and liver folate levels are decreased in diet-induced obese mice. Chronic high-fat diet consumption impairs expression of hepatic PCFT and RFC. NRF-1 regulates hepatic folate transporters expression and folate levels.

Herein a new class of iminoboronates obtained from 2-acetylbenzene boronic acids and aminophenols is presented. The N,O-ligand topology enabled the formation of an additional B-O bond that locks the boron center in a tetrahedral geometry. This molecular arrangement decisively contributes to improve the construct's stability in biocompatible conditions and retaining the iminoboronate reversibility in more acidic environments. 2-Acetylbenzene boronic acid was reacted with a fluorescent amino-coumarin to yield a stable and non-fluorescent N,O-iminoboronate. This mechanism was further used to assemble a folate receptor targeting conjugate that selectively delivered the fluorescent amino-coumarin to MDA-MB-231 human breast cancer cells.

Annonaceous acetogenins (ACGs) are one of the most active constituents isolated from Annona species with potent antitumor activity. However, the poor solubility and severe side effect greatly limit their use in clinic. In this study, folic acid (FA) modified annonaceous acetogenins nanosuspensions (FA-PEG-ACGs-NSps) had been successfully prepared using DSPE-PEG-FA and soybean lecithin (SPC) as stabilizers. The resultant FA-PEG-ACGs-NSps had a mean particle size of 119.7 nm, a zeta potential of –23.0 mV and a high drug payload of 49.68%. The obtained ACGs-NSps had a good stability in various physiological media, and showed sustained drug release. Compared to common ACGs nanoparticles (PEG-ACGs-NSps), FA-PEG-ACGs-NSps showed significantly enhanced in vitro cytotoxicity against folate receptor-positive HeLa cell lines (IC50, 0.483 μg/mL vs. 0.915 μg/mL, p

Drug efflux transporters, in particular P-glycoprotein (Pgp), limit the success of chemotherapy. We previously found that synthetic doxorubicin conjugated with nitric oxide (NO)-releasing group overcomes resistance by inducing a NO-mediated inhibition of Pgp. Here we produced the first liposomal formulations of this nitrooxy-doxorubicin decorated with folic acid (FA), termed LNDF, in order to improve their active targeting against Pgp-expressing tumors. Folate was inserted onto liposomes surface using two different methods and the formulations were compared with respect to their technological features and in vitro behavior. By analyzing human and murine breast cancer cells with different expression of FA receptor (FAR) and Pgp, we demonstrated that LNDF are internalized in a FAR-dependent manner and achieve maximal anti-tumor efficacy against FAR-positive/Pgp-positive cells. Upon uptake of LNDF, nitrooxy-doxorubicin was delivered within nucleus, where it induced cell cycle arrest and DNA damages, and mitochondria, where it impaired the mitochondrial energy metabolism and triggered mitochondria-dependent apoptosis. LNDF reduced the growth of FAR-positive/Pgp-positive tumors and prevented tumor formation in mice, whereas doxorubicin and Caelyx® failed. LNDF cardiotoxicity was comparable to Caelyx®. The sensitivity to LNDF was maintained in tumors exposed to repeated cycles of the drug and in cells derived from the exposed tumors, excluding the onset of secondary resistance. By combining an innovative multitarget cargo drug, conceived to achieve high efficacy against Pgp-expressing cells, and appropriate strategies of liposome formulation and decoration, we produced a therapeutic tool that may represent a significant advancement in the treatment of FAR-positive/Pgp-positive tumors.

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

Aim: We endeavored to create a folate-targeted liposome (Fol-liposome) that could selectively target areas of inflammation. Materials & methods: Fol-liposomes were prepared with encapsulated DiD fluorophore or betamethasone (BM) to image and treat an adjuvant-induced rat model of rheumatoid arthritis. Results: Fol-liposomes selectively accumulated in arthritic rat paws to a greater extent than nontargeted liposomes. When these Fol-liposomes were used to encapsulate BM and administered to arthritic rats, animals exhibited less paw swelling, lower arthritis scores, a reduction in bone erosion, less splenomegaly and better maintenance of body weight when compared with nontreated or nontargeted BM-containing liposome groups. Conclusion: Fol-liposomes can selectively deliver imaging and therapeutic agents to sites of inflammation in a rat model of rheumatoid arthritis.

