Detection of folate receptor-positive circulating tumor cells as a biomarker for diagnosis, prognostication, and therapeutic monitoring in breast cancer.

OBJECTIVES: This study is to explore the clinical significance of folate receptor-positive circulating tumor cells (FR+ CTC) in the early diagnosis and disease progress in patients with breast cancer. METHODS: Folate receptor-positive circulating tumor cells was enriched from peripheral blood of the patients with immunomagnetic separation method and quantitated by folate receptor on the CTC with the ligand-targeted PCR. RESULTS: The levels of FR+ CTC were significantly higher in breast cancer patients compared with healthy controls. Detective rate of FR+ CTC was decreased in 19 of 27 patients underwent the surgery in 2 weeks post-operation compared with pre-operation; statistical analysis showed the difference was significant. We also found that the combination of FR+ CTC, CEA, CA125, and CA153 can significantly improve the diagnostic efficiency for breast cancer. CONCLUSIONS: This study showed the detective rate of FR+ CTC is significantly increased in the patients with breast cancer, and the detective level is associated with disease progress.

One-pot synthesis of folic acid modified carbonized polymer dots with red emittision for selective imaging of cancer cells.

Carbonized polymer dots (CPDs), as a novel fluorescent material, have broad application prospects in the fields of bio-imaging, bio-sensors, disease diagnosis and photovoltaic devices due to their low cost, low toxicity, easy modification and little environmental impact. In this paper, folic acid (FA) modified CPDs (FA-CPDs) are synthesized from p-Phenylenediamine (p-PD) and FA molecules using a traditional one pot hydrothermal reaction in order to detect cancer cells containing a folate receptor (FR). The synthesized FA-CPDs were characterized by transmission electron microscopy, Fourier transfrom infrared spectroscopy, x-ray photoelectron spectroscopy, x-ray diffraction, UV-vis and fluorescence techniques. The red fluorescence emission is realized by doping phosphorus atoms into the carbonized polymer. Upon excitation at 513 nm, the maximum emission wavelength of FA-CPDs aqueous solution was obtained at 613 nm. Moreover, the as-prepared FA-CPDs exhibit excellent excitation-independent behavior and good stability with high quantum yield (QY) at about 30.6%. The binding of FA-CPDs with FRs on cancer cells produces target recognition and enters the cells through endocytosis. Additionally, it is worth noting that FA-CPDs have good biocompatibility and imaging in HeLa cells has been successfully achieved. Therefore, our FA-CPDs have potential applications as biocompatibility probes for cancer diagnosis and treatment.

Ligand-targeted polymerase chain reaction for the detection of folate receptor-positive circulating tumour cells as a potential diagnostic biomarker for pancreatic cancer.

OBJECTIVES: To detect folate receptor (FR)-positive circulating tumour cells (FR+ CTCs) by using ligand-targeted polymerase chain reaction (LT-PCR) in periampullary cancer patients and to investigate the diagnostic value of FR+ CTCs in distinguishing pancreatic cancer (PC) from benign pancreatic disease. MATERIALS AND METHODS: CTCs were enriched from 3 mL of peripheral blood and portal vein blood by immunomagnetic depletion of leucocytes and were then detected by LT-PCR. The diagnostic performance of FR+ CTCs in PC was investigated by receiver-operating characteristic curve analysis. RESULTS: In total, 57 consecutive patients, including 46 patients with PC, five patients with non-pancreatic periampullary cancer (non-PC) and six patients with benign pancreatic diseases, were enrolled. FR+ CTC levels were significantly higher in patients with malignant diseases (PC and non-PC) than in patients with benign pancreatic diseases (P < .01). There was no notable difference in CTC levels between patients with PC and those with non-PC (P > .05). The combination of FR+ CTCs with carbohydrate antigen 19-9 (CA19-9) had better diagnostic efficiency than each of these two markers alone, with high sensitivity (97.8%) and specificity (83.3%). CONCLUSIONS: LT-PCR is feasible and reliable for detecting FR+ CTCs in patients with periampullary cancer. FR+ CTCs, especially when used in combination with CA19-9, have potential as a biomarker for the diagnosis of PC.

