Assessment of the in Vitro Effects of Folate Core-Shell Conjugated Iron Oxide Nanoparticles as a Potential Agent for Acute Leukemia Treatment.

BACKGROUND AND OBJECTIVE: There is a growing need to comprehend the potential outcomes of nanoparticles (NPs) on human well-being, including their potential for detecting and treating leukemia. This study examined the role of iron folate core-shell and iron oxide nanoparticles in inducing apoptosis and altering the expression of the B-cell lymphoma 2 (Bcl-2), Bcl-2 associated X-protein (Bax), and Caspase-3 genes in leukemia cells. METHODS: The obtained iron oxide and iron folate core-shell nanoparticles were analyzed using a variety of analytical techniques, including ultraviolet-visible (UV-Vis) absorption spectroscopy, Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), dynamic light scattering (DLS), zeta potential, and transmission electron microscopy (TEM). Additionally, FTIR and UV-Vis were used to characterize doxorubicin. The MTT test was utilized to investigate the cytotoxicity of iron oxide and iron folate core-shell nanoparticles. The expression of the apoptotic signaling proteins Bcl-2, Bax, and Caspase-3 was evaluated using the real-time reverse transcription polymerase chain reaction (RT-qPCR) method. Additionally, flow cytometry was performed to gauge the degrees of necrosis and apoptosis. RESULTS: UV-Visible spectroscopy analysis showed that the generated iron oxide and iron folate core-shell NPs had a distinctive absorption curve in the 250-300 nm wavelength range. The XRD peaks were also discovered to index the spherical form with a size of less than 50 nm, which validated the crystal structure. The FTIR analysis determined the bonds and functional groups at wavenumbers between 400 and 4000 cm-1. A viable leukemia treatment approach is a nanocomposite consisting of iron and an iron folate core-shell necessary for inhibiting and activating cancer cell death. The nearly resistant apoptosis in the CCRF-CEM cells may have resulted from upregulating Bax and Casepase-3 while downregulating Bcl-2 expression. CONCLUSIONS: Our study documents the successful synthetization and characterization of iron oxide, which has excellent anticancer activities. A metal oxide conjugation with the nanoparticles' core-shell enhanced the effect against acute leukemia.

Folic acid-chitosan coated stylosin nanostructured lipid carriers: fabrication, in vitro-in vivo assessment in breast malignant cells.

Synthesis of targeted nanostructure lipid carriers for stylosin (STY-CFN-NPs) delivery to MCF-7 cells. STY-CFN-NPs were formulated via the homogenization and ultra-sonication technique. After evaluating the amount of drug encapsulation and FA binding, the toxicity effect of the STY and STY-CFN-NPs on MCF-7 cells was measured by the MTT method. Cell cycle analysis, AO/PI staining and qPCR to assess the inducing of apoptosis as well as Tubo cancer cell inoculated mouse model for antitumor properties of STY-CFN-NPs were used. Significant increases in nanoparticle size and changes in zeta potential were observed after FA-CS coating on nanoparticles. Slow release of the STY within 144 h as well as the acceptable rate for STY encapsulation efficiency (92.4% and FA binding (52.5%) to the STY-CFN-NPs (PS: 66.26 ± 3.02 nm, ZP: 29.54 ± 1.01 mV and PDI: 0.32 ± 0.01) was reported. STY-CFN-NPs exhibited higher toxicity compared to STY suspension and treatment with STY-CFN-NPs was lead to increased apoptotic cells, stopped cells in the SubG1 phase, and also increased caspase and BAX expression and decreased BCL-2 and BCL-XL expression in in vitro and decreased the size of murine tumors (54.57% in 16 days) in in vivo. The results showed STY-CFN-NPs have good potential for breast cancer management.

Assessment of Health Claims Related to Folic Acid in Food Supplements for Pregnant Women According to the European Regulation.

Pregnant women are a vulnerable group with increased nutritional requirements. The daily intake of folic acid, a crucial vitamin for embryonic development, must be reinforced through supplementation, as sometimes diets are not well equilibrated. As consumers increasingly rely on food supplements, it is vital to properly inform them about the health benefits provided by supplements' consumption to ensure their safe use. The objective of this work was to assess the compliance level of health claims related to folic acid in food supplements commercialized in Spain according to the European regulation. Authors performed (1) a review of health-related claims approved for folic acid in Europe, (2) a market research of food supplements commercialized in Spain with those claims, and (3) a selection of food supplements for chemical analysis in the lab to assess these claims. The results showed that nine health-related claims are currently approved for folic acid in Europe. The analytical results for folic acid content in the selected samples were consistent with the declared values and within the tolerance ranges established in the European Guidance document. All samples included accurate dosages and met the legal requirements (European Regulations 1924/2006, 432/2012, 1169/2011) for all approved claims for folic acid.

Synthesis, radiolabelling, and biological assessment of folic acid-conjugated G-3 99mTc-dendrimer as the breast cancer molecular imaging agent.

Hence, in this study, the authors aimed to develop a dendrimer-based imaging agent comprised of poly(ethylene glycol) (PEG)-citrate, technetium-99 m (99mTc), and folic acid. The dendrimer-G3 was synthesised and conjugated with folic acid, which confirmed by Fourier transform infrared, proton nuclear magnetic resonance, dynamic light scattering, and transition electron microscopy. 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-Tetrazolium-5-Carboxanilide cytotoxicity assay kit was used to measure the cellular toxicity of dendrimer. Imaging and biodistribution studies were conducted on the mice bearing tumour. The results showed that the fabricated dendrimer-G3 has a size of 90 ± 3 nm, which was increased to 100 ± 4 nm following the conjugation with folic acid. The radiostablity investigation showed that the fabricated dendrimers were stable in the human serum at various times. Toxicity assessment confirmed no cellular toxicity against HEK-293 cells at 0.25, 0.5, 1, 2, 4, and 8 mg/µl concentrations. The in vivo studies demonstrated that the synthesised dendrimers were able to provide a bright SPECT image applicable for tumour detection. In conclusion, the authors' study documented the positive aspects of PEG-citrate dendrimer conjugated with folic acid as the SPECT contrast agent for breast cancer detection.

Technology for Triple Fortification of Salt with Folic Acid, Iron, and Iodine.

As many of the maternal and child health complications result from folic acid, iron, and iodine deficiencies; it makes sense to combat these simultaneously. We have developed cost-effective technology to deliver these three micronutrients simultaneously through salt. Our goal was to retain at least 70% of the micronutrients during 6 months of storage. The fortified salt was formulated by spraying a solution that contained 2% iodine and 0.5% or 1% folic acid onto salt and adding encapsulated ferrous fumarate. The formulated triple fortified salt contained 1,000 ppm iron, 50 ppm iodine, and 12.5 or 25 ppm folic acid. The spray solution and the salt were stored for 2 and 6 months respectively at 25, 35, and 45 °C 60 to 70% relative humidity. Even at 45 °C, over 70% of both iodine and folic acid were retained in the salt. The best formulation based on the color of the salt and stability of iodine and folic acid contained 12.5 ppm folic acid, 50 ppm iodine, and 1,000 ppm iron. These results indicate that iron, iodine, and folic acid can be simultaneously delivered to a vulnerable population through salt using the technology described. Also, the quality control of the process can be developed around pteroic acid that was detected as a primary degradation product of folic acid. PRACTICAL APPLICATION: The technology developed is already transferred to India for industrial scale up. When fully operational, the technology will simultaneously solve iron, iodine, and folic acid deficiencies in vulnerable populations at a very low cost.

Quantitative Assessment of Nanoparticle Biodistribution by Fluorescence Imaging, Revisited.

Fluorescence-based whole-body imaging is widely used in the evaluation of nanoparticles (NPs) in small animals, often combined with quantitative analysis to indicate their spatiotemporal distribution following systemic administration. An underlying assumption is that the fluorescence label represents NPs and the intensity increases with the amount of NPs and/or the labeling dyes accumulated in the region of interest. We prepare DiR-loaded poly(lactic- co-glycolic acid) (PLGA) NPs with different surface layers (polyethylene glycol with and without folate terminus) and compare the distribution of fluorescence signals in a mouse model of folate-receptor-expressing tumors by near-infrared fluorescence whole-body imaging. Unexpectedly, we observe that fluorescence distribution patterns differ far more dramatically with DiR loading than with the surface ligand, reaching opposite conclusions with the same type of NPs (tumor-specific delivery vs predominant liver accumulation). Analysis of DiR-loaded PLGA NPs reveals that fluorescence quenching, dequenching, and signal saturation, which occur with the increasing dye content and local NP concentration, are responsible for the conflicting interpretations. This study highlights the critical need for validating fluorescence labeling of NPs in the quantitative analysis of whole-body imaging. In light of our observation, we make suggestions for future whole-body fluorescence imaging in the in vivo evaluation of NP behaviors.

Folic acid Targeted Polymeric Micelles Based on Tocopherol Succinate- Pulluan as an Effective Carrier for Epirubicin: Preparation, Characterization and In-vitro Cytotoxicity Assessment.

BACKGROUND: Chemotherapy still encounters a serious drawback, the lack of selectivity of anticancer drugs toward neoplastic cells, thus, the normal cells are affected by the cytotoxic action of the drugs. This causes a narrow therapeutic index in most anticancer drugs. OBJECTIVE: We describe the preparation of pullulan-tocopherol succinate-folic acid (Pu-TS-FA) micelles for the first time to targeted delivery of Epirubicin (EPI) to Hela and MCF-7 cell lines. METHODS: We confirmed the structure of conjugate using spectroscopic methods. The degree of substitution for both folic acid and tocopherol succinate was calculated using 1HNMR. We prepared the micelles via direct dissolution method. All the physicochemical properties of micelles including size, zeta potential, polydispersity index (PDI), critical micelle concentration (CMC), entrapment efficiency (EE %) and release efficiency (RE24%) were determined. The morphology of particles was studied using transmission electron microscopy (TEM), and the in-vitro cell cytotoxicity of EPI loaded micelles was studied using MTT assay on MCF-7 and Hela cell lines. RESULTS: The optimized micelles showed the particle size of 149.5 nm, the zeta potential of -6.49 mV, a polydispersity index of 0.259 ± 0.07, LE% of 88 %, and RE24% of 63 ± 2.45 % with a relatively low CMC 194.87 µg/ml. TEM showed the relatively uniform spherical structure for particles and in vitro MTT assay showed that EPI loaded micelles were more toxic on Hela cell line than MCF7 as expected. CONCLUSION: Since the Pu-TS-FA micelle could improve the anticancer activity of epirubicin and would be a promising candidate for EPI treatment of cancers.

Multimodal molecular 3D imaging for the tumoral volumetric distribution assessment of folate-based biosensors.

The aim of this study was to characterize the in vivo volumetric distribution of three folate-based biosensors by different imaging modalities (X-ray, fluorescence, Cerenkov luminescence, and radioisotopic imaging) through the development of a tridimensional image reconstruction algorithm. The preclinical and multimodal Xtreme imaging system, with a Multimodal Animal Rotation System (MARS), was used to acquire bidimensional images, which were processed to obtain the tridimensional reconstruction. Images of mice at different times (biosensor distribution) were simultaneously obtained from the four imaging modalities. The filtered back projection and inverse Radon transformation were used as main image-processing techniques. The algorithm developed in Matlab was able to calculate the volumetric profiles of 99mTc-Folate-Bombesin (radioisotopic image), 177Lu-Folate-Bombesin (Cerenkov image), and FolateRSense™ 680 (fluorescence image) in tumors and kidneys of mice, and no significant differences were detected in the volumetric quantifications among measurement techniques. The imaging tridimensional reconstruction algorithm can be easily extrapolated to different 2D acquisition-type images. This characteristic flexibility of the algorithm developed in this study is a remarkable advantage in comparison to similar reconstruction methods.

Synthesis and biological assessment of folate-accepted developer (99m)Tc-DTPA-folate-polymer.

A novel cancer-targetable folate-poly(2-hydroxyethyl methacrylate) (PFDH) copolymer containing DTPA segment was prepared by conventional chemical synthesis and labeled with (99m)Tc subsequently. The (99m)Tc-labled PFDH could be produced easily with high radiochemical yield of 91% and radiochemical purity of 95%. The LogP octanol-water value for the (99m)Tc-labled PFDH was -2.19 and the radiotracer was stable in phosphate-buffered saline and human serum for 2h (>95% in PBS or ∼90% in human serum). To investigate (99m)Tc-labled PFDH tumor targeting, the in vitro and in vivo stability, cell uptake, in vivo biodistribution, and SPECT imaging were evaluated, respectively. These preliminary results strongly suggest that the novel folate conjugated dendrimer maybe developed to be potential for delivery of therapeutic radionuclides.

