Low-dose daily folic acid (400 µg) supplementation does not affect regulation of folate transporters found present throughout the terminal ileum and colon of humans: a randomized clinical trial.

BACKGROUND: Folic acid supplementation during the periconceptional period reduces the risk of neural tube defects in infants, but concern over chronic folic acid exposure remains. An improved understanding of folate absorption may clarify potential risks. Folate transporters have been characterized in the small intestine, but less so in the colon of healthy, free-living humans. The impact of folic acid fortification or supplementation on regulation of these transporters along the intestinal tract is unknown. OBJECTIVE: The objective was to characterize expression of folate transporters/receptor (FT/R) and folate hydrolase, glutamate carboxypeptidase II (GCPII), from the terminal ileum and throughout the colon of adults and assess the impact of supplemental folic acid. METHODS: In this 16-wk open-labeled randomized clinical trial, adults consumed a low folic acid-containing diet, a folate-free multivitamin, and either a 400 µg folic acid supplement or no folic acid supplement. Dietary intakes and blood were assessed at baseline, 8 wk, and 16 wk (time of colonoscopy). Messenger RNA (mRNA) expression and protein expression of FT/R and GCPII were assessed in the terminal ileum, cecum, and ascending and descending colon. RESULTS: Among 24 randomly assigned subjects, no differences in dietary folate intake or blood folate were observed at baseline. Mean ± SD red blood cell folate at 16 wk was 1765 ± 426 and 911 ± 242 nmol/L in the 400 and 0 µg folic acid group, respectively (P < 0.0001). Reduced folate carrier, proton-coupled folate transporter, and folate-receptor alpha expression were detected in the terminal ileum and colon, as were efflux transporters of breast cancer resistance protein and multidrug resistance protein-3. Other than a higher mRNA expression of FR-alpha and GCPII in the 400 µg supplement group in the ascending colon, no treatment differences were observed (P < 0.02). CONCLUSIONS: Folate transporters are present throughout the terminal ileum and colon; there is little evidence that a low dose of folic acid supplementation affects colonic absorption. This trial was registered at clinicaltrials.gov as NCT03421483.

Laboratory assessment of folate (vitamin B9) status.

Folate (vitamin B9) plays a crucial role in fundamental cellular processes, including nucleic acid biosynthesis, methyl group biogenesis and amino acid metabolism. The detection and correction of folate deficiency prevents megaloblastic anaemia and reduces the risk of neural tube defects. Coexisting deficiencies of folate and vitamin B12 are associated with cognitive decline, depression and neuropathy. Folate deficiency and excess has also been implicated in some cancers. Excessive exposure to folic acid, a synthetic compound used in supplements and fortified foods, has also been linked to adverse health effects. Of at least three distinct laboratory markers of folate status, it is the total abundance of folate in serum/plasma that is used by the majority of laboratories. The analysis of folate in red cells is also commonly performed. Since the folate content of red cells is fixed during erythropoiesis, this marker is indicative of folate status over the preceding ~4 months. Poor stability, variation in polyglutamate chain length and unreliable extraction from red cells are factors that make the analysis of folate challenging. The clinical use of measuring specific folate species has also been explored. 5-Methyltetrahydrofolate, the main form of folate found in blood, is essential for the vitamin B12-dependent methionine synthase mediated remethylation of homocysteine to methionine. As such, homocysteine measurement reflects cellular folate and vitamin B12 use. When interpreting homocysteine results, age, sex and pregnancy, specific reference ranges should be applied. The evaluation of folate status using combined markers of abundance and cellular use has been adopted by some laboratories. In the presence of discordance between laboratory results and strong clinical features of deficiency, treatment should not be delayed. High folate status should be followed up with the assessment of vitamin B12 status, a review of previous results and reassessment of folic acid supplementation regime.

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

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

Folate-targeting annonaceous acetogenins nanosuspensions: significantly enhanced antitumor efficacy in HeLa tumor-bearing mice.

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 < .05), which could be effectively reversed simply by pretreatment of free FA. In vivo experiments demonstrated that FA-PEG-ACGs-NSps brought more drug molecules into tumors and greatly improved the antitumor efficacy (TIR, 76.45% vs. 25.29%, p < .001). Therefore, DSPE-PEG-FA is considered as a proper stabilizer with active targeting effect for ACGs-NSps to reduce toxicity, enlarge the safe dosage range and apply in clinic for the treatment of folate-positive tumors. Therefore, FA-PEG-ACGs-NSps may be a prospective drug delivery system for ACGs to improve their therapeutic window and find application in clinic to treat FR over-expressed tumors.

Folate-conjugated liposomes target and deliver therapeutics to immune cells in a rat model of rheumatoid arthritis.

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.