Synthesis of folic acid functionalized gold nanoclusters for targeting folate receptor-positive cells.

We report on the synthesis of water-soluble gold nanoclusters capped with polyethylene glycol (PEG)-based ligands and further functionalized with folic acid for specific cellular uptake. The dihydrolipoic acid-PEG-based ligands terminated with -OMe, -NH2 and -COOH functional groups are produced and used for surface passivation of Au nanoclusters (NCs) with diameters <2 nm. The produced sub 2 nm Au NCs possess long-shelf life and are stable in physiologically relevant environments (temperature and pH), are paramagnetic and biocompatible. The paramagnetism of Au NCs in solution is also reported. The functional groups on the capping ligands are used for direct conjugation of targeting molecules onto Au NCs without the need for post synthesis modification. Folic acid (FA) is attached via an amide group and effectively target cells expressing the folate receptor. The combination of targeting ability, biocompatibility and paramagnetism in FA-functionalized Au NCs is of relevance for their exploitation in nanomedicine for targeted imaging.

Real-time quartz crystal microbalance cytosensor based on a signal recovery strategy for in-situ and continuous monitoring of multiple cell membrane glycoproteins.

A real-time quartz crystal microbalance (QCM) cytosensor based on a signal recovery strategy was first developed for in-situ and continuous monitoring of multiple cell membrane glycoproteins. In this work, gold nanoparticles (AuNPs) were linked with ligands to fabricate ligand-functionalized mass nanoprobes with signal amplification for increasing monitoring sensitivity. The mass nanoprobes bound to cell surface could be eluted with glycine-hydrochloric acid buffer, which led to a quick recovery of resonance frequency. Using the strategy, folate receptors (FR), CD44 molecule and epidermal growth factor receptor (EGFR) on cell membrane as the models were monitored continuously. The quantification result of MDA-MB-231 cells showed a range of linearity of 3.0 × 104 to 1.0 × 106 cells and a detection limit of 5.0 × 103 cells. Furthermore, the multianalyte cytosensor exhibited three sensitive and recoverable frequency shifts during continuous monitoring for in-situ and continuous evaluation of the expression levels of FR, CD44 and EGFR on cell membrane, which exhibited that the average numbers of molecules of FR, CD44 and EGFR per MDA-MB-231 cell were 0.5 × 106, 0.2 × 106 and 1.4 × 105 with the relative standard deviation of 4.8%, 4.5% and 5.1%, respectively. Compared with monolithic multichannel QCM, the multianalyte cytosensor based on a single microbalance could not only exclude acoustic interference but also reduce instrumental cost. This work provided a simple and efficient QCM cytosensor for in-situ and continuous monitoring of multiple cell membrane glycoproteins that offered a new avenue for early diagnosis of cancer.

Folic acid-conjugated green luminescent carbon dots as a nanoprobe for identifying folate receptor-positive cancer cells.

Early diagnosis is pivotal in subsequent prognosis and treatment of cancer. Herein, folic acid-conjugated carbon dots (FA-CDs) as a fluorescent nanoprobe were fabricated for identifying cancer cells visually. Green luminescent carbon dots (CDs) from active dry yeast (ADY) were readily prepared in scale-up to reach macroscopic production with a high yield of ~50% via a facile and rapid microwave approach. The as-prepared CDs were further combined with folic acid (FA) by covalent bonding to fabricate the FA-CDs for identification of cancer cells over-expressing folate receptor (FR). Experimental outcomes demonstrated that the resultant FA-CDs noninvasively entered into cancer cells via receptor-mediated endocytosis and could differentiate FR-positive HepG2 cells from a cell mixture by fluorescence imaging, which suggests a promising prospect of the FA-CDs as an efficient probe for cancer diagnosis and succeeding personalized therapy.

Demonstration of the lack of cytotoxicity of unmodified and folic acid modified graphene oxide quantum dots, and their application to fluorescence lifetime imaging of HaCaT cells.

The authors describe the synthesis of water-soluble and fluorescent graphene oxide quantum dots via acid exfoliation of graphite nanoparticles. The resultant graphene oxide quantum dots (GoQDs) were then modified with folic acid. Folic acid receptors are overexpressed in cancer cells and hence can bind to functionalized graphene oxide quantum dots. On excitation at 305 nm, the GoQDs display green fluorescence with a peak wavelength at ~520 nm. The modified GoQDs are non-toxic to macrophage cells even after prolonged exposure and high concentrations. Fluorescence lifetime imaging and multiphoton microscopy was used (in combination) to image HeCaT cells exposed to GoQDs, resulting in a superior method for bioimaging. Graphical abstract Schematic representation of graphene oxide quantum dots, folic acid modified graphene oxide quantum dots (red), and the use of fluorescence lifetime to discriminate against green auto-fluorescence of HeCaT cells.

Folate receptor-targeted ultrasonic PFOB nanoparticles: Synthesis, characterization and application in tumor-targeted imaging.

In this study, we aimed to determine an effective strategy for the synthesis of folate receptor (FR) targeted-nanoparticles (FRNPs). The nanoparticles used as ultrasound contrast agents (UCAs) were composed of a liquid core of perfluorooctyl bromide (PFOB) liposome and a targeted shell chemically conjugated with folic acid (FA) and polyethylene glycol (PEG). This was done in order to avoid recognition and clearance by the mononuclear phagocyte system [also known as the reticuloendothelial system (RES)] and enhance the targeting capability of the nanoparticles to tumors overexpressing folate receptor (FR). The FRNPs exhibited an average particle size of 301±10.8 nm and surface potential of 39.1±0.43 mV. Subsequently, in vitro, FRNPs labeled with FITC fluorescence dye were visibly uptaken into the cytoplasm of FR-overexpressing cancer cells (Bel7402 and SW620 cells), whereas the A549 cells expressing relatively low levels of FR just bound with few FRNPs. These results demonstrated that FRNPs have a high affinity to FR-overexpressing cancer cells. Additionally, in in vivo experiments, FRNPs achieved a greater enhancement of tumor ultrasound imaging and a longer enhancement time in FR-overexpressing tumors and the Cy7-labeled FRNPs exhibited a relatively high tumor-targeted distribution in FR­overexpressing tumors. Targeted ultrasound and fluorescence imaging revealed that FRNPs have the ability to target FR-overexpressing tumors and ex vivo fluorescence imaging was then used to further verify and confirm the presence of FRNPs in tumor tissues with histological analysis of the tumor slices. On the whole, our data demonstrate that the FRNPs may prove to be a promising candidate for the early diagnosis for FR-overexpressing tumors at the molecular and cellular levels.

One-step synthesis of photoluminescent carbon dots with excitation-independent emission for selective bioimaging and gene delivery.

Photoluminescent carbon dots (C-dots), as new members of the quantum sized carbon analogues have attracted significant attention due to their unique size, less toxicity, good compatibility and relatively easy surface modification. In this work, we report a simple, low-cost and one-step hydrothermal carbonization approach to synthesize the positively charged C-dots using PEI and FA. From the photoluminescence (PL) measurements, the as-prepared C-dots exhibit good stability and intense PL with the high quantum yield (QY) at Ca. 42%. Significantly, The as-prepared C-dots integrate the advantages of C-dots and PEI: the presence of C-dots can effectively decrease the cytotoxicity of PEI, the C-dots can be applied in biological system for selective imaging of folate receptor (FR)-positive cancerous cells from normal cells, while the cationic PEI with positive charges can make them link to plasmid DNA and efficiently transfect the therapeutic plasmid into cells. Therefore, the as-prepared with the facile synthesis method can be a promising photoluminescent probe for cancer diagnosis and gene therapy.

Label-free femtomolar cancer biomarker detection in human serum using graphene-coated surface plasmon resonance chips.