Combination therapies are widely investigated cancer treatment modalities. Carbon based systems such as graphene oxide (GO), plasmonic nanoparticles such as silver nanoparticles (AgNPs), and the folate analog, methotrexate (MTX), have been separately studied for their potential anticancer effects. In this study, we combined these systems to develop AgNPs-embedded GO with conjugated MTX (MTX-GO/AgNPs) and studied their folate receptor-targeted anticancer effects. Results revealed successful formation of AgNPs on GO along with MTX conjugation as suggested by UV/visible, TEM, AFM, FTIR, and XRD analysis. Folate receptor-positive MCF-7 cells were more prone to cytotoxic effects of MTX-GO/AgNPs compared to folate receptor-negative HepG2 cells. Folic acid analog MTX interacts with folate receptors expressed in MCF-7 cells, improving cellular uptake and subsequent anticancer effects of the system. Importantly, AgNPs enhanced the total ROS production within the treated cells leading to improve cellular apoptosis, as evidenced by western blot. Moreover, near infrared (NIR)-induced photothermal effects of GO improved the anticancer activity of the system. Therefore, the combinational therapy system MTX-GO/AgNPs can be potentially applied for effective folate receptor-targeted treatment of cancers.

Non-viral delivery of nucleic acids for therapies based on RNA interference requires a rational design and optimal self-assembly strategies. Nucleic acid particles need to be small, stable and functional in terms of selective cell uptake and controlled release of encapsulated nucleic acids. Here we report on small (∼38 nm) monomolecular nucleic acid/lipid particles (mNALPs) that contain single molecules of short double-stranded oligonucleotides covered by a tight, highly curved lipid bilayer. The particles consist of DOPE, DOTAP, DOPC and DSPE-PEG(2000) and are assembled with 21 bp double-stranded DNA or small interfering RNA by solvent exchange on a hydrodynamic-focusing microfluidic chip. In comparison to vortex mixing by hand this method increases the encapsulation efficiency by 20%, and yields particles with a narrower size distribution, negligible aggregate formation and high stability in blood plasma and serum. Modification of mNALPs with folate-conjugated PEG-lipids results in specific binding and uptake by epithelial carcinoma KB cells overexpressing folate receptors. Binding is significantly reduced by competitive inhibition using free folate and is not observed with non-targeted mNALPs, revealing high specificity. The functionalized mNALPs show gene silencing in the presence of chloroquine, an endosome-destabilizing agent. Together, the robust self-assembly of small-sized mNALPs with their high stability and receptor-specific cell uptake demonstrate that the tight, PEG-grafted lipid-bilayer encapsulation may offer a promising approach towards the delivery of short double-stranded oligonucleotides.

Previous studies have demonstrated that circulating DNA-encapsulated microbubbles (MBs) combined with focused ultrasound (FUS) can be used for local blood-brain barrier (BBB) opening and gene delivery. However, few studies focused on how to increase the efficiency of gene delivery to brain tumors after the released gene penetrating the BBB. Here, we proposed the use of folate-conjugated DNA-loaded cationic MBs (FCMBs). When combined with FUS as a trigger for BBB opening, FCMBs were converted into nanometer-sized vesicles that were transported to the brain parenchyma. The FCMBs can selectively aggregate around tumor cells that overexpressed the folate receptor, thus enhancing gene delivery via folate-stimulated endocytosis. Our results confirmed that FCMBs can carry DNA on the surface of the MB shell and have good targeting ability on C6 glioma cells. In addition, the optimized FUS parameters for FCMBs-enhanced gene delivery were confirmed by cell experiments (center frequency = 1 MHz; acoustic pressure = 700 kPa; pulse repetition frequency = 5 Hz; cycle number = 10000; exposure time = 1 min; FCMBs concentration = 4 × 10 7 MB/mL). In vivo data also indicated that FCMBs show better gene transfection efficiency than MBs without folate conjugation and the traditional approach of directly injecting the gene. This study described our novel development of multifunctional MBs for FUS-triggered gene delivery/therapy.