Folate deprivation induces cell cycle arrest at G0/G1 phase and apoptosis in hippocampal neuron cells through down-regulation of IGF-1 signaling pathway.

Folate deficiency contributes to impaired adult hippocampal neurogenesis, yet the mechanisms remain unclear. Here we use HT-22 hippocampal neuron cells as model to investigate the effect of folate deprivation (FD) on cell proliferation and apoptosis, and to elucidate the underlying mechanism. FD caused cell cycle arrest at G0/G1 phase and increased the rate of apoptosis, which was associated with disrupted expression of folate transport and methyl transfer genes. FOLR1 and SLC46A1 were (P<0.01) down-regulated, while SLC19A1 was up-regulated (P<0.01) in FD group. FD cells exhibited significantly (P<0.05) higher protein content of BHMT, MAT2b and DNMT3a, as well as increased SAM/SAH concentrations and global DNA hypermethylation. The expression of the total and all the 3 classes of IGF-1 mRNA variants was significantly (P<0.01) down-regulated and IGF-1 concentration was decreased (P<0.05) in the culture media. IGF-1 signaling pathway was also compromised with diminished activation (P<0.05) of STAT3, AKT and mTOR. CpG hypermethylation was detected in the promoter regions of IGF-1 and FOLR1 genes, while higher SLC19A1 mRNA corresponded to hypomethylation of its promoter. IGF-1 supplementation in FD media significantly abolished FD-induced decrease in cell viability. However, IGF-1 had limited effect in rescuing the cell phenotype when added 24h after FD. Taken together, down-regulation of IGF-1 expression and signaling is involved in FD-induced cell cycle arrest and apoptosis in HT-22 hippocampal neuron cells, which is associated with an abnormal activation of methyl transfer pathway and hypermethylation of IGF-1 gene promoter.

Design, preparation, and in vitro characterization of a trimodally-targeted nanomagnetic onco-theranostic system for cancer diagnosis and therapy.

In this study, the aim was to introduce and characterize a new trimodally-targeted nanomagnetic onco-theranostic system for simultaneous early diagnosis and efficient treatment of cancer. The onco-theranostic system was designed as it could target the tumor site through three targeting approach, i.e. magnetic, folic acid receptor, and pH sensitivity, and concurrently, due to the presence of superparamagnetic iron oxide nanoparticles (SPIONs) with super paramagnetic characteristics could be useful as MRI contrast agent for early cancer diagnosis. To achieve this goal, SPIONs were coated with chitosan and folic acid-conjugated chitosan via ionic gelation method in order to obtain non-targeted nanomagnetic onco-diagnostic (NT/NOD) and targeted nanomagnetic onco-diagnostic (T/NOD) systems. Finally, doxorubicin was loaded successfully into NT/NOD and T/NOD in order to obtain nanomagnetic onco-theranostic (NT/NOT) and targeted nanomagnetic onco-theranostic (T/NOT) systems. The entrapment efficiency and drug loading of T/NOT was determined to be 62.33 ± 5.20% and 10.26 ± 1.36%, respectively. MTT assay revealed that all systems were biocompatible within the concentration range investigated. Also, the T/NOT system showed the lowest IC50 comparing with free doxorubicin and NT/NOT system. In addition, uptake studies and competitive inhibition study verified the folate receptor mediated endocytosis of targeted system by MCF-7 as a folate receptor-positive cell line. The finding revealed that the extent of drug release from theranostic systems was pH-sensitive as it was higher at acidic media compared to that of in the neutral condition. Finally, T2-weighted phantom images, with an acceptable and dose-dependent resolution, proved the potential of T/NOT system as promising T2 MR contrast agent for diagnostic purpose. These finding proved that the prepared T/NOT system have great potential as a novel tumor-targeting nanotheranostic agent for simultaneous MRI imaging and treatment of folate receptor-positive cancers. Further studies are needed to test their behavior in vivo.

Doxorubicin loaded polymeric gold nanoparticles targeted to human folate receptor upon laser photothermal therapy potentiates chemotherapy in breast cancer cell lines.

The current research focuses on the application of folate conjugated and doxorubicin loaded polymeric gold nanoparticles (GNPs) for the targeted treatment of folate receptor overexpressing breast cancers, augmented by adjunctive laser photothermal therapy. Herein, GNPs surface modified with folate, drug doxorubicin and polyethylene glycol were engineered and were used as vehicles for folate receptor targeted delivery of doxorubicin into cancer cells. Subsequently, the GNPs were photo-excited using laser light for mediating hyperthermia in the cancer cells. In vitro studies were performed to validate the efficacy of the combined modality of folate conjugated and doxorubicin loaded polymeric GNP mediated chemotherapy followed by photothermal therapy in comparison to treatment with free drug; and the combination modality showed better therapeutic efficacy than that of plain doxorubicin treatment in MDA-MB-231 breast cancer cells that express increased levels of surface folate receptors when compared to MCF-7 breast cancer cells that express low levels of folate receptor. The mechanism of cell death was investigated using fluorescent microscopy. Immunoassays showed the up-regulation of the pro-apoptotic protein p53 and down-regulation of the anti-apoptotic protein Bcl-2. Collectively, these results suggest that the folate tagged doxorubicin loaded GNPs are an attractive platform for targeted delivery of doxorubicin and are agents suitable for photothermal cancer therapy.

The effect of ligand density on in vivo tumor targeting of nanographene oxide.

Recently, the application of nanographene oxide (nGO) as a drug delivery system has significantly increased. But, the rational engineering of nGO surface to improve its in vivo targeting and biodistribution remains mostly unexplored. In this study, we have prepared folic acid conjugated Pluronic for non-covalent functionalization of nanographene oxide (nGO) sheets and active tumor targeting. To modulate the ligand density on the nGO surface, different ratios of folate conjugated Pluronic and unmodified Pluronic were combined and used for coating nGO sheets. The surface density of targeting ligand linearly increased as the relative amount of folate conjugated Pluronic was increased. The association of functionalized nGOs with folate receptor overexpressing human epithelial mouth carcinoma cells (KB cells) was evaluated by flow cytometry. Cellular uptake of nGO by KB cells increased steadily with the increase in ligand density. In contrast, the in vivo experiment in mouse xenograft model did not show the steady increase in tumor targeting by increasing ligand density. Upon intravenous administration into KB tumor-bearing mice, tumor accumulation of nGO did not show a significant targeting effect up to 25% of ligand coating density. However, a strong and similar tumor accumulation of nGO was observed for both 50% and 100% folate coatings. Thus, a significant difference in tumor accumulation of nGO was observed between the low folate density groups and high folate density groups, suggesting the existence of a critical ligand density for tumor targeting. The significant difference of tumor targeting of nGO depending on ligand density also resulted in the dramatic change in photothermal tumor ablation by the irradiation of NIR laser.

Performance, serum biochemical responses, and gene expression of intestinal folate transporters of young and older laying hens in response to dietary folic acid supplementation and challenge with Escherichia coli lipopolysaccharide.

The present study was conducted to investigate the effects of dietary folic acid (FA) supplementation on performance, serum biochemical indices, and mRNA abundance of intestinal folate transporters in young and older laying hens after acute lipopolysaccharide (LPS) challenge. Two experiments were conducted separately involving 48 Shaver White young laying hens (24 wk of age) in experiment 1 and 48 Shaver White older laying hens (58 wk of age) in experiment 2. Birds were fed 2 diets in a complete randomized design. The diets were wheat-soybean meal based, with or without supplemental 4 mg of FA/kg of diet. Birds were fed for 8 wk, during which time feed consumption and egg production were monitored. At the end of each feeding experiment, 6 hens from each dietary treatment were injected intravenously with 8 mg/kg of BW of either Escherichia coli LPS or sterile saline. Four hours after injection, blood and intestinal samples were collected for further analysis. Compared with the control, dietary FA supplementation increased egg weight and egg mass and decreased serum glucose levels in the young laying hens, and reduced serum uric acid in the older laying hens (P < 0.05). Relative to saline injection, plasma homocysteine, serum calcium, and phosphorus levels were found to be lower in both young and older laying hens after LPS challenge (P < 0.05). Other serum biochemical variables and the mRNA expression of 2 folate transport genes in the small and large intestine were differentially affected by LPS challenge, and some of those responses varied with the age of the birds. Additionally, interactions between diet and LPS challenge were specifically found in the older laying hens. In summary, in addition to improving production performance, there were effects of dietary FA supplementation and its interaction with LPS challenge on biochemical constituents, and age played a role in the development of responses to diet and bacterial LPS infections.

Development of new folate-based PET radiotracers: preclinical evaluation of 68Ga-DOTA-folate conjugates.

PURPOSE: A number of (111)In- and (99m)Tc-folate-based tracers have been evaluated as diagnostic agents for imaging folate receptor (FR)-positive tumours. A (68)Ga-folate-based radiopharmaceutical would be of great interest, combining the advantages of PET technology and the availability of (68)Ga from a generator. The aim of the study was to develop a new (68)Ga-folate-based PET radiotracer. METHODS: Two new DOTA-folate conjugates, named P3026 and P1254, were synthesized using the 1,2-diaminoethane and 3-{2-[2-(3-amino-propoxy)-ethoxy]-ethoxy}-propylamine as a spacer, respectively. Both conjugates were labelled with (67/68)Ga. Binding affinity, internalization and externalization studies were performed using the FR-positive KB cell line. Biodistribution and PET/CT imaging studies were performed in nude mice, on a folate-deficient diet, bearing KB and HT1080 (FR-negative) tumours, concurrently. The new radiotracers were evaluated comparatively to the reference molecule (111)In-DTPA-folate ((111)In-P3139). RESULTS: The K(d) values of (67/68)Ga-P3026 (4.65 ± 0.82 nM) and (67/68)Ga-P1254 (4.27 ± 0.42 nM) showed high affinity for the FR. The internalization rate followed the order (67/68)Ga-P3026 > (67/68)Ga-P1254 > (111)In-P3139, while almost double cellular retention was found for (67/68)Ga-P3026 and (67/68)Ga-P1254, compared to (111)In-P3139. The biodistribution data of (67/68)Ga-DOTA-folates showed high and receptor-mediated uptake on the FR-positive tumours and kidneys, with no significant differences compared to (111)In-P3139. PET/CT images, performed with (68)Ga-P3026, showed high uptake in the kidneys and clear visualization of the FR-positive tumours. CONCLUSION: The DOTA-folate conjugates can be efficiently labelled with (68)Ga in labelling yields and specific activities which allow clinical application. The characteristics of the (67/68)Ga-DOTA-folates are comparable to (111)In-DTPA-folate, which has already been used in clinical trials, showing that the new conjugates are promising candidates as PET radiotracers for FR-positive tumours.

Cerebral folate deficiency.

Cerebral folate deficiency (CFD) is defined as any neurological syndrome associated with a low cerebrospinal fluid (CSF) concentration of 5-methyltetrahydrofolate (5MTHF) in the presence of normal peripheral folate status. CFD has a wide clinical presentation, with reported signs and symptoms generally beginning at around 4 months of age with irritability and sleep disturbances. These can be followed by psychomotor retardation, dyskinesia, cerebellar ataxia and spastic diplegia. Other signs may include deceleration of head growth, visual disturbances and sensorineural hearing loss. Identification of CFD is achieved by determining 5MTHF concentration in CSF. Once identified, CFD can in many cases be treated by administering oral folinic acid. Supplementation with folic acid is contraindicated and, if used, may exacerbate the CSF 5MTHF deficiency. Generation of autoantibodies against the folate receptor required to transport 5MTHF into CSF and mutations in the folate receptor 1 (FOLR1) gene have been reported to be causes of CFD. However, other mechanisms are probably also involved, as CFD has been reported in Aicardi-Goutiere's and Rett syndromes and in mitochondriopathies. Several metabolic conditions and a number of widely used drugs can also lead to a decrease in the concentration of CSF 5MTHF, and these should be considered in the differential diagnosis if a low concentration of 5MTHF is found following CSF analysis.

Depleted folate pool and dysfunctional mitochondria associated with defective mitochondrial folate proteins sensitize Chinese ovary cell mutants to tert-butylhydroperoxide-induced oxidative stress and apoptosis.

The functional role of mitochondrial (mt) folate-associated proteins in mammalian cells is not clearly understood. We investigated the respiratory function and apoptosis phenotype of Chinese hamster ovary (CHO) mutant cells with defective mt serine hydroxymethyltransferase (SHMT) activities (glyA) or with defective mt folate transporter (glyB) in the absence/presence of oxidant challenge. The mechanisms underlying their aberrant phenotypes were explored. Compared with CHOK1 wild-type cells, both mutants carried dysfunctional mitochondria with reduced respiratory complex IV activity and depolarized mt membrane potential (P<.05). Elevated superoxide levels and accumulated mtDNA large deletions were observed in glyB in association with a depleted compartmental folate pool (P<.05). tert-Butylhydroperoxide (tBH) treatment at 50 microM for 72 h significantly depleted mt and cytosolic folate levels, impaired antioxidant defenses, and aggravated mt oxidative dysfunction in both mutants (P<.05), more severely in glyB. Only tBH-treated glyB cells displayed an elevated ratio of mt Bax/Bcl-2, activation of procaspases 9 and 3, and apoptosis promotion. The apoptotic phenotype of tBH-treated glyB could be partially corrected by folate supplementation (10-1000 microM), which enriched compartmental folate levels, restored antioxidant defenses, eliminated mt oxidative injuries, and normalized mt membrane function. Our data identify previously unrecognized roles of mt folate-associated proteins in the protection of mitochondria against oxidative insults. Defective mt folate transporter sensitized glyB cells to elevated oxidative stress and tBH-induced apoptosis, partly mediated by depleted compartmental folate and mt dysfunction. Defective mt SHMT sensitized glyA to respiratory dysfunction and tBH-induced oxidative injury without apoptosis promotion.