Sensitive and selective detection of cancer biomarkers is vital for the successful diagnosis of early stage cancer and follow-up treatment. Surface Plasmon Resonance (SPR) in combination with different amplification strategies is one of the analytical approaches allowing the screening of protein biomarkers in serum. Here we describe the development of a point-of-care sensor for the detection of folic acid protein (FAP) using graphene-based SPR chips. The exceptional properties of CVD graphene were exploited to construct a highly sensitive and selective SPR chip for folate biomarker sensing in serum. The specific recognition of FAP is based on the interaction between folic acid receptors integrated through π-stacking on the graphene coated SPR chip and the FAP analyte in serum. A simple post-adsorption of human serum:bovine serum albumin (HS:BSA) mixtures onto the folic acid modified sensor resulted in a highly anti-fouling interface, while keeping the sensing capabilities for folate biomarkers. This sensor allowed femtomolar (fM) detection of FAP, a detection limit well adapted and promising for quantitative clinical analysis.

Immobilization free electrochemical biosensor for folate receptor in cancer cells based on terminal protection.

The determination of folate receptor (FR) that over expressed in vast quantity of cancerous cells frequently is significant for the clinical diagnosis and treatment of cancers. Many DNA-based electrochemical biosensors have been developed for FR detection with high selectivity and sensitivity, but most of them need complicated immobilization of DNA on the electrode surface firstly, which is tedious and therefore results in the poor reproducibility. In this study, a simple, sensitive, and selective electrochemical FR biosensor in cancer cells has been proposed, which combines the advantages of the convenient immobilization-free homogeneous indium tin oxide (ITO)-based electrochemical detection strategy and the high selectivity of the terminal protection of small molecule linked DNA. The small molecule of folic acid (FA) and an electroactive molecule of ferrocence (Fc) were tethered to 3'- and 5'-end of an arbitrary single-stranded DNA (ssDNA), respectively, forming the FA-ssDNA-Fc complex. In the absence of the target FR, the FA-ssDNA-Fc was degraded by exonuclease I (Exo I) from 3'-end and produced a free Fc, diffusing freely to the ITO electrode surface and resulting in strong electrochemical signal. When the target FR was present, the FA-ssDNA-Fc was bound to FR through specific interaction with FA anchored at the 3'-end, effectively protecting the ssDNA strand from hydrolysis by Exo I. The FR-FA-ssDNA-Fc could not diffuse easily to the negatively charged ITO electrode surface due to the electrostatic repulsion between the DNA strand and the negatively charged ITO electrode, so electrochemical signal reduced. The decreased electrochemical signal has a linear relationship with the logarithm of FR concentration in range of 10fM to 10nM with a detection limit of 3.8fM (S/N=3). The proposed biosensor has been applied to detect FR in HeLa cancer cells, and the decreased electrochemical signal has a linear relationship with the logarithm of cell concentration ranging from 100-10000cell/mL. Compared with the traditional heterogeneous electrochemical FR biosensors, the proposed biosensor owns the merits of the simplicity and high specificity, presenting the great potential application in the area of early diagnosis of cancers.

In vivo monitoring of activated macrophages and neutrophils in response to ischemic osteonecrosis in a mouse model.

Ischemic osteonecrosis (IO) is caused by disruption of the blood supply to bone. It is a debilitating condition with pathological healing characterized by excessive bone resorption and delayed osteogenesis. Although the majority of research has focused on the role of osteoblasts and osteoclasts in the disease progression, we hypothesize that innate immune cells, macrophages and neutrophils, play a significant role. With the recent development of real-time imaging probes for neutrophils and macrophages, the purpose of this study was to investigate the kinetic immune cell response in a mouse model of IO. Our results show that induction of IO leads to a significant accumulation of activated neutrophils and macrophages at the affected tissue by 48 h after surgery. Additionally, the accumulation of these immune cells remained elevated in comparison to sham controls for up to 6 weeks, indicative of chronic inflammation. Immunohistochemistry confirmed the immune cell infiltration into the necrotic bone marrow and the increased presence of TNFα-positive cells, demonstrating, for the first time, a direct response of these cells to ischemia induced necrotic bone. These new findings support a hypothesis that IO is an osteoimmunologic condition where innate immune cells play a significant role in the chronic inflammation.