Novel bioconjugates (Agm6-M-PEG-FA) for active oligonucleotide (ON) delivery have been developed by conjugating a cationic oligo-guanidyl star-like shaped “head” (Agm6-M) to a polymeric “tail” (PEG) terminating with folic acid (FA) as targeting agent or methoxy group (Agm6-M-PEG-FA and Agm6-M-PEG-OCH3, respectively). Gel electrophoresis showed that the bioconjugates completely associated with ONs at 3 nitrogen/phosphate (N/P) ratio. Studies performed with folate receptor (FR)-overexpressing HeLa cells, showed that optimal cell up-take was obtained with the 75:25 w/w Agm6-M-PEG-OCH3:Agm6-M-PEG-FA mixture. Dynamic light scattering and transmission electron microscopy showed that the polyplexes had size

Valproic acid is an established structural and neurodevelopmental teratogen. Recently, we demonstrated that valproate alters the barrier function of perfused term human placentas. Here, we conducted a pilot study to evaluate the effects of subchronic valproate exposure on carrier expression in cultured placental villous explants from early human pregnancies. Placental tissue of gestational age 6-13 weeks was collected from elective pregnancy terminations in women without known epilepsy. The effects of valproate (42, 83, or 166 μg/mL) on the mRNA expression of 37 major placental carriers and related genes were evaluated by a customized gene expression array (n = 5, 5 days). Five-day exposure to valproate was associated with high variability in gene expression. However, two main gene clusters were identified, including a cluster of three major folate carriers. Exposure to low therapeutic levels of valproate (42 μg/mL) was associated with a tendency toward reduced mRNA expression of genes encoding folate and amino acid and fatty acid carriers (P = 0.065, paired analysis). Our initial findings suggest that valproate can affect the function of the human placenta during early pregnancy.

Recent studies have demonstrated that the oviductal environment plays an active role in modulating the epigenetic marks of the preimplantation embryo genome, but the molecular factors that mediate this epigenetic effect are unknown. Folate is a well-known epi-nutrient that can impact on cell epigenetic machinery during embryonic and fetal development. However, the study of this epi-nutrient in the oviduct is still limited. The present study was conducted to confirm the presence and physiological concentration of folate in bovine oviductal fluid (OF) and to determine if bovine oviduct epithelial cells (BOECs) are able to regulate the uptake of this micronutrient. Samples of OF from ipsi- and contralateral oviducts were collected at different stages of the estrous cycle and folate levels were determined using a competitive receptor binding immunoassay. In addition, gene expression of folate receptors (FOLR1, FOLR2) and transporters (SLC19A1, SLC46A1) were analyzed in BOECs from ampulla and isthmus regions during different stages of the estrous cycle using RT-qPCR. In vitro culture assays were also performed to evaluate whether expression of these genes responds to hormonal stimulation. Our results demonstrated presence of folate in the OF, showing changes of its concentration in the ipsilateral oviduct during the estrous cycle and significantly lower levels at the postovulatory stage. Moreover, gene expression of folate receptors and transporters was detected in BOECs, showing regional and cycle-dependent changes. In particular, differential expression of FOLR1 mRNA was observed in BOECs from the isthmus region, reaching significantly higher levels during the postovulatory stage. Under in vitro culture conditions, gene expression of folate receptors and transporters was maintained in BOEC explants and a particular susceptibility to steroid hormone stimulation was observed. In conclusion, the present study confirms the presence of folate in the bovine oviduct and proves the existence of a fine-tuned regulation of the expression of its receptors and transporters, highlighting the importance to expand the knowledge about this epi-nutrient in the oviductal context. Fil: García, Elina V.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina. Universidad Nacional de Tucumán; Argentina Fil: Oliva, María E.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina Fil: Leblanc, Jean Guy Joseph. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina Fil: Barrera, Antonio Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina

Multifunctional pH-responsive folate receptor mediated targeted polymer nanoparticles (TPNps) were developed for docetaxel (DTX) delivery based on poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol)poly (β-amino ester) (P123-PAE) and poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol)-folate (P123-FA) copolymers. The DTX was loaded into the TPNps with a decent drug loading content of 15.02 ± 0.14 wt%. In vitro drug release results showed that the DTX was released from the TPNps at a pH-dependent manner. Tetrazolium dye (MTT) assay revealed that the bland polymer nanoparticles displayed almost nontoxicity at 200 μg/mL concentration. However, the DTX-loaded TPNps showed high anti-tumor activity at low IC50 (0.72 μg/mL) for MCF-7 cells following 48 h incubation. Cellular uptake experiments revealed that the TPNps had higher degree of cellular uptake than nontargeted polymer nanoparticles, indicating that the nanoparticles were internalized into the cells via FA receptor-mediated endocytosis. Moreover, the cellular uptake pathways for the FA grafted polymer were involved in energy-dependent, clathrin-mediated and caveolae-mediated endocytosis. The cell killing effect and cellular uptake of the DTX-TPNps by the MCF-7 cells were all enhanced by about two folds at pH 5.5 when compared with pH 7.4. The TPNps also significantly prolonged the in vivo retention time for the DTX. These results suggest that the biocompatible pH responsive folate-modified polymer nanoparticles present a promising safe nanosystem for intracellular targeted delivery of DTX.

Here we describe an in vitro primary culture system for Caenorhabditis elegans germline stem cells. This culture system was used to identify a bacterial folate as a positive regulator of germ cell proliferation. Folates are a family of B-complex vitamins that function in one-carbon metabolism to allow the de novo synthesis of amino acids and nucleosides. We show that germ cell proliferation is stimulated by the folate 10-formyl-tetrahydrofolate-Glun both in vitro and in animals. Other folates that can act as vitamins to rescue folate deficiency lack this germ cell stimulatory activity. The bacterial folate precursor dihydropteroate also promotes germ cell proliferation in vitro and in vivo, despite its inability to promote one-carbon metabolism. The folate receptor homolog FOLR-1 is required for the stimulation of germ cells by 10-formyl-tetrahydrofolate-Glun and dihydropteroate. This work defines a folate and folate-related compound as exogenous signals to modulate germ cell proliferation.

In order to develop novel (68) Ga-labeled PET tracers for folate receptor imaging, two DOTA-conjugated Pteroyl-Lys derivatives, Pteroyl-Lys-DOTA and Pteroyl-Lys-DAV-DOTA, were designed, synthesized and radiolabeled with (68) Ga. Biological evaluations of the two radiotracers were performed with FR-positive KB cell line and athymic nude mice bearing KB tumors. Both (68) Ga-DOTA-Lys-Pteroyl and (68) Ga-DOTA-DAV-Lys-Pteroyl exhibited receptor specific binding in KB cells in vitro. The tumor uptake values of (68) Ga-DOTA-Lys-Pteroyl and (68) Ga-DOTA-DAV-Lys-Pteroy were 10.06 ± 0.59%ID/g and 11.05 ± 0.60%ID/g at 2 h post-injection, respectively. Flank KB tumor was clearly visualized with (68) Ga-DOTA-DAV-Lys-Pteroyl by Micro-PET imaging at 2 h post-injection, suggesting the feasibility of using (68) Ga-labeled Pteroyl-Lys conjugates as a novel class of FR targeted probes.

An appropriate codelivery system for chemotherapeutic agents and nucleic acid drugs will provide a more efficacious approach for the treatment of cancer. Combining gene therapy with chemotherapeutics in a single delivery system is more effective than individual delivery systems carrying either gene or drug. In this work, we developed folate (FA) receptor targeted magnetic-mesoporous silica nanoparticles for the codelivery of VEGF shRNA and doxorubicin (DOX) (denoted as M-MSN(DOX)/PEI-FA/VEGF shRNA). Our data showed that M-MSN(DOX)/PEI-FA could strongly condense VEGF shRNA at weight ratios of 30:1, and possesses higher stability against DNase I digestion and sodium heparin. In vitro antitumor activity assays revealed that HeLa cell growth was significantly inhibited. The intracellular accumulation of DOX by confocal microscopy and fluorescence spectrophotometry showed that M-MSN(DOX)/PEI-FA were more easily taken up than nontargeted M-MSN(DOX). Quantitative PCR and ELISA data revealed that M-MSN/PEI-FA/VEGF shRNA induced a significant decrease in VEGF expression as compared to cells treated with either the control or other complexes. The invasion and migration phenotypes of the HUVECs were significantly decrease after coculture with MSN/PEI-FA/VEGF shRNA nanocomplexes-treated HeLa cells. The approach provides a potential strategy to treat cancer by a singular nanoparticle delivery system.

Since the introduction of nanocarriers, the delivery of chemotherapeutic agents for treatment of patients with cancer has been possible with better effectiveness. The latest findings are also support that further enhancement in therapeutic effectiveness of these nanocarriers can be attained, if surface decoration with proper targeting agents is considered. This study aimed at treating a variety of 4T1 murine breast cancer cell line, mainly demonstrating high folate and TRAIL receptor expression of cancerous cells. The therapeutic efficacy of paclitaxel loaded Cremophore EL (Taxol®), paclitaxel loaded waterborne polyurethane nanomicelles (PTX-PU) and paclitaxel loaded waterborne polyurethane nanomicelles conjugated with folate (PTX-PU-FA) and TRAIL (PTX-PU-TRAIL) on treating 4T1 cell was also compared. The findings that worth noting are: PTX-PU outperformed Taxol® in a Balb/C mouse model, furthermore, tumor growth was adequately curbed by folate and TRAIL-decorated nanomicelles rather than the unconjugated formulation. Tumors of mice treated with PTX-PU-FA and PTX-PU-TRAIL shrank substantially compared to those treated with Taxol®, PTX-PU and PTX-PU-TRAIL (average 573 mm3 versus 2640, 846, 717 mm3 respectively), 45 days subsequent to tumor inoculation. The microscopic study of hematoxylin-eosin stained tumors tissue and apoptotic cell fraction substantiated that the most successful therapeutic effects have been observed for the mice treated with PTX-PU-FA (about 90% in PTX-PU-FA versus 75%, 60%, 15% in PTX-PU-TRAIL, PTX-PU, and Taxol® group respectively). Using folate-targeted nanocarriers to treat cancers characterized by a high level of folate ligand expression is well substantiated by the findings of this study.

To improve the water solubility and tumor targeting ability of docetaxel (DTX), and thus enhance the drug's antitumor efficacy and safety, a novel folate receptor (FR)-targeted cyclodextrin drug delivery vehicle (FA-CD) was successfully synthesized. The synthesis of the designed cyclodextrin was confirmed by Fourier transform infrared spectroscopy (FTIR), proton nuclear magnetic resonance (1H NMR), and differential scanning calorimetry (DSC). The in vitro cytotoxicity was investigated using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and the results showed that no significant differences (p0.05) appeared in cytotoxicity between the different cyclodextrins in the different cell lines. Besides, the DTX/FA-CD inclusion complex was prepared. The cellular uptake and competition assays were examined using the HepG2, HeLa, and KB cell lines, which have different levels of folate receptor expression. Interestingly, the Cy5.5/FA-CD complex had higher uptake in the HepG2, HeLa, and KB cells, compared with non-targeted Cy5.5/CD complex (p0.001). The time-dependent drug uptake into KB cells observed by LSCM confirmed the drug delivery via endocytic routes. Data from the competition assays, especially in KB cells, showed that a significant inhibitory effect (p0.001) was obtained when the concentration of FA was increased, and suggested that the Cy5.5/FA-CD was internalized through a FR-mediated mechanism. Moreover, the in vitro bioactivity assay also demonstrated efficient antitumor activity, and the order of the cell viabilities (% of control) was OBHepG2HeLaKB for DTX/FA-CD (p0.001). For DTX/CD, however, it displayed minimum antitumor behavior in all cell types. An apoptosis study by FCM and LSCM also revealed that the FA-modified complexes were more effective in inducing apoptosis in FR-expressing cells. Finally, an in vivo biodistribution study in KB-bearing healthy mice revealed that the DTX/FA-CD complex has enhanced tumor-targeting efficacy and diminished systemic side effects. These results suggest that the novel FR-targeted cyclodextrin complex is a promising alternative as an anticancer drug delivery system.

