Therapeutic and Diagnostic Potential of Folic Acid Receptors and Glycosylphosphatidylinositol (GPI) Transamidase in Prostate Cancer.

Due to the proliferation-induced high demand of cancer cells for folic acid (FA), significant overexpression of folate receptors 1 (FR1) is detected in most cancers. To our knowledge, a detailed characterization of FR1 expression and regulation regarding therapeutic and diagnostic feasibilities in prostate cancer (PCa) has not been described. In the present study, cell cultures, as well as tissue sections, were analyzed using Western blot, qRT-PCR and immunofluorescence. In addition, we utilized FA-functionalized lipoplexes to characterize the potential of FR1-targeted delivery into PCa cells. Interestingly, we detected a high level of FR1-mRNA in healthy prostate epithelial cells and healthy prostate tissue. However, we were able to show that PCa cells in vitro and PCa tissue showed a massively enhanced FR1 membrane localization where the receptor can finally gain its function. We were able to link these changes to the overexpression of GPI-transamidase (GPI-T) by image analysis. PCa cells in vitro and PCa tissue show the strongest overexpression of GPI-T and thereby induce FR1 membrane localization. Finally, we utilized FA-functionalized lipoplexes to selectively transfer pDNA into PCa cells and demonstrate the therapeutic potential of FR1. Thus, FR1 represents a very promising candidate for targeted therapeutic transfer pathways in PCa and in combination with GPI-T, may provide predictive imaging in addition to established diagnostics.

Enhanced anticancer activity of (-)-epigallocatechin-3-gallate (EGCG) encapsulated NPs toward colon cancer cell lines.

(-)-Epigallocatechin-3-gallate (EGCG), a bioactive polyphenol of green tea, has chemo-preventive effects against various cancer cells. Nanoparticles (NPs) carrying different ligands are able to specifically interact with their receptors on different cancer cells that can provide effective release of cytotoxic drugs. In the present study, we have prepared EGCG entrapped NPs using PLGA (poly(d,l-lactide-co-glycolide)). Polyethylene glycol (PEG) and folic acid (FA) via double emulsion solvent evaporation (DESE) method obtained PLGA-EGCG (P-E), PLGA-PEG-EGCG (PP-E), and PLGA-PEG-FA-EGCG (PPF-E). Nanoformulations had been characterized with 1H NMR and FT-IR techniques, AFM, and DLS. PPF-E NPs showed an average size of 220 nm. Analysis of zeta potential confirmed the stability of NPs. HCT-116, HT-29, HCT-15, and HEK 293 cells were treated with both the prepared NPs and free EGCG (0-140 µM). Result showed PPF-E NPs had improved delivery, uptake and cell cytotoxicity toward human folic acid receptor-positive (FR+) colorectal cancer (CRC) cells as mainly on HCT-116 compared to HT-29, but not on the folic acid-negative cells (FR-) as HCT-15. PPF-E NPs enhanced intracellular reactive oxygen species (ROS) level in absence of N-acetyl-l-cysteine (NAC), elevated DNA fragmentation level, and increased apoptotic cell death at higher doses compared to other two NPs and free EGCG. In conclusion, PPF-E NPs exerted greater efficacy than PP-E, P-E, and free EGCG in HCT-116 cells.

Preparation and evaluation of doxorubicin-loaded red blood cell membrane chitosan-targeted nanoparticles / 中国药房

OBJECTIVE To prepare and evalu ate doxorubicin-loaded red blood cell membrane chitosan-targeted nanoparticles of targeting tumor cell folate acid （FA）receptor（FA-RBC-DOX-CS-NPs）. METHODS Doxorubicin-loaded chitosan nanoparticles （DOX-CS-NPs） were prepared by ion cross-linking method. FA and amino polyethylene glycol phospholithin （NH2- PEG2000-DSPE）were covalently linked to modify the red blood cell membrane to construct FA-RBC-DOX-CS-NPS. FA-RBC- DOX-CS-NPs were characterized and investigated on in vitro drug release characteristics ，antitumor activity and endocytosis ability （investigation with human breast cancer MCF- 7 cells）. RESULTS Average particle size of FA-RBC-DOX-CS-NPs was （254.200± 2.651）nm，and polydispersity index was 0.199±0.031；Zeta potential was （－10.100±0.213）mV. FA-RBC-DOX-CS-NPs released fast in the tumor microenvironment （pH6.5）. Cellular experiments showed that ，the nanoparticles could inhibit the activity of MCF- 7 cell proliferation and improve the efficiency of endocytosis. CONCLUSIONS FA-RBC-DOX-CS-NPs are prepared successfully. The nanoparticles have good tumor cell targeting and endocytosis ability ，and can realize the enrichment of drugs in tumor cells.

Research Progress on the Relationship between Folic Acid Transporter of Placenta and Pregnancy Complications / 中国药学杂志

The mother can deliver nutrients to the fetus only through the placenta during pregnancy. Folic acid plays an important role in placenta development and fetal growth, and is involved in cell division, embryogenesis and fetal growth. The ability of transplacental folic acid is one of the limiting steps of folate application. Folic acid and its active forms arrive at the fetus by three transporters: folic acid receptor, reduced folic acid carrier and proton coupled folic acid transporter. The expression and transport capacity of transporters were changed significantly with pregnancy complications such as hypertension, fetal growth restriction and premature delivery. Antiepileptic drugs, progesterone, antihypertensive drugs, drug abuse, and heavy metals can alter the transport of folic acid and across the placental barrier of other drugs by regulating the placental folate transporter.

Flower-like curcumin-loaded folic acid-conjugated ZnO-MPA- ßcyclodextrin nanostructures enhanced anticancer activity and cellular uptake of curcumin in breast cancer cells.

Non-spherical structures are beneficial to advance drug delivery effectiveness compared with common spherical ones, due to increased drug loading capability, improved bonding to a vascular wall, enhanced cellular uptake efficacy and prolonged circulation times. In this study, flower-like Zinc oxide-ßcyclodextrin (ßCD) nanostructures functionalized by 3-mercaptopropionic acid (MPA) as a non-spherical delivery system was successfully synthesized for aqueous delivery of curcumin (CUR) to enhance its targeting, bioavailability, and release profile. Terminal carboxyl functional groups were used for the conjugation of folic acid (FA) with the aim of active targeting to folate overexpressing breast cancer cells. The in vitro experimental study and mathematical modeling of CUR release revealed a sustained release with Fickian diffusion as the major release mechanism. MTT, colony formation and Annexin-V FITC/PI assays showed the superior anticancer effect of the system compared to free CUR against breast cancer cell line MDA-MB-231 by promoting the apoptotic respond with no cytotoxic effect on HEK293 normal cells. The efficacy of targeting strategy with FA moieties was demonstrated using the augmented cellular uptake of the FA-conjugated system on overexpressed folate receptor alpha (FRα) cells (MDA-MB-468 breast cancer cell line). Furthermore, loading of CUR to the delivery systems significantly lowered the MIC values (2.5 to 5-fold) against S. aureus and E. coli the infections of which are serious problems in cancer patients. According to the results of this study, the system can serve as a promising non-spherical delivery vehicle for enhancing bioavailability and targeting of hydrophobic anticancer agents in the future.

Development of biosurfactant-based graphene quantum dot conjugate as a novel and fluorescent theranostic tool for cancer.

BACKGROUND: Biosurfactants are amphipathic molecules of microbial origin that reduce surface and interfacial tension at gas-liquid-solid interfaces. Earlier, the biosurfactant was isolated and characterized in our laboratory from Candida parapsilosis. The property of the biosurfactant is further explored in this study by using quantum dots (QDs) as nanocarrier. MATERIALS AND METHODS: Graphene quantum dots (GQDs) were synthesized by bottom-up approach through pyrolysis of citric acid. GQDs were conjugated with both biosurfactant and folic acid (FA) using carbodiimide chemistry. The prepared GQD bioconjugate was studied for diagnostic and therapeutic effects against cancer cells. RESULTS AND DISCUSSION: Photoluminescence quantum yield (QY) of plain GQDs was measured as 12.8%. QY for biosurfactant conjugated GQDs and FA-biosurfactant conjugated GQDs was measured as 10.4% and 9.02%, respectively, and it was sufficient for targeting cancer cells. MTT assay showed that more than 90% of cells remained viable at concentration of 1 mg/mL, hence GQDs seemed to be non-toxic to cells. Biosurfactant conjugated GQDs caused 50% reduction in cellular viability within 24 hours. FA conjugation further increased the specificity of bioconjugated GQDs toward tumor cells, which is clearly evident from the drug internalization studies using confocal laser scanning microscopy. A higher amount of drug uptake was observed when bioconjugated GQDs were decorated with FA. CONCLUSION: The ability of GQD bioconjugate could be used as a theranostic tool for cancer. It is foreseen that in near future cancer can be detected and/or treated at an early stage by utilizing biosurfactant conjugated GQDs. Therefore, the proposed study would provide a stepping stone to improve the life of cancer patients.

Folic acid receptor-targeted human serum albumin nanoparticle formulation of cabazitaxel for tumor therapy.

BACKGROUND: We previously developed cabazitaxel (CTX)-loaded human serum albumin nanoparticles (NPs-CTX) via a self-assembly method, and these NPs showed efficacy in prostate cancer therapy. Many studies have shown that the levels of folic acid (FA) receptor on the surface of various tumor cells are high. Therefore, FA-modified NPs-CTX may have enhanced antitumor effects compared with unmodified NPs-CTX. METHODS: NPs-CTX were first prepared via self-assembly, and FA was conjugated on the surface of NPs-CTX through the -NH2 groups of the NPs to produce FA-NPs-CTX. The FA-NPs-CTX were evaluated in tumor cells with high FA receptor (FR) expression in vitro and in vivo. RESULTS: Both NPs-CTX and FA-NPs-CTX exhibited good stability and morphology. Drug release from the NPs was not affected by FA conjugation. Compared with CTX dissolved in a mixture of Tween 80 and 13% ethanol (w/w) at a ratio of 1:4 (v/v) (Tween-CTX), the two nanoformulations had lower lytic activity against normal red blood cells. However, FA-NPs-CTX showed greater inhibition of tumor cells with overexpressed FR, compared with NPs-CTX, in the cytotoxicity experiments. Moreover, the cellular uptake of FA-NPs-CTX was enhanced through FR-mediated endocytosis in HeLa cells in vitro and HeLa xenograft tumors in vivo. Although Tween-CTX exhibited tumor growth inhibition similar to FA-NPs-CTX in vivo, this inhibition also caused adverse side effects; the median lethal dose (LD50) of Tween-CTX to mice was 5.68 mg/kg, while FA-NPs-CTX-treated mice survived at doses exceeding 400 mg/kg. CONCLUSION: The results showed that FA-NPs-CTX caused inhibition of tumor growth in a manner similar to that of Tween-CTX; however, the safety and tolerability of CTX were greatly improved by FA conjugation compared with those of Tween-CTX. In summary, FA-NPs-CTX have great potential in CTX delivery, and this formulation is a promising candidate for the treatment of cancers with high FR levels.

Characterization and evaluation of a folic acid receptor-targeted cyclodextrin complex as an anticancer drug delivery system.

To improve the water solubility and tumor targeting ability of docetaxel (DTX), and thus enhance the drug's antitumor efficacy and safety, a novel folate receptor (FR)-targeted cyclodextrin drug delivery vehicle (FA-CD) was successfully synthesized. The synthesis of the designed cyclodextrin was confirmed by Fourier transform infrared spectroscopy (FTIR), proton nuclear magnetic resonance (1H NMR), and differential scanning calorimetry (DSC). The in vitro cytotoxicity was investigated using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and the results showed that no significant differences (p>0.05) appeared in cytotoxicity between the different cyclodextrins in the different cell lines. Besides, the DTX/FA-CD inclusion complex was prepared. The cellular uptake and competition assays were examined using the HepG2, HeLa, and KB cell lines, which have different levels of folate receptor expression. Interestingly, the Cy5.5/FA-CD complex had higher uptake in the HepG2, HeLa, and KB cells, compared with non-targeted Cy5.5/CD complex (p<0.001). The time-dependent drug uptake into KB cells observed by LSCM confirmed the drug delivery via endocytic routes. Data from the competition assays, especially in KB cells, showed that a significant inhibitory effect (p<0.001) was obtained when the concentration of FA was increased, and suggested that the Cy5.5/FA-CD was internalized through a FR-mediated mechanism. Moreover, the in vitro bioactivity assay also demonstrated efficient antitumor activity, and the order of the cell viabilities (% of control) was OB>HepG2>HeLa>KB for DTX/FA-CD (p<0.001). For DTX/CD, however, it displayed minimum antitumor behavior in all cell types. An apoptosis study by FCM and LSCM also revealed that the FA-modified complexes were more effective in inducing apoptosis in FR-expressing cells. Finally, an in vivo biodistribution study in KB-bearing healthy mice revealed that the DTX/FA-CD complex has enhanced tumor-targeting efficacy and diminished systemic side effects. These results suggest that the novel FR-targeted cyclodextrin complex is a promising alternative as an anticancer drug delivery system.

An special epithelial staining agents: folic acid receptor-mediated diagnosis (FRD) effectively and conveniently screen patients with cervical cancer.

High-quality screening with cytology has markedly reduced mortality from cervical cancer. However, it needs experienced pathologists to review and make the final decisions. We have developed folic acid receptor-mediated diagnosis (FRD) kits to effectively and conveniently screen patients with cervical cancer. We conduct present study aim to assess clinical significances of FRD in screening cervical cancer. A total of 169 patients were enrolled at Chinese People's liberation Army (PLA) general hospital. We compared diagnostic significances of FRD with thinprep cytology test (TCT). Meanwhile, colposcopy was also performed to confirm any lesion suspicious for cervical cancer. The sensitivity and specificity of FRD were 71.93% and 66.07% in diagnosis cervical cancer, respectively. Meanwhile, the positive predictive values (PPV), negative predictive values (NPV), Youden index were 51.90%, 82.22%, 0.38, respectively. On the other hand, the sensitivity and specificity of TCT in diagnosis cervical cancer were 73.68% and 61.61% respectively. PPV, NPV and Youden index for TCT were 49.41%, 82.14% and 0.35 respectively. Overall, FRD have high values of sensitivity, specificity and Youden index. However, this difference failed to statistical significance. FRD have comparable diagnostic significance with TCT. Therefore, FRD might serve as one effective method to screen cervical cancer. Especially for those patients living in remote regions of China, where cytology was unavailable.

Pharmacodynamics of folic acid receptor targeted antiretroviral nanotherapy in HIV-1-infected humanized mice.

Long-acting nanoformulated antiretroviral therapy (nanoART) can sustain plasma drug levels and improve its biodistribution. Cell targeted-nanoART can achieve this and bring drug efficiently to viral reservoirs. However, whether such improvements affect antiretroviral responses remains unknown. To these ends, we tested folic acid (FA)-linked poloxamer407-coated ritonavir-boosted atazanavir (FA-nanoATV/r) nanoparticles for their ability to affect chronic HIV-1 infection in humanized mice. Following three, 100mg/kg FA-nanoATV/r intramuscular injections administered every other week to infected animals, viral RNA was at or below the detection limit, cell-associated HIV-1p24 reduced and CD4+ T cell counts protected. The dosing regimen improved treatment outcomes more than two fold from untargeted nanoATV/r. We posit that these nanoformulations have potential for translation to human use.