Phase I clinical trial of 99mTc-etarfolatide, an imaging agent for folate receptor in healthy Japanese adults.

OBJECTIVE: Technetium etarfolatide ((99m)Tc-EF) is a radioactive diagnostic imaging agent that was developed to assess the expression of folate receptors in tumors. Administering folic acid prior to the administration of (99m)Tc-EF has been shown to improve SPECT images. Here, we conducted a phase I clinical trial to assess the safety, pharmacokinetics, and radiation dosimetry of (99m)Tc-EF injection following pre-administration of folic acid in healthy Japanese male adults. METHODS: Six healthy Japanese male adults were enrolled in the study. Folic acid was intravenously administered, followed 1-3 min later by an intravenous injection of (99m)Tc-EF (740 MBq ± 20 %). Assessments of subjective symptoms and objective findings, electrocardiograms, physical examination, and laboratory tests were performed before and up to 7 days after the injection to assess the safety of (99m)Tc-EF. Blood and urine collections and whole-body planar imaging were conducted at various time points up to 24 h after the injection to assess the pharmacokinetics of (99m)Tc-EF. The internal radiation dosimetry was calculated based on the pharmacokinetics results using the MIRD method. RESULTS: Five adverse events were observed in three subjects (50 %) after administration of the folic acid and (99m)Tc-EF, while these events were mild and non-serious. Of those five events, three were considered to be related to the administered agents. The radioactivity in blood rapidly decreased and showed a biphasic profile. The activity of (99m)Tc-EF at 5 min post injection was largest in the bone marrow, followed by the liver and kidneys, and had decreased within 24 h in all organs/tissues without appreciable retention. The pharmacokinetics results suggested that (99m)Tc-EF was mainly eliminated by kidney. The results also suggested that when administered at 925 MBq of (99m)Tc-EF, which is the maximum dose generally used for clinical trials in other countries, the corresponding effective dose of (99m)Tc-EF is equal to or less than those determined for the current radioactive diagnostic imaging agents. CONCLUSIONS: The results of this study assessing the safety and radiation dosimetry of (99m)Tc-EF with folic acid pre-administration suggested that folic acid and (99m)Tc-EF should be appropriate for further studies. No pharmacokinetics concerns were noted.

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.

Expression of high affinity folate receptor in breast cancer brain metastasis.

High affinity folate receptor (HFR) can be overexpressed in breast cancer and is associated with poor prognosis, however the expression in breast cancer brain metastases (BCBM) is unknown. The aim of this study was to analyze the rate of HFR expression in BCBM and its role in the prognosis of this high-risk cohort. We analyzed 19 brain metastasis (BM) and 13 primary tumors (PT) from a total of 25 patients. HFR status was assessed by immunohistochemistry. Median follow-up was 4.2 years (range 0.6-18.5). HFR was positive in 4/19 BM (21.1%) and in 1/13 PT (7.7%). Positive samples had low H-scores (range 1-50). 56% of patients had apocrine differentiation. OS was similar between patients with positive HFR (median OS 48 months) and negative HFR (median OS 69 months) (P = 0.25); and between patients with apocrine differentiation (median OS 63 months) and those without apocrine differentiation (median OS 69 months) (P = 0.49). To the best of our knowledge, this is the first analysis of HFR expression in BCBM. While previous studies associated the presence of HFR with worse prognosis; in our cohort HFR was positive in only 21.1% of BM with low levels of positivity. Neither HFR nor apocrine features had impact in OS.

A "turn-on" silver nanocluster based fluorescent sensor for folate receptor detection and cancer cell imaging under visual analysis.

A novel terminal protection based label-free and "turn-on" fluorescent sensor for detection of folate receptors (FRs) and HeLa cells is developed by fluorescence resonance energy transfer (FRET) between single-walled carbon nanotubes (SWCNTs) and silver nanoclusters (AgNCs). Multilevel visual analysis (m(2)VA) was firstly proposed and applied in optimizing the experimental parameters.

