Development of a novel multi-functional integrated bioconjugate effectively targeting K-Ras mutant pancreatic cancer / 药物分析学报

Folate receptor(FR)overexpression occurs in a variety of cancers,including pancreatic cancer.In addi-tion,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-HSA-LDP-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.

The experimental study on intraperitoneal injection of targeted degradable nanoparticles 177Lu-FA-DOTA-PEG-PLGA in the treatment of ovarian cancer peritoneal metastases and ascites / 中华放射医学与防护杂志

Objective:To observe the distribution of 177Lu-FA-DOTA-PEG-PLGA nanoparticles in vivo, and evaluate the therapeutic effect of nanoparticles intraperitoneal injection on ovarian cancer peritoneal metastases and ascites. Methods:Nanoparticles were prepared and injected into human ovarian cancer xenograft nude mice model by tail vein. Micro-SPECT/CT imaging was performed at different times (4, 24, 72 h and 7 d) after injection to observe the distribution of nanoparticles in vivo. Nude mouse models of intraperitoneal metastases of human ovarian cancer were randomly divided into negative control group (normal saline), chemotherapy group (cisplatin 3 mg/kg, twice a week) and nanoparticle group (18.5 MBq), with 4 mice in each group. After 7 days, intraperitoneal tumor growth was evaluated by in vivo fluorescence imaging. The relative tumor inhibition rate was counted. Tumor cell apoptosis rate was detected by TUNEL method, and the proliferation activity tumor Ki67 was detected by immunohistochemical method. The ascites volume of each group was compared after treatment. Results:Micro-SPECT/CT imaging showed the radioactive uptake of the transplanted tumor, and the 24 h tumor muscle uptake ratio (T/M) was the highest, about 2.81±0.49. Intravital fluorescence imaging showed that, after intraperitoneal administration, the fluorescence intensity of abdominal tumor in particle group, chemotherapy group and control group was (1.45±0.19)×10 10, (2.21±0.36)×10 10 and (2.63±0.79)×10 10( F=6.09, P=0.029), respectively. The relative tumor growth inhibition (TGI) of the particle group and the chemotherapy group were 35.6% and 18.6%, respectively. The tumor cell apoptosis rates in particle group and chemotherapy group were higher than those in control group ( F=9.96, P=0.009). Ki67 indexes in particle group and chemotherapy group were lower than those in control group ( F=9.93, P=0.013). The ascites volume in particle group and chemotherapy group were both smaller than those in control group ( F=13.43, P=0.006). Conclusions:177Lu-FA-DOTA-PEG-PLGA nanoparticles can be used for the targeted imaging of ovarian cancer. After intraperitoneal injection, nanoparticles show local retention, degradation and absorption and thus inhibit the growth of peritoneal metastases and ascites of ovarian cancer, which provides a new idea for the diagnosis and treatment of advanced ovarian cancer with peritoneal metastasis.

stablishment of a diagnostic model for clinical stage Ⅰ non-small cell lung cancer: A study based on clinical imaging features combined with folate receptor-positive circulating tumor cells tests / 中国胸心血管外科临床杂志

@#Objective    To analyze the correlation between folate receptor-positive circulating tumor cells (FR+CTC) and the benign or malignant lesions of the lung, and to establish a malignant prediction model for pulmonary neoplasm based on clinical data, imaging and FR+CTC tests. Methods    A retrospective analysis was done on 1 277 patients admitted to the Affiliated Hospital of Qingdao University from September 2018 to December 2019, including 518 males and 759 females, with a median age of 57 (29-85) years. They underwent CTC examination of peripheral blood and had pathological results of pulmonary nodules and lung tumors. The patients were randomly divided into a trial group and a validation group. Univariate and multivariate analyses were performed on the data of the two groups. Then the nomogram prediction model was established and verified internally and externally. Receiver operating characteristic (ROC) curve was used to test the differentiation of the model and calibration curve was used to test the consistency of the model. Results    Totally 925 patients suffered non-small cell lung cancer and 113 patients had benign diseases in the trial group; 219 patients suffered non-small cell lung cancer and 20 patients had benign diseases in the verification group. The FR+CTC in the peripheral blood of non-small cell lung cancer patients was higher than that found in the lungs of the patients who were in favorite conditions (P<0.001). Multivariate analysis showed that age≥60 years, female, FR+CTC value>8.7 FU/3 mL, positive pleural indenlation sign, nodule diameter, positive burr sign, consolidation/tumor ratio<1 were independent risk factors for benign and malignant lung tumors with a lesion diameter of ≤4 cm. Thereby, the nomogram prediction model was established. The area under the ROC curve (AUC) of the trial group was 0.918, the sensitivity was 86.36%, and the specificity was 83.19%. The AUC value of the verification group was 0.903, the sensitivity of the model was 79.45%, and the specificity was 90.00%, indicating nomogram model discrimination was efficient. The calibration curve also showed that the nomogram model calibration worked well. Conclusion    FR+CTC in the peripheral blood of non-small cell lung cancer patients is higher than that found in the lungs of the patients who carry benign pulmonary diseases. The diagnostic model of clinical stage Ⅰ non-small cell lung cancer established in this study owns good accuracy and can provide a basis for clinical diagnosis.

Effect of diallyl trisulfide on the expression of folate receptor α in human gastric cancer cell lines BGC823 and AGS and the related regulatory mechanism / 肿瘤研究与临床

Objective:To investigate the effect of diallyl trisulfide (DATS) on the expression of folate receptor α (FRα) in human gastric cancer cells and the related regulatory mechanism.Methods:Human gastric cancer cell lines BGC823 and AGS were selected, BGC823 cells were treated with 10, 20, 40, 80 μmol/L DATS for 48 hours, and AGS cells were treated with 5, 10, 20, 40 μmol/L DATS for 48 hours. Cells treated with 6 μmol/L histone deacetylase (HDAC) inhibitor trichostatin A (TSA) were used as positive control for epigenetic study, and cells untreated with DATS were used as negative control. The apoptosis of gastric cancer cells induced by DATS was detected by flow cytometry. After BGC823 and AGS cells were treated by DATS for 48 hours, they were replaced with DATS-free cell culture medium and cultured for different time to detect the changes in FRα protein expression. BGC823 and AGS cells were treated with 6 μmol/L TSA or 40 μmol/L DATS. The protein expression levels of FRα, HDAC1 and HDAC2, and histone H3 lysine 9 acetylation (H3K9ac) and histone H4 lysine 5 acetylation (H4K5ac) were detected by Western blot. BGC823 cells were inoculated into BALB/c nude mice to establish tumor bearing model. The nude mice in DATS group were injected intraperitoneally with 10 mg/kg DATS for 16 days, and then the protein in tumor-bearing tissues was extracted to detect the expression of target protein, while the control group was injected with equal dose of 0.9% NaCl solution.Results:The expressions of FRα protein in BGC823 and AGS cells were up-regulated in a dose-dependent way after gradient concentrations of DATS treatment ( F = 65.68, P < 0.01; F = 26.65, P < 0.01). After changing the cell culture medium without DATS, the expressions of FRα protein in BGC823 and AGS cells gradually decreased and returned to the initial levels ( F = 74.57, P < 0.01; F = 30.92, P < 0.01). With the increase of DATS concentration, the apoptosis rates of BGC823 and AGS cells increased ( F = 32.95, P < 0.01; F = 38.97, P < 0.01). After TSA treatment, FRα protein expressions in BGC823 and AGS cells were up-regulated by 4.5 times ( t = -12.62, P < 0.01) and 3.6 times ( t = -10.00, P < 0.01). After 40 μmol/L and 20 μmol/L DATS treatment, the expression level of FRα protein in BGC823 and AGS cells was up-regulated (both P < 0.01), the expressions of HDAC1 and HDAC2 were inhibited (all P < 0.01), and the levels of H3K9ac and H4K5ac acetylation modification increased (all P < 0.01). The results of tumor-bearing nude mice experiment showed that the volume of transplanted tumor in DATS group was smaller than that in the control group [(214±39) mm 3 vs. (577±98) mm 3], and the difference was statistically significant ( t = 4.86, P < 0.01). Compared with the control group, FRα protein expression in the transplanted tumor tissues of DATS group was up-regulated about 2 times ( t = -5.29, P < 0.01), and the expression levels of HDAC1 and HDAC2 proteins were down-regulated ( t = 9.36, P < 0.01; t = 9.88, P < 0.01). Conclusions:DATS up-regulates the expression of FRα protein in human gastric cancer BGC823 and AGS cells in a dose-dependent and reversible manner. The mechanism may be related to the effect of DATS on histone acetylation modification in tumor cells.

Diagnostic value of folate receptor-positive circulating tumor cell detection in subcentimeter pulmonary nodules / 肿瘤研究与临床

Objective@#To investigate the value of the folate receptor (FR)-positive circulating tumor cell (CTC) detection in the diagnosis of benign and malignant subcentimeter pulmonary nodules(the maximum diameter ≤10 mm).@*Methods@#Thirty-seven patients with subcentimeter pulmonary nodules (the chest CT showed the maximum diameter was ≤10 mm) in the Xuanwu Hospital of Capital Medical University from July to December 2018 were collected. Among them, 22 cases were diagnosed with early stage lung adenocarcinoma by postoperative pathological diagnosis and another 15 cases were benign lung lesion. Venous blood samples from these patients were collected before surgery and then utilized to detect FR+ CTC level (defined unit as FU/3 ml) by novel ligand-targeted polymerase chain reaction (LT-PCR), and the enzyme-linked immunosorbent assay was used to detect the levels of tumor markers, including carcinoembryonic antigen (CEA), neuron-specific enolase(NSE), cytokeratin 19 fragment CYFRA21-1, carbohydrate antigen 125 (CA125), CA199, pro-gastrin releasing peptide (pro-GRP), etc. The t-test was used to compare the measurement values between the groups. The CTC value 8.70 FU/3 ml described in the detection kit instruction was used as the threshold. The binary logistic regression was used to analyze the risk factors of malignant pulmonary nodules. The kappa consistency test was used to identify the consistency of the diagnosis results obtained by the FR+ CTC level and the pathological results of surgically resected specimens. The receiver operating characteristic curve (ROC) was drawn to evaluate the efficiency of each index for the diagnosis of benign and malignant subcentimeter pulmonary nodules.@*Results@#The level of FR+ CTC in patients with early stage lung cancer was higher than that in patients with benign lung lesion, and the difference was statistically significant [(11.0±3.0) FU/3 ml vs. (7.0±3.7) FU/3 ml, t=-3.327, P = 0.001]. The level of FR+ CTC was not related to the age, gender and smoking history of patients (all P>0.05). Logistic regression analysis indicated that high-level FR+ CTC was one of the risk factors for malignant pulmonary nodules (OR = 37.333, 95% CI 3.994-349.010, P = 0.002). The kappa consistency test indicated that the level of FR+ CTC used for the diagnosis of lung subcentimeter nodules presented a certain accuracy (κ = 0.627, P < 0.01). ROC illustrated that the FR+ CTC was better than CEA, NSE and CYFRA21-1 when it was used as an indicator for the diagnosis of malignant pulmonary nodules. The area under the curve(AUC) of FR+ CTC was 0.830 (95% CI 0.639-0.968), and the diagnostic sensitivity and specificity were 72.7% (95% CI 49.6%-88.4%) and 93.3% (95% CI 66.0%-99.7%), respectively. When FR+ CTC, CEA, NSE and CYFRA21-1 were combined for lung cancer diagnosis, the AUC, sensitivity and specificity were 0.776 (95% CI 0.614-0.938), 86.4% and 73.3%, respectively.@*Conclusion@#The detection of FR+ CTC has a high value in the diagnosis of benign and malignant subcentimeter pulmonary nodules.

Appcation of detection of circulating tumor cells and exosome mir-21 in diagnosis of lung ground glass opacity / 吉林大学学报(医学版)

Objective: To evaluate the practicability and feasibility of detection of circulating tumor cells (CTC) combined with exosome miR-21 in the diagnosis of ground glass opacity (GGO), and to provide the evidence for the diagnosis of GGO. Methods: Seventy patients were selected according to the diagnostic and exclusion criteria of GGO. All the patients underwent thoracoscopic segmentectomy. The patients diagnosed as lung cancer after operation were regarded as case group (n=24), and the patients diagnosed as benign lesion after operation were regarded as control group (n=46). Before operation, CTC was captured from the peripheral blood by negative enrichment of immunomagnetic beads. CTC was labeled with tumor-specific folate ligand-oligonucleotide conjugate. The exosomes of the same specimen were detected, purified and identified, and the expression levels of serum exosome miR-21 of the patients in two groups were detected. The diagnostic efficacies of CTC and exosome miR-21 in the GGO paitents were evaluated by ROC curves. Results: The positive rate of CTC in the patients in case group was higher than that in control group (P0. 05). The purified samples showed exosome characteristics. The expression level of serum exosome miR-21 in the patients in case group was higher than that in control group (P<0. 05). The area under ROC curve (AUC) of CTC was 0. 891, and the AUC of exosome miR-21 was 0.711, which showed good specificity. The sensitivity and specificity of the combined detection were 85.64% and 86.79%, respectively. Conclusion: The detection of CTC combined with exosome miR-21 can be used in the clinical diagnosis of early lung cancer with high sensitivity and specificity.

Effect of macrophage FRβ expression on bleomycin induced pulmonary fibrosis in mice / 重庆医学

Objective To investigate the effect of macrophage FRβ expression on bleomycin induced pulmonary fibrosis(PF) in mice.Methods The rats were divided into the normal group,control group and experimental group,6 cases in each group.The mice were performed the PF induction.The experimental group was treated with immunotoxin,the control group was given the contrast protein and the normal group was not treated.The mouse left lung was used for histological analysis,and the right lung was used for hydroxyproline analysi s.The effect of macrophage FRβ expression on bleomycin induced pulmonary fibrosis in mice and the pulmonary macrophage FRβ expression in the patients with idiopathic pulmonary fibrosis(UIP) were detected.Results The macrophage FRβ expression mainly existed in the patients with UIP and pulmonary fibrosis area of PF mice induced by bleomycin;the survival rate was significantly increased by giving the mice immunotoxin with nose(P=0.003),and the level of total hydroxyproline and fibrosis of PF mice induced by bleomycin was decreased(P=0.009,0.014);immunohistochemistry results showed that immunotoxin could reduce the cells number of lung tumor necrosis factor(TNF)-α,chemotactic CCL2 and CCL12 cells inPF mice induced by bleomycin(P=0.000).Conclusion The FRβ expression of macrophages plays a pathogenic role in IPF,and the targeted therapy of FRβ expression in macrophages may be an effective method for the treatment of IPF.

Experimental study on preparation and targeting in vitro of nanobubbles with two-fold amount of folate / 中华超声影像学杂志

Objective To investigate the selective targeting ability of a novel folate-modified nanobubbles with two-fold amount of folate [(FOL)2-NBs] . Methods DSPE-PEG2000-AD-(FOL)2with two-fold of folate per DSPE-PEG2000 chain was synthesized and then tested by 1 H nuclear magnetic resonance (1H NMR) . The novel (FOL)2-NBs was prepared using the mechanical shaking method based on lipid-stabilized perfluoropropane . The bubble size was measured by Malvern laser particle size analyzer and the contrast enhancement ability was also detected with imaging machine using a self-made agarose mold . The experiment of selective targeting ability was also carried out in human breast cancer MCF-7 cell with over-expression of folate receptor ( FR) using fluorescence activated cell sorting ( FACS) . Results The result of 1H NMR proved that DSPE-PEG2000-AD-( FOL )2was successfully synthesized ,and the purity reached up to 90% . The novel prepared ( FOL) 2-NBs showed superior contrast enhancement ability with a particle size of ( 286 .87 ± 22 .96) nm . Compared with the conventional NBs ,the novel ( FOL) 2-NBs exhibited improved selective cellular targeting ability proven by FACS . Conclusions A novel nanobubble with improved selective targeting ability is successfully prepared and shows great potential in extravascular imaging and curation in FR overexpressed tumors .

The different types and stages of folate receptor alpha expression in endometrial carcinoma and assessment on its diagnostic performance / 实用医学杂志

Objective To explore the folate receptor alpha(FRA)expression in different types and stages of in endometrial carcinoma(EC). The sensitivity and specificity of FRA testing were compared with CA125 to evaluate its diagnostic performance.Methods Serum from 50 cases of EC patients and 30 cases of normal patients was collected.Tissue from 83 cases of typeⅠEC and 30 cases of normal endometrium and typeⅡEC were collected. The expression of serum FRA was detected by ELISA.The expression ofserum CA125 was detected by electrochemi-luminescence.The expression of FRA and CA125 in tissues was detected by immunohistochemistry. Results The rate of elevated FRA expression of in typeⅠEC tissue was higher than that in typeⅡEC(P < 0.05). The rate of elevated FRA expression in type I EC was higher than that in the early stage(P < 0.05). The ROC curve showed that the sensitivity and specificity of FRA was higher than these of CA125′s. Further,they are higher in the com-bined serum FRA and CA125.Conclusions This study shows that the FRA expression level in endometrial carci-noma varies in different subtypes,indicating the potential different pathogeneses. The rate of elevated FRA expression in type I endometrial carcinoma was higher than that in the early stage. This provides the possibility for the application of FRA targeted fluorescent developer in advanced endometrial carcinoma meticulous surgery. Combination of FRA and CA125 have better diagnostic value in detecting EC.

Efficacy enhancement of folate-receptor targeted phasetransition contrast agent for HIFU ablation of tumor in vivo / 中华超声影像学杂志

Objective To explore the value of Folate-receptor targeted phaseransition nanoparticle contrast agent (FR PNPCA) in increasing efficacy of high intensity focused ultrasound (HIFU) ablation of xerograft tumor in vivo.Methods Firstly,the FR-PNPCA and non targeted phasetransition nanoparticle contrast agent (PNPCA) were prepared by a two step emulsification method respectively.Then 36 nude mice bearing xerograft ovary tumor were randomly divided into 3 groups and underwent HIFU ablation respectively.Group A was the control group,which was irradiated at 2 h after the injection of saline by the tail vein.Group B was the untargeted group,which was irradiated at 2 h after the injection of PNPCA.Group C was the targeted group,which was irradiated at 2 h after the injection of FR-PNPCA.The gray scale changes of irradiating areas in tumors were observed during ablation.Some mice were sacrificed at 1 h after ablation and the tumors were resected to observe the coagulative necrosis by TTC staining and HE staining.Lastly,The remain mice were sacrificed at 24 h after ablation,and the tumor sections were analyzed with immunohistochemical method including PCNA and TUNEL to explore the effect of HIFU ablation on tumor cell proliferating and necrosis.Results Obvious gray-scale changes could be seen in all tumors in group B and C immediately after HIFU irradiation,but almost no changes were founded in group A.The differences of gray-scale change values and squares between group B and C were statistically significant (P ＜0.05).Coagulative necrosis with irregular shape and clear boundary was observed in group B and C after irradiation,and the range of necrosis in group C was much larger than that in group B (P ＜ 0.01),but little necrosis was founded in group A.In addition,the differences of proliferating rate and necrosis rate between group B and C were statistically significant (P ＜0.05).Conclusions FR-PNPCA can not only significantly enhance the efficacy,but also can help monitoring the process during HIFU ablation of tumor ablation.Therefore,it is a promising synergistic agent for HIFU ablation.

Distribution in the body of targeted ursolic acid liposomes and their inhibitory effect on mice transplanted tumor / 中国药学杂志

OBJECTIVE: To study the distribution of folate receptor targeted ursolic acid liposomes and evaluate its antitumor effects on human epidermoid carcinoma xenografts in nude mice. METHODS: Folate derivative was used as targeting molecule and conjugated with mPEG-DSPE for obtaining liposome with long circulation features. This novel targeted agent was prepared by a thin-film dispersion method and ursolic acid was loaded into the liposomes as the anticancer drugs. Tissue distribution of liposomes in heart, liver, spleen, stomach, kidney and tumor were investigated. The antitumor effect of the targeted ursolic acid liposomes (F-PEG-L-UA) on human epidermoid carcinoma xenografts in nude mice was also studied. RESULTS: Compared with free ursolic acid liposome, UA liposome was slowly removed from blood circulation and the concentration of F-PEG-L-UA in liver, kidney and tumor tissue is significantly higher than other groups. The growth speed of tumor in the group of F-PEG-L-UA was significantly slowed down compared with other groups. The tumor volume in F-PEG-L-UA group was about 50% