folic acid has emerged as a promising ligand for the selective delivery of imaging and therapeutic agents to target cells , such as cancer cells and activated macrophages , in inflammatory sites ( 1 ) . as a selective targeting ligand ,
folic acid has several advantages such as 1 ) high affinity to its target , folate receptors ( frs ) , even after conjugation with diagnostic / therapeutic agents , 2 ) ease of conjugation with a variety of imaging and therapeutic agents , and 3 ) very low or undetectable expression of its receptors on normal cells , despite its high expression on cancer cells and activated macrophages ( 1 ) .
folic acid primarily enters non - pathogenic , normal cells through the reduced folate carrier to effect its functions ( 2 ) , but folic acid linked with conjugating agents only enters cells through the fr ( 23 ) .
fr is a cell surface glycosylphosphatidylinositol ( gpi)-anchored glycoprotein , and there are three isoforms in humans : fr- , - , and - ( 45 ) .
fr- is overexpressed on many types of cancer cells , including ovary , lung , breast , kidney , brain , endometrium , and colon cancer , whereas fr- is overexpressed on activated macrophages which are implicated in inflammatory pathologies , including rheumatoid arthritis , psoriasis , crohn 's disease , and systemic lupus erythematosus ( 6 ) .
for this reason , frs have been regarded as promising molecular targets for both diagnostics and therapeutic treatment of a variety of human diseases . in this review
, we will introduce the characteristics and structures of frs and discuss their expression profiles on normal , as well as pathogenic , cells .
furthermore , we will give an overview of recent studies describing strategies to target fr- selectively expressed on activated macrophages for the diagnosis and therapy of human inflammatory diseases .
folic acid , also known as folate and vitamin b9 , is essential for cells to generate dna , rna , and metabolic amino acids that are required for their proliferation and division ( 78 ) .
because eukaryotic cells are incapable of synthesizing folic acid , it is delivered into cells through either the reduced folate carrier , which is present in all cell types , or the fr , which is expressed in limited cells ( 9 ) . although folic acid is transported into the cells through either system , folate conjugates designed for diagnostics and therapeutics ( fig .
the fr is a cell surface glycoprotein of molecular weight in the range of 38~45 kda with a high affinity for folic acid ( kd<10~10 ) , and is attached to the plasma membrane by a glycosylphosphatidylinositol ( gpi ) anchor ( 45 ) .
human fr ( hfr ) is encoded by a family of hfr genes , and three isoforms , hfr- , - , and - , have been reported to date ( 4 ) .
the gene which is predicted to encode hfr- was also found from genome data mining ; however hfr--expressing cells and tissues have not yet been identified ( 10 ) . hfr- and - are membrane - associated proteins , whereas hfr- is a secreted protein because it does not have the signal peptide for the gpi anchor at its c - terminus ( 1112 ) .
hfrs share 68~79% amino acid sequence identity and have n - glycosylation sites that are critical for their proper folding ( 1213 ) . hfr- and - transport folic acid into cells via receptor - mediated endocytosis .
although all hfrs have been reported to have high binding affinity with folic acid , relative affinities of hfr- and - for folate conjugates are significantly different , in the range of 2~100 fold ( 14 ) .
studies of chimeric hfr constructs showed that amino acid sequences , such as leu49 in hfr- , and ala49 , val104 , and glu166 in hfr- , are critical for the differential ligand specificities of hfr- and - ( 1516 ) .
frs are expressed on the cells of different tissues depending on the types of fr isoforms .
fr- is expressed in the epithelial cells of normal tissues , such as type i and ii alveolar cells in the lungs , choroid plexus , ovary , fallopian tube , uterus , epididymis , submandibular salivary and bronchial glands , placental trophoblasts , and the basolateral membrane of retinal pigment epithelial cells ( 51718 ) .
because the fr- expressed in these normal tissues is distributed on the luminal surfaces of the tissues , it is protected from fr - targeted folate conjugates administered intravenously .
however , fr- is expressed in proximal kidney tubules , which are exposed to folate conjugates filtered from the blood stream .
interestingly , fr- is overexpressed on many malignant tumor cells of epithelial origin , including lung , ovarian , cervical , endometrial , brain , and breast cancers ( 45 ) , and significant correlation has been observed between the fr- expression level and the grade of the tumor ( 1920 ) .
although many previous studies have focused on the expression of fr- on the cells of neoplastic tissues , recent studies have shown that fr- is expressed on different types of cells .
fr- is a marker protein in normal hematopoiesis of myelomonocytic lineage cells ( 5 ) , and is expressed in neutrophils , cd34 hematopoietic progenitor cells , placenta , spleen , and thymus ( 212223 ) .
expression of fr- is also found in pathogenic cells , such as acute myelogenous leukemia ( aml ) cells and chronic myelogenous leukemia ( cml ) cells ( 2124 ) .
interestingly , fr- is highly expressed on activated , but not normal and resting macrophages , which are implicated in the pathogenesis of human inflammatory diseases , such as rheumatoid arthritis , psoriasis , crohn 's disease , systemic lupus erythematosus , atherosclerosis , diabetes , ulcerative colitis , osteoarthritis , glomerulonephritis , and sarcoidosis ( 6 ) .
the expression of fr- , indeed , was confirmed in synovial macrophages obtained from patients suffering from rheumatoid arthritis and in a mouse rheumatoid arthritis model ( 2526 ) .
fr- is expressed in normal and malignant hematopoietic cells in the spleen , bone marrow , and thymus as well as ovarian , cervical , and uterine cancer cells ( 1113 ) .
although fr- mrna was detected in lymphoid leukemia cells , a secreted form of fr- protein has not been detected in the serum of patients with lymphoid leukemia ( 4 ) .
because activated macrophages , which are regarded as key players in the pathogenesis of inflammatory diseases , express a high level of fr- on their surface , folate - conjugated imaging agents have been designed and synthesized to detect pathogenic activated macrophages in the lesions of inflammatory diseases by selectively targeting fr-. folic acid has been linked to a variety of dyes or radiopharmaceuticals , such as fitc , texas red , alexa fluor , oregon green , technetium ( tc ) , gallium ( ga ) , and indium ( in ) ( 272829 ) , and these folate imaging agents have been used for the detection of fr - positive cancer cells and pathogenic activated macrophages . because these folate - conjugated imaging agents successfully imaged cancer cells in vitro , in vivo , and
even clinically , they were further used for the imaging of activated macrophages in the lesions of inflammatory diseases for their diagnosis .
imaging studies of inflammatory diseases were initially conducted on rheumatoid arthritis to examine whether folate - conjugated imaging agents could selectively detect inflamed arthritic joints .
first , arthritic dogs were treated with a folate radiotracer , tc - ec20 ( fig .
1c ) , to image expected sites of inflammation , and dramatic uptake of tc - ec20 into arthritic joints was observed , whereas normal control dogs displayed background levels of radioactivity ( 30 ) .
subsequent imaging studies were conducted in adjuvant - induced arthritic rats , and tc - ec20 was used for imaging inflamed arthritic tissues .
the resulting images revealed accumulation of tc - ec20 in inflamed joints , livers , and spleens of arthritic rats , but not in those of healthy control rats ( 26 ) .
importantly , treatment of the arthritic rats with excess free folic acid completely blocked the uptake of tc - ec20 into their joints and organs , strongly indicating that uptake of tc - ec20 is dominantly fr - mediated ( 26 ) .
depletion of macrophages from arthritic rats with liposomal clodronate treatment also blocked the uptake of tc - ec20 , and the binding of folate - conjugated agents was observed only in macrophages in the total cell populations obtained from the livers of atherosclerotic mice ( 31 ) .
an autoradiography study revealed that tc - ec20 was much more accumulated in the atherosclerotic lesions of apoe-/- mice fed on a high fat diet than their counterparts fed on a normal diet ( 31 ) .
folate - conjugated fluorescent dyes and radioactive agents specifically targeted asthmatic lung macrophages in a murine asthma model , whereas little uptake by macrophages presented in healthy lung tissue ( 32 ) .
these observations reveal that only fr - expressing macrophages are selective targets for folate - conjugated imaging agents in animal models of human inflammatory diseases .
fr - targeted imaging using folate radiopharmaceuticals was also conducted in human patients suffering from both cancer and inflammatory disease .
a patent with ovarian cancer was administered folate - diethylenetriaminepentaacetic acid ( dtpa)-in to detect cancer tissues , and uptake was observed within the ovarian cancer tissues ( 33 ) .
uptake of folate - dtpa - in and tc - ec20 was also detected in inflamed knee joints of a patient suffering from osteoarthritis and rheumatoid arthritis , whereas no uptake was observed in healthy knees ( 13435 ) .
these fr - targeted in vivo imaging studies of macrophage - mediated inflammatory diseases in animal models and human patients are summarized in table i , and all data strongly support the idea that folate - conjugated imaging agents can selectively target fr - positive activated macrophages in inflamed tissues .
furthermore , this fr - targeted imaging of activated macrophages could also be applied to other macrophage - mediated inflammatory diseases .
based on successful fr - targeted imaging of cancers and activated macrophages in inflamed tissues using folate - conjugated imaging agents , folate - drug conjugates were designed and used to treat cancers and inflammatory diseases .
initially , conjugation of conventional chemotherapeutic agents to folic acid was regarded as a good strategy for fr - targeted therapy of human diseases .
however , many chemotherapeutic agents are hydrophobic , and hydrophobic chemotherapeutic conjugates exert considerably less target cell specificity than watersoluble ones , therefore , hydrophilic linkers are required to connect folic acid and its chemotherapeutic cargo ( 36 ) . in addition , it is critical to conjugate the therapeutic agents to folic acid without decreasing the affinity of folic acid for its folate receptor .
most importantly , designing linkers that release free drug inside the cells after fr - mediated endocytosis is a major challenge limiting the therapeutic efficacy of folate - drug conjugates ( 36 ) . with these guidelines
, it is clear that an alternative strategy must be developed to improve the potential of fr - targeted therapy of diseases .
consequently , a new strategy named  fr - targeted immunotherapy ,  which minimizes the limitations mentioned above , was developed to treat human diseases ( fig .
a highly immunogenic , low molecular weight hapten linked to folate beautifully decorates fr positive cancer cells or activated macrophages , leading to rapid elimination of these targeted cells by macrophages , nk cells , and complement components in the body 's immune system through antibody - dependent cellular cytotoxicity ( adcc ) and complement - dependent cellular cytotoxicity ( cdc ) ( fig .
2 ) . given the therapeutic efficacy of fr - targeted immunotherapy against cancers ( 37383940 ) , immunotherapy using folate - hapten conjugates was also applied for the targeting of activated macrophages to treat several animal models of inflammatory diseases .
adjuvant- or collagen - induced arthritic rodents , previously immunized with a hapten ( fitc ) , were treated with fr - targeted immunotherapy , resulting in the alleviation of arthritic symptoms by eliminating fr - positive activated macrophages in the inflamed lesions of the rodents ( 41 ) .
fr - targeted immunotherapy also showed therapeutic effects on systemic lupus erythematosus in a murine disease model by alleviating disease symptoms and extending the life span of treated animals ( 42 ) .
various folate - hapten conjugates were further used for fr - targeted immunotherapy of inflammatory diseases .
initial results with a folate - fitc conjugate showed a good therapeutic effect in animal models of rheumatoid arthritis and systemic lupus erythematosus ( 4142 ) .
folate conjugates of dinitrophenyl ( dnp ) and trinitrophenyl ( tnp ) were used for fr - targeted immunotherapy of rheumatoid arthritis in a rodent animal model , and these folate - hapten conjugates exerted therapeutic effects ( 43 ) .
fr - targeted recombinant immunotoxin was also used for fr - targeted immunotherapy of atherosclerosis .
administration of atherosclerotic apoe-/- mice with recombinant immunotoxin targeted fr - positive activated macrophages and alleviated the symptoms of atherosclerosis in the animal model ( 44 ) .
one concern that could be raised is whether fr - targeted immunotherapy eliminates the macrophage population in our body during treatment , resulting in significant loss of the body 's immunity against pathogen infection .
however , the conjugates used for the studies of fr - targeted immunotherapy mentioned above were well - tolerated by all animals , and they did not show any symptoms of pathogen infection or side effects during the immunotherapy .
this strongly suggests that the possibility of the destruction of the body 's immune system during fr - targeted immunotherapy is negligible , and fr - targeted immunotherapy effectively removes only fr - positive activated macrophages that play a critical role in the pathogenesis of inflammatory diseases .
given that therapeutic fr - targeted immunotherapy was successful for the treatment of macrophage - mediated inflammatory diseases such as rheumatoid arthritis , systemic lupus erythematosus , and atherosclerosis ( table i ) , this strategy is promising and could be further applied for the treatment of other types of macrophage - mediated inflammatory diseases .
inflammation is essential for host defense against pathogen invasion ; however , uncontrolled or repeated chronic inflammation leads to pathogenic conditions and diseases such as inflammatory diseases and cancers ( 4546474849 ) .
macrophages are critical immune cells to initiate inflammatory responses ( 50 ) , and increasing numbers of studies have successfully proven that activated macrophages , which express high levels of fr- , are found in the inflamed tissues of a variety of inflammatory diseases and are actively involved in the pathogenesis of these diseases .
this strongly suggests that selective targeting of frs using folate conjugated imaging or therapeutic agents could be a good strategy for the diagnosis , as well as therapeutic treatment , of macrophage - mediated inflammatory diseases . in spite of these successful studies proving the utility of frs as diagnostic and therapeutic targets for inflammatory diseases ,
moreover , few therapeutic drugs developed based on the strategy of selective fr targeting have been reported so far for these diseases , which raises the necessity of developing new potential drugs targeting frs with strong efficacies and minimal toxicities . in conclusion , selective targeting of frs , especially fr- on activated macrophages , could be a promising strategy for the diagnosis and treatment of macrophage - mediated inflammatory diseases , and there will be a high demand for the development of fr - targeted efficacious and safe therapeutics .
 inflammation , an innate immune response mediated by macrophages , forms the first line of defence to protect our body from the invasion of various pathogens . although inflammation is a defensive response , chronic inflammation has been regarded as the major cause of many types of human diseases such as inflammatory / autoimmune diseases , cancers , neurological diseases , and cardiovascular diseases . 
 folate receptor ( fr ) is a cell surface glycosylphosphatidylinositol ( gpi)-anchored glycoprotein , and its three isoforms , fr- , fr- , and fr- , are found in humans . 
 interestingly , frs are highly expressed on a variety of cells , including cancer cells and activated macrophages , whereas their expression on normal cells is undetectable , indicating that fr - targeting could be a good selective strategy for the diagnosis and therapeutic treatment of cancers and activated macrophage - mediated inflammatory diseases . 
 previous studies successfully showed fr - targeted imaging of many types of cancers in animal models as well as human patients . 
 recently , a number of emerging studies have found that activated macrophages , which are critical players for a variety of inflammatory diseases , highly express frs , and selective targeting of these fr - positive activated macrophages is a good approach to diagnose and treat inflammatory diseases . in this review , 
 we describe the characteristics and structure of frs , and further discuss fr - targeted diagnostics and therapeutics of human diseases , in particular , activated macrophage - mediated inflammatory diseases . 