The detection of disease markers is considered an important step for early diagnosis of cancer. We design in this work a novel electrochemical sensing platform for the sensitive and selective detection of folic acid protein (FP). The platform is fabricated by electrophoretic deposition (EPD) of reduced graphene oxide (rGO) onto a gold electrode and post-functionalization of rGO with folic acid. Upon FP binding, a significant current decrease can be measured using differential pulse voltammetry (DPV). Using this scheme, a detection limit of 1pM is achieved. Importantly, the method also allows the detection of FP in serum being thus an appealing approach for the sensitive detection of biomarkers in clinical samples.

Spindle defect and chromosome misalignment occuring in oocyte meiosis induce nondisjunction. Nondisjunction causes Down syndrome, also known as trisomy 21. Folic acid (FA) is an essential nutrient composition for fetal growth and development. It has been reported that FA nutritional status is associated with the risk of Down syndrome. However, to our knowledge, little is known about the effect of FA deficiency on abnormal oocytes (spindle defects, chromosome misalignments and immature oocyte) in vivo. In the present study, we investigate the effects of FA deficiency on oocyte meiosis in female mice. In order to induce FA deficiency in mice, female Crl:CD1 mice were fed a FA-free diet for 58 d. The diet also contained an antibiotic which has functions on limiting FA formation by intestinal microorganisms. The level of FA deficiency was determined by measuring the concentration of FA in the liver, hemocyte, uterus, ovary, and urine. FA concentrations in these samples from the FA-deficient group were 50-90% lower. Despite this, the frequency of abnormal oocytes was no different between the FA-deficient and control groups (20.0% vs 14.6%). According to the past research, FA transporter was strongly expressed in oocytes. Hence, it is possible that FA-free diets may not affect the concentration of oocyte FA in mice. To sum up these data, our study concluded that FA deficiency did not adversely affect oocyte meiosis.

The activation of tumor suppressor p53 protein through inhibition of its interaction with the oncogenic Murine Double Minute 2 (MDM2) protein presents a novel therapeutic strategy against cancer. Accordingly, several small-molecule inhibitors have been developed that mimic three hydrophobic groups of p53 involved in p53-MDM2 binding and thus block the p53-binding pocket on MDM2. Interestingly, presence of a fourth, solvent-exposed hydrophilic moiety in these MDM2 inhibitors is shown to enhance their binding to MDM2 by protecting the inhibitor-MDM2 binding interface from surrounding solvent. In this context, we hypothesized that vitamin folic acid (FA) may prove to be suitable as the hydrophilic cover for enhancing activity of present MDM2 inhibitors. The proposed conjugation of FA to MDM2 inhibitors may also lead to their enhanced and selective uptake by cancer cells, owing to significantly higher expression of the FA receptors on cancer cells compared to normal cells. Therefore, based on our novel hypothesis we designed FA-conjugated MDM2 inhibitors and investigated their binding with MDM2 protein as well as the FA receptor. Specifically, a molecular modeling approach combining flexible receptor docking and molecular mechanics energy minimization calculations revealed highly favorable interactions of FA-conjugated MDM2 inhibitors with both MDM2 protein and the FA receptor as compared to native crystal ligands. Furthermore, these binding interactions were found to be stable using 50,000 ps molecular dynamics simulations. In summary, the newly-designed molecules of this kind, with better MDM2 target binding and enhanced cellular uptake potential, may prove highly useful against cancer and thus warrant further experimental investigations.

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 (10–30MBq/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 ≥20MBq (≥28Gy). Application of radiofolates with an absorbed renal dose ≥23Gy 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.

DNA has been used to build nanostructures with potential biomedical applications. However, their use is limited by the lack of information on the mechanism of entry, intracellular fate and degradation rate of nanostructures inside cells. We generated octahedral DNA nanocages functionalized with folic acid and investigated the cellular uptake mediated by two distinctive internalization pathways, using two cellular systems expressing the oxidized low-density lipoprotein receptor-1 (LOX-1) and the α isoform of the folate receptor (αFR), respectively. Here, we report that DNA nanocages are very efficiently and selectively internalized by both receptors with an efficiency at least 30 times higher than that observed in cells not expressing the receptors. When internalized by LOX-1, nanocages traffic to lysosomes within 4 hours and are rapidly degraded. When the uptake is mediated by αFR, DNA nanocages are highly stable (>48 hours) and accumulate inside cells in a time-dependent way. These data demonstrate that the selection of the cellular receptor is crucial for targeting specific sub-cellular compartments and for modulating the DNA nanocage intracellular half-life, indicating that vitamin-mediated uptake may constitute a protected pathway for intracellular drug delivery.

Folic-acid-based radioconjugates have been developed for nuclear imaging of folate receptor (FR)-positive tumors, however, high renal uptake was unfavorable in view of a therapeutic application. Previously, it was shown that pre-injection of pemetrexed (PMX) increased the tumor-to-kidney ratio of radiofolates several-fold. In this study, PMX was combined with the currently best performing radiofolate ([177Lu]cm13), which is outfitted with an albumin-binding entity. Biodistribution studies were carried out in mice bearing KB or IGROV-1 tumor xenografts, both FR-positive tumor types. SPECT/CT was performed with control mice injected with [177Lu]folate only and with mice that received PMX in addition. Control mice showed high uptake of radioactivity in KB and IGROV-1 tumor xenografts, but retention in the kidneys was also high, resulting in tumor-to-kidney ratios of ~0.85 (4 h p.i.) and ~0.60 (24 h p.i.) or ~1.17 (4 h p.i.) and ~1.11 (24 h p.i.) respectively. Pre-injection of PMX improved the tumor-to-kidney ratio to values of ~1.13 (4 h p.i.) and ~0.92 (24 h p.i.) or ~1.79 (4 h p.i.) and ~1.59 (24 h p.i.), respectively, due to reduced uptake in the kidneys. It was found that a second injection of PMX&mdash, 3 h or 7 h after administration of the radiofolate&mdash, improved the tumor-to-kidney ratio further to ~1.03 and ~0.99 or ~1.78 and ~1.62 at 24 h p.i. in KB and IGROV-1 tumor-bearing mice, respectively. SPECT/CT scans readily visualized the tumor xenografts, whereas accumulation of radioactivity in the kidneys was reduced in mice that received PMX. In this study, it was shown that PMX had a positive impact in terms of reducing the kidney uptake of albumin-binding radiofolates, hence, the administration of PMX resulted in ~1.3&ndash, 1.7-fold higher tumor-to-kidney ratios. This is, however, a rather moderate effect in comparison to the previously shown effect of PMX on conventional radiofolates (without albumin binder), which led to 5&ndash, 6-fold increased tumor-to-kidney ratios. An explanation for this result may be the different pharmacokinetic profiles of PMX and long-circulating radiofolates, respectively. Despite the promising potential of this concept, it is believed that a clinical translation would be challenging, particularly when PMX had to be injected more than once.

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

The objective of the present study is to enhance the permeation of bioactive molecules across highly lipophilic insurmountable blood brain barrier (BBB) using folic acid (FA) functionalized poly(lactic-co-glycolic acid) (PLGA) nanoparticles (FA-PNPs) coated with non-ionic surfactant, Polysorbate 80 (P80). The developed Mtx-Tf loaded folic acid anchored PNPs formulation depicted particle size, zeta potential and entrapment efficiency of 109 ± 2.3 nm, –9.38 ± 0.9 mV and 71.0 ± 0.6%, respectively. In vitro release showed biphasic release pattern with initial >35% release in 8 h followed by sustained release, up to 65% by the end of 168 h. SRB cyto-toxicity assay depicted P80-Mtx-Tf-FA-PNPs possessing better cellular cyto-toxicity (C6 Glioma) as compared to counter test formulations, i.e. 100 μM nano-encapsulated drug-conjugate concentration incubated for 72 h resulted in apoptosis of ∼100% of the cellular population. Qualitatively, ∼85% of P80-FITC-FA-PNPs have been found to be internalized by C6 glioma cells post 5 h of incubation. Quantitative cellular uptake assay demonstrated a collinear dependence on drug-conjugate concentration. In vivo administration of P80-Mtx-Tf-FA-PNPs depicted significant decrease, i.e. 73.6% in the tumour spheroid volume. Serum analysis data pointed towards the significant relevance of P80-coated FA-PNPs in lowering the drug diffusion rate by 88.4% (after 30 min) and three-fold enhanced retention time of drug in systemic circulation, thereby felicitating an effective prognostic treatment. This study clearly demonstrated reduced systemic toxicity, enhanced biocompatibility and anti-tumour efficacy of formulated NPs.

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

WOS: 000457049400086, PubMed ID: 29458269, Doxorubicin is widely used anticancer drug; however, use of doxorubicin is limited. Under externally applied magnetic field, magnetic agents can help to transport drug directly to tumor. Folate receptor is overexpressed in ovarian carcinomas. In this study, we aimed to develop magnetically responsive and folate receptor-targeted biomimetic drug delivery system for ovarian cancer therapy. Doxorubicin-loaded and glucose/gluconic acid-coated magnetic nanoparticles were synthesized and erythrocyte membrane vesicles were used for coating of nanoparticles. Folate ligand was anchored to surface so as to target receptor. Hydrodynamic size of nanocarrier was found as 91.2 +/- 20.8nm. The results showed that delivery system has controlled drug release profile and biocompatible features. In folate-free medium, folate receptor-targeted nanocarrier showed 10.33-fold lower IC50 values for A2780 cells and 3.93-fold lower for OVCAR3 cells compared to non-targeted nanoparticles and demonstrated more cytotoxicity against ovarian cancer cells. Moreover, magnetically and folate receptor-targeted doxorubicin delivery system was significantly more effective for therapy of xenografted nude mice than free doxorubicin based on tumor shrinkages and biochemical parameters. In conclusion, it can be suggested that folate ligand-attached and biomimetically designed magnetic drug delivery system have advantages and potential for targeted ovarian cancer therapy., Scientific and Technological Research Council of Turkey (TUBITAK)Turkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK) [113Z165]; Ege University Scientific Research Project OfficeEge University [2014 BIL 006, 2015 FEN 055], The authors thank the Scientific and Technological Research Council of Turkey (TUBITAK) (Project ID: 113Z165) and Ege University Scientific Research Project Office (Project ID: 2014 BIL 006 and 2015 FEN 055) for their financial supports.

BACKGROUND Folate plays a fundamental role for fetal development, participating in cell division, embryogenesis, and fetal growth. The fetus depends on maternal supply of folate across the placenta. The objective of this study was to compare the expression of Folate Receptor-α (FR-α), Reduced Folate Carrier (RFC), and Proton Coupled Folate Transporter (PCFT) in placentas from pregnancies complicated with birth defects (BD) and controls. METHODS Case-control study, including placentas of BD-complicated pregnancies (n = 25) and a control group (n = 25). We determined the placental expression of FR-α, RFC, and PCFT by immunohistochemistry. Optical density was measured to obtain a relative quantification of the expression. RESULTS The expression of PCFT was greater in placentas from pregnancies complicated with BD than in those from the control group (p