Stability, Pharmacokinetics, Biodistribution and Safety Assessment of Folate-Conjugated Pullulan Acetate Nanoparticles as Cervical Cancer Targeted Drug Carriers.

It is recognized that the stability and journey in the body of nanoparticles are important issues for drug formulations. In this study, we prepared folate-conjugated pullulan acetate nanoparticles (FPANs) and epirubicin loaded FPANs (FPA/EPI) using dialysis method. The storage stability of FPANs and FPA/EPI at 4 degrees C could be up to 3 months. Using folate receptor overexpressed Hela cells, dose dependent cellular uptake and receptor-mediated endocytosis of FPA/EPI were confirmed. From the in vivo pharmacokinetics test, compared to free EPI, half-life time (t½) of FPA/EPI was extended 1.57 times and the area under-the-curve (AUC) increased 3.95 times as well. In addition, biodistribution data showed that, EPI concentration in tumor in FPA/EPI group was 2.01 times higher than that in free EPI group after 96 h; The concentration of drug in liver treated by FPA/EPI was 5.7-11.6 times, while in heart, kidney, especially in stomach and intestine were much lower than those in free EPI group from 24 to 96 h. Furthermore, blank FPANs showed no apparent acute toxicity at dose up to 125 mg/kg. All results suggested that FPA/EPI showed a promising potential on treating cervical carcinoma and its metastatic hepatocellular carcinoma in future because of the high stability, less toxicity and tumor targeting.

Gold nanoprobes-based resonance Rayleigh scattering assay platform: Sensitive cytosensing of breast cancer cells and facile monitoring of folate receptor expression.

A rapid, facile assay for sensitive cytosensing of breast cancer cells should help to guide potential medical evaluation for breast cancer. Here, we report development of novel resonance Rayleigh scattering (RRS) cytosensor for cell recognitions and folate (FA) receptor expression analyses on living cells. Using FA-conjugated gold nanoparticles (FA-AuNPs) as nanoprobes, the constructed nanoprobes-assembled recognition interface could increase the binding capacity for cell recognition, amplify Au-aggregates-enhanced RRS signal, and then enhance the sensitivity for membrane antibody assay. FA-AuNPs-based RRS measurements enabled a distinct 34-times-enhancement in RRS intensities after incubation with human breast cancer cells, compared with normal cells. Receptor-targeted cytosensor was used to quantitatively detect human breast cancer MCF-7, liver cancer HepG2 and normal cells, which expressing different amount of FA receptor, respectively. The detection limit for MCF-7 cells was 12 cells/mL with good selectivity and reproducibility. Furthermore, the proposed cytosensor allowed for dynamic evaluation of FA receptor expression on different living cells after dihydroartemisinin stimulus. This assay platform shows the good potential for clinical diagnostics and antibody-targeted drug screening.

In vitro control release, cytotoxicity assessment and cellular uptake of methotrexate loaded liquid-crystalline folate nanocarrier.

Folate molecules self-assemble in the form of stacks to form liquid-crystalline solutions. Nanocarriers from self-assembled folates are composed of highly ordered structures, which offer high encapsulation of drug (95-98%), controlled drug release rates, active cellular uptake and biocompatibility. Recently, we have shown that the release rates of methotrexate can be controlled by varying the size of nanoparticles, cross-linking cation and cross-linking concentration. The present study reports the in vitro cytotoxic behavior of methotrexate loaded liquid-crystalline folate nanoparticles on cultured HeLa cells. Changing drug release rates can influence cytotoxicity of cancer cells. Therefore, to study the correlation of release rate and cytotoxic behavior, the effect of release controlling parameters on HeLa cells was studied through MTT assay. It is reported that by controlling the methotrexate release, the survival rates of HeLa cells can be controlled. Released methotrexate kills HeLa cells as effectively as free methotrexate solution. The co-culture based in vitro cellular uptake study through fluorescence microscopy on folate receptor positive and negative cancer cells shows that the present nanocarrier has the potential to distinguish cancer cells from normal cells. Overall, the present study reports the in vitro performance of self-assembled liquid-crystalline folate nanoparticles, which will be a platform for further in vivo studies and clinical trials.

Bioaccessibility of vitamin A, vitamin C and folic acid from dietary supplements, fortified food and infant formula.