A ratiometric colorimetric detection of the folate receptor based on terminal protection of small-molecule-linked DNA.

Development of strategies for the sensitive and selective detection of the folate receptor (FR) that are simple and low cost is of great importance for assessing cancer therapeutics due to its crucial role in physiological, pharmacological and pathological processes. In this paper, gold nanoparticle (AuNP)-based novel ratiometric colorimetry for the detection of the folate receptor (FR) is proposed based on terminal protection of small-molecule-linked DNA. The single-stranded DNA (ssDNA) terminally tethered to folic acid (FA) is protected from degradation by exonuclease I (Exo I) when the FA moiety is bound to FR. The hybridization between FR-protected DNA and DNA-functionalized Au NPs generated a red-to-purple colour change, allowing the visual detection of FR. The detection limit of FR can be as low as 0.33 ng mL(-1) with the naked eye. It provides a promising strategy for visual detection of the binding event of FA to its protein receptor-FR with advantages such as simplicity, high selectivity, and a wide linear range.

Gold nanocluster-based fluorescence biosensor for targeted imaging in cancer cells and ratiometric determination of intracellular pH.

The dysregulated pH is working as a mark of cancer. It is a challenge for developing a biosensor for targeted imaging in cancer cells and monitoring of intracellular pH. Here, a ratiometric fluorescence biosensor for pH determination was developed with targeted imaging into folate acceptor (FR)-rich cancer cells at the same time. AuNCs protected by bovine serum albumin (BSA) worked as reference fluorophore and fluorescein-isothiocyanate (FITC) acted as the response signal for pH. For targeted imaging of cancer cells, the AuNCs were simultaneously conjugated with folic acid (FA). The developed ratiometric biosensor can monitor pH with a wide linear range from 6.0-7.8 with a pKa at 6.84. Under every different pH condition, the probe showed high selectivity over various metal ions and amino acids with its fluorescence ratio stayed almost constant (<5%). It also showed good cyclic accuracy when pH switched between 6.0 and 8.0, as well as low cytotoxicity. The AuNC-based inorganic-organic nanohybrid biosensor showed good cell-permeability, low cytotoxicity, and long-term photostability. Accordingly, the pH biosensor was employed to gain targeted imaging in FR(+ve) Hela cells with FR(-ve) lung carcinoma cells A549 as comparison, and achieved to monitor the pH changes in Hela cells.

Distinguish cancer cells based on targeting turn-on fluorescence imaging by folate functionalized green emitting carbon dots.

Developing efficient methods for visual detection of cancer cells has the potential to contribute greatly to basic biological research and early diagnosis of cancer. Here, we report facile and one-step synthesis of green fluorescence carbon dots (CDs) with the help of a new passivating agent--poly(acrylate sodium) (PAAS). Based on the as-prepared CDs, a novel turn-on fluorescence probe was designed for targeting imaging of cancer cells via hydrogen-bond interaction between folic acid and CDs (FA-CDs). Intracellular experiments indicated that FA-CDs probe could accurately distinguish folate receptor (FR)-positive cancer cells in different cell mixtures with turn-on mode. In particular, combining the targeting of FA-CDs probe with the excellent photostability of CDs has inestimable meaning for fluorescence-assisted surgical resection and acquisition real-time information about tumor cells. Obviously, the as-prepared FA-CDs probe may have great potential as a high-performance platform for accurately recognizing special cancer cells, which may provide new tools for cancer prognosis and therapy.

A target-triggered exponential amplification-based DNAzyme biosensor for ultrasensitive detection of folate receptors.

We develop a new method for ultrasensitive detection of folate receptors (FRs) using a target-triggered isothermally exponential amplification reaction (EXPAR)-based DNAzyme biosensor. This method exhibits excellent specificity and high sensitivity with a detection limit as low as 0.23 fM and a large dynamic range of 6 orders of magnitude from 1 fM to 1 nM. It might be further applied for the detection of various small molecule-binding proteins by simply changing the linked small molecule moiety of the hairpin probes.