Folate receptor-targeted near-infrared photodynamic therapy for folate receptor-overexpressing tumors

BACKGROUND Photodynamic therapy(PDT)is a minimally invasive form of cancer therapy,and the development of a novel photosensitizer(PS)with optimal properties is important for enhancing PDT efficacy.Folate receptor(FR)membrane protein is frequently overexpressed in 40%of human cancer and a good candidate for tumor-specific targeting.Specific active targeting of PS to FR can be achieved by conjugation with the folate moiety.A folate-linked,near-infrared(NIR)-sensitive probe,folate-Si-rhodamine-1(FolateSiR-1),was previously developed and is expected to be applicable to NIR-PDT.AIM To investigate the therapeutic efficacy of NIR-PDT induced by FolateSiR-1,a FRtargeted PS,in preclinical cancer models.METHODS FolateSiR-1 was developed by conjugating a folate moiety to the Si-rhodamine derivative through a negatively charged tripeptide linker.FR expression in the designated cell lines was examined by western blotting(WB).The selective binding of FolateSiR-1 to FR was confirmed in FR overexpressing KB cells(FR+)and tumors by fluorescence microscopy and in vivo fluorescence imaging.Low FR expressing OVCAR-3 and A4 cell lines were used as negative controls(FR-).The NIR light(635±3 nm)-induced phototoxic effect of FolateSiR-1 was evaluated by cell viability imaging assays.The time-dependent distribution of FolateSiR-1 and its specific accumulation in KB tumors was determined using in vivo longitudinal fluorescence imaging.The PDT effect of FolateSiR-1 was evaluated in KB tumor bearing mice divided into four experimental groups:(1)FolateSiR-1(100μmol/L)alone;(2)FolateSiR-1(100μmol/L)followed by NIR irradiation(50 J/cm2);(3)NIR irradiation(50 J/cm2)alone;and(4)no treatment.Tumor volume measurement and immunohistochemical(IHC)and histological examinations of the tumors were performed to analyze the effect of PDT.RESULTS High FR expression was observed in the KB cells by WB,but not in the OVCAR-3 and A4 cells.Substantial FR-specific binding of FolateSiR-1 was observed by in vitro and in vivo fluorescence imaging.Cell viability imaging assays showed that NIR-PDT induced cell death in KB cells.In vivo longitudinal fluorescence imaging showed rapid peak accumulation of FolateSiR-1 in the KB tumors 2 h after injection.In vivo PDT conducted at this time point caused tumor growth delay.The relative tumor volumes in the PDT group were significantly reduced compared to those in the other groups[5.81±1.74(NIR-PDT)vs 12.24±2.48(Folate-SiR-1),vs 11.84±3.67(IR),vs 12.98±2.78(Untreated),at Day 16,P<0.05].IHC analysis revealed reduced proliferation marker Ki-67-positive cells in the PDT treated tumors,and hematoxylin-eosin staining revealed features of necrotic-and apoptotic cell death.CONCLUSION FolateSiR-1 has potential for use in PDT,and FR-targeted NIR-PDT may open a new effective strategy for the treatment of FR-overexpressing tumors.

Progress in research on the relationship between autism and folate receptor autoantibody expression

Autism spectrum disorder(ASD)is a serious neurodevelopmental disorder,the etiology and mechanism of which are not yet clear.Studies have shown that folate deficiency can lead to abnormalities in the de-velopment of the central nervous system.Patients with autism spectrum disorders develop folate-alpha recep-tor autoantibodies.Folate-alpha receptor autoantibodies block folate transport,leading to a deficiency of folate in nerve tissues.Folate is effective in treating patients with folate-alpha receptor autoantibodies.

Folate receptor-mediated boron-10 containing carbon nanoparticles as potential delivery vehicles for boron neutron capture therapy of nonfunctional pituitary adenomas

Invasive nonfunctional pituitary adenomas （NFPAs） are difficult to completely resect and often develop tumor recurrence after initial surgery. Currently, no medications are clinically effective in the control of NFPA. Although radiation therapy and radiosurgery are useful to prevent tumor regrowth, they are frequently withheld because of severe complications. Boron neutron capture therapy （BNCT） is a binary radiotherapy that selectively and maximally damages tumor cells without harming the surrounding normal tissue. Folate receptor （FR）-targeted boron-10 containing carbon nanoparticles is a novel boron delivery agent that can be selectively taken up by FR-expressing cells via FR-mediated endocytosis. In this study, FR-targeted boron-10 containing carbon nanoparticles were selectively taken up by NFPAs cells expressing FR but not other types of non-FR expressing pituitary adenomas. After incubation with boron-10 containing carbon nanoparticles and following irradiation with thermal neutrons, the cell viability of NFPAs was significantly decreased, while apoptotic cells were simultaneously increased. However, cells administered the same dose of FR-targeted boron-10 containing carbon nanoparticles without neutron irradiation or received the same neutron irradiation alone did not show significant decrease in cell viability or increase in apoptotic cells. The expression of Bcl-2 was down-regulated and the expression of Bax was up-regulated in NFPAs after treatment with FR-mediated BNCT. In conclusion, FR-targeted boron-10 containing carbon nanoparticles may be an ideal delivery system of boron to NFPAs ceils for BNCT. Furthermore, our study also provides a novel insight into therapeutic strategies for invasive NFPA refractory to conventional therapy, while exploring these new applications of BNCT for tumors, especially benign tumors.

Molecular imaging of advanced atherosclerotic plaques with folate receptor-targeted 2D nanoprobes

Vulnerable atherosclerotic plaques are responsible for most cardiovascular diseases(CVDs).Folate receptor(FR)positive activated macrophages were thought to be a prominent component in the development of vulnerable plaque.The objective of this study is to develop folate conjugated two-dimensional(2D)Pd@Au nanomaterials(Pd@Au-PEG-FA)for targeted multimodal imaging of the FRs in advanced atherosclerotic plaques.Pharmacokinetic and imaging studies(single photon emission computed tomography(SPECT),computed tomography(CT)and photoacoustic(PA)imaging)were performed to confirm the prolonged blood half-life and enrichment of radioactivity in atherosclerotic plaques.Strong signals were detected in vivo with SPECT,CT and PA imaging in heavy atherosclerotic plaques,which were significantly higher than those of the normal aortas after injection of Pd@Au-PEG-FA.Blocking studies with preinjection of excess FA could effectively reduce the targeting ability of Pd@Au-PEG-FA in atherosclerotic plaques,further demonstrating the specific binding of Pd@Au-PEG-FA for plaque lesions.Histopathological characterization revealed that the signal of probe was in accordance with the high-risk plaques.In summary,the Pd@Au-PEG-FA has favorable pharmacokinetic properties and provides a valuable approach for detecting high-risk plaques in the presence of FRs in atherosclerotic plaques.

Unmodified and positively charged gold nanoparticles for sensitive colorimetric detection of folate receptor via terminal protection of small molecule-linked ss DNA

The detection of protein/small molecule interactions plays important roles in drug discovery and protein/metabolite interactions in biology. In this work, by coupling the terminal protection of small molecule-linked ss DNA strategy with the unmodified and positively charged gold nanoparticle((+)Au NP) nanoprobes, we have developed a sensitive and simple colorimetric sensor for the detection of folate receptor, a highly expressed protein in many kinds of malignant tumors. The target folate receptor binds the folate moieties of the folate-linked ss DNA through high affinity interactions and protects the protein-bound ss DNA from digestion by exonuclease I. The protected ss DNA thus adsorbs the((+)Au NP) through electrostatic interactions, leading to a red-to-blue color change of the sensing solution for sensitive colorimetric detection of folate receptor at the sub-nanomolar level. Besides, this colorimetric sensor shows high selectivity toward folate receptor against other control proteins. The developed sensor avoids the modification/conjugation of the Au NP nanoprobes and the involvement of any expensive instruments for signal transduction in protein detection. Featured with these obvious advantages, the colorimetric sensor strategy demonstrated herein can be easily expanded for sensitive and convenient detection of various protein/small molecule interactions.

Folate-chitosan-gemcitabine core-shell nanoparticles targeted to pancreatic cancer

Objective：Human pancreatic cancer is one of the most common clinical malignancies.The effect of comprehensive treatment based on surgery is general.The effects of chemotherapy were not obvious mainly because of lack of targeting and chemoresistance in pancreatic cancer.This study aimed to investigate the effects of folate receptor （FR）-mediated gemcitabine FA-Chi-Gem nanoparticles with a core-shell structure by electrostatic spray on pancreatic cancer.Methods：In this study,the levels of expression of FR in six human pancreatic cancer cell lines were studied by immunohistochemical analysis.The uptake rate of isothiocyanate-labeled FA-Chi nanoparticles in FR high expression cell line COLO357 was assessed by fluorescence microscope and the inhibition rate of FA-Chi-Gem nanoparticles on COLO357 cells was evaluated by MTT assay.Moreover,the biodistribution of PEG-FA-ICGDER02-Chi in the orthotopic pancreatic tumor model was observed using near-infrared imaging and the human pancreatic cancer orthotopic xenografts were treated with different nanoparticles and normal saline control.Results：The expression of FR in COLO357 was the highest among the six pancreatic cancer cell lines.The FR mainly distributed on cell membrane and fewer in the cytoplasm in pancreatic cancer.Moreover,the absorption rate of the FA-Chi-Gem nanoparticles was more than the Chi nanoparticles without FA modified.The proliferation of COLO357 was significantly inhibited by FA-Chi-Gem nanoparticles.The PEG-FA-ICGDER02-Chi nanoparticles were enriched in tumor tissue in human pancreatic cancer xenografts,while non-targeted nanoparticles were mainly in normal liver tissue.PEG-FA-Gem-Chi significantly inhibited the growth of human pancreatic cancer xenografts （PEG-FA-Gem-Chi vs.Gem,t=22.950,P=0.000）.Conclusions：PEG-FA-FITC-Chi nanoparticles might be an effective targeted drug for treating human FR-positive pancreatic cancer.

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

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

Development of a novel multi-functional integrated bioconjugate effectively targeting K-Ras mutant pancreatic cancer

Folate receptor(FR)overexpression occurs in a variety of cancers,including pancreatic cancer.In addition,enhanced macropinocytosis exists in K-Ras mutant pancreatic cancer.Furthermore,the occurrence of intensive desmoplasia causes a hypoxic microenvironment in pancreatic cancer.In this study,a novel FR-directed,macropinocytosis-enhanced,and highly cytotoxic bioconjugate folate(F)-human serum albumin(HSA)-apoprotein of lidamycin(LDP)-active enediyne(AE)derived from lidamycin was designed and prepared.F-HSA-LDP-AE consisted of four moieties:F,HSA,LDP,and AE.F-HSA-LDP presented high binding efficiency with the FR and pancreatic cancer cells.Its uptake in wild-type cells was more extensive than in K-Ras mutant-type cells.By in vivo optical imaging,F-HSA-LDP displayed prominent tumor-specific biodistribution in pancreatic cancer xenograft-bearing mice,showing clear and lasting tumor localization for 360 h.In the MTT assay,F-HSA-LDP-AE demonstrated potent cytotoxicity in three types of pancreatic cancer cell lines.It also induced apoptosis and caused G2/M cell cycle arrest.F-HSALDP-AE markedly suppressed the tumor growth of AsPc-1 pancreatic cancer xenografts in athymic mice.At well-tolerated doses of 0.5 and 1 mg/kg,(i.v.,twice),the inhibition rates were 91.2%and 94.8%,respectively(P<0.01).The results of this study indicate that the F-HSA-LDP multi-functional bioconjugate might be effective for treating K-Ras mutant pancreatic cancer.

Characterization of folic acid,5-methyltetrahydrofolate and synthetic folinic acid in the high-affinity folate transporters:impact on pregnancy and development

Objectives:Folates are B vitamins that are essential for several molecular,cellular,and biological processes,including nucleotide synthesis,methylation,and methionine cycling.The physiological impacts of these processes on health also extend to cell proliferation,folate deficiency anemia,and reduction of the risk of birth defects during pregnancy.The primary objective of this study was to characterize the binding affinities of different folate forms,folic acid(FA),5-methyltetrahydrofolate(5MTHF),and folinic acid,to the folate receptorsαandβ,and to the bovine milk folate binding protein.These three dietary forms of folate are found in enriched grains(FA),various fruits and leafy vegetables(folinic acid),and red blood cells(5MTHF).Methods:The half maximal inhibitory concentration values and binding curves of each of these folates for each receptor were determined.Results:Our results indicated that FA had the highest affinity for all folate receptors,followed by 5MTHF,and lastly,by folinic acid,examined by several orders of magnitudes.Conclusion:These data are expected to provide new insights into the therapeutic applications of the different forms of folate in a variety of diseases.

FOLR1-induced folate deficiency reduces viral replication via modulating APOBEC3 family expression

Folate receptor alpha(FOLR1)is vital for cells ingesting folate(FA).FA plays an indispensable role in cell pro-liferation and survival.However,it is not clear whether the axis of FOLR1/FA has a similar function in viral replication.In this study,we used vesicular stomatitis virus(VSV)to investigate the relationship between FOLR1-mediated FA deficiency and viral replication,as well as the underlying mechanisms.We discovered that FOLR1 upregulation led to the deficiency of FA in HeLa cells and mice.Meanwhile,VSV replication was notably sup-pressed by FOLR1 overexpression,and this antiviral activity was related to FA deficiency.Mechanistically,FA deficiency mainly upregulated apolipoprotein B mRNA editing enzyme catalytic subunit 3B(APOBEC3B)expression,which suppressed VSV replication in vitro and in vivo.In addition,methotrexate(MTX),an FA metabolism inhibitor,effectively inhibited VSV replication by enhancing the expression of APOBEC3B in vitro and in vivo.Overall,our present study provided a new perspective for the role of FA metabolism in viral infections and highlights the potential of MTX as a broad-spectrum antiviral agent against RNA viruses.

Designed fabrication of active tumor targeting covalent organic framework nanotherapeutics via a simple post-synthetic strategy

Developing agents that can accurately differentiate tumors from normal healthy tissues is of utmost importance for safe cancer therapy.Active targeting has been considered as an effective technique for tumor recognition.In this work,we demonstrate a folate-functionalized nanoscale covalent organic framework(FATD nCOF)highly specific to cancer cells through active targeting of their enriched folate receptors(FRs).The FATD nCOF prepared by simple post-synthetic modification of the COF surface defeats disperses well in water and exhibits a high loading capacity for various anticancer drugs.The biocompatible FATD nCOF is selectively internalized by FR-harboring cancer cells and consequently augments the efficacy of the loaded drug,Withaferin A(Wi-A),for targeted cancer cell killing.In biomolecular mechanism studies,Wi-A-loaded FATD(FATD@Wi-A)nanocomposites show remarkably a higher rate of apoptosis in FR-enriched cancer cells.Comparative analyses of FR-positive and FR-negative tumor xenografts reveal enhanced selective antitumor activity of FATD@Wi-A nanotherapeutics.Taken together,the study findings suggest that FATD nCOF holds great promise for active targeting of tumors in vivo.Our simple yet effective technology might be valuable for creating new state-of-the-art COFs for chemical and biomedical applications.