In the Netherlands, vitamin intake occurs mainly via food and for some vitamins also via fortified food. In addition, some people take dietary supplements. Information on the bioavailability of vitamins is important for a good estimation of the actual exposure to vitamins. Furthermore, for a reliable intake estimation, it is important to know the accurateness of the claimed vitamin concentration on the product label. In the current study, the amount of vitamin A, vitamin C, and folic acid in different products and their maximum bioavailability (bioaccessibility) were investigated. In about half of the products, the amount of vitamins significantly deviated from the declared amounts. The vitamin bioaccessibility ranged from <1% to 100%. When assessing the dietary intake exposure of vitamins, it is important to take into account both the possible deviation from the declared level and (the variability of) the bioaccessibility of the vitamin in the products.

Evaluation of food and nutrient intake assessment using concentration biomarkers in European adolescents from the Healthy Lifestyle in Europe by Nutrition in Adolescence study.

Accurate food and nutrient intake assessment is essential for investigating diet-disease relationships. In the present study, food and nutrient intake assessment among European adolescents using 24 h recalls (mean of two recalls) and a FFQ (separately and the combination of both) were evaluated using concentration biomarkers. Biomarkers included were vitamin C, ß-carotene, DHA+EPA, vitamin B12 (cobalamin and holo-transcobalamin) and folate (erythrocyte folate and plasma folate). For the evaluation of the food intake assessment 390 adolescents were included, while 697 were included for the nutrient intake assessment evaluation. Spearman rank and Pearson correlations, and validity coefficients, which are correlations between intake estimated and habitual true intake, were calculated. Correlations were higher between frequency of food consumption (from the FFQ) and concentration biomarkers than between mean food intake (from the recalls) and concentration biomarkers, especially for DHA+EPA (r 0·35 v. r 0·27). Most correlations were higher among girls than boys. For boys, the highest validity coefficients were found for frequency of fruit consumption (0·88) and for DHA+EPA biomarker (0·71). In girls, the highest validity coefficients were found for fruit consumption frequency (0·76), vegetable consumption frequency (0·74), mean fruit intake (0·90) and DHA+EPA biomarker (0·69). After exclusion of underreporters, correlations slightly improved. Correlations between usual food intakes, adjusted for food consumption frequency, and concentration biomarkers were higher than correlations between mean food intakes and concentration biomarkers. In conclusion, two non-consecutive 24 h recalls in combination with a FFQ seem to be appropriate to rank subjects according to their usual food intake.

Morphological properties of nanoporous folic acid materials and in vitro assessment of their biocompatibility.

BACKGROUND: Mesoporous silica-based particles are of potential interest for the development of novel therapeutic targeted delivery vehicles. Their ability to load and release large quantities of active pharmaceutical products with varying properties, combining controlled and targeted release functions make them unique amongst nanotechnology-based carrier systems. MATERIALS & METHODS: In this study, nanoporous folic acid-templated materials (NFM-1) were prepared and the synthetic strategies for the control of textural and morphology properties of NFM-1 are described. The potential biocompatibility of NFM-1 particles with different morphology (gyroid shaped, fibers and rod-shaped) was assessed using a panel of human cell lines. RESULTS: The results reveal that NFM-1 morphology has an impact on cell viability such that particles showing higher aspect ratios possess increased cytotoxicity. CONCLUSION: These studies provide useful information for the development of novel mesoporous materials for biomedical applications, including cell-specific drug delivery.

Assessment of diffuse transmission mode in near-infrared quantification--part I: The press effect on low-dose pharmaceutical tablets.

Quantitative applications for pharmaceutical solid dosage forms using near-infrared (NIR) spectroscopy are central to process analytical technology (PAT) manufacturing designs. A series of studies were conducted to evaluate the use of NIR transmission mode under various pharmaceutical settings. The spectral variability in relation to tablet physical parameters were investigated using placebo tablets with different thickness and porosity steps and both variables showed an exponential relationship with the detected transmittance signal drop. The drug content of 2.5% m/m folic acid tablets produced under extremely different compaction conditions was predicted and found to agree with UV assay results after inclusion of extreme physical outliers to the training sets. NIR transmission was also shown to traverse a wide section of the tablet by comparing relative blocking intensities from different regions of the tablet surface and >90% of the signal was detected through a central area of 7 mm diameters of the tablet surface. NIR Quantification of both film thickness and active ingredient for film-coated tablets are examined in part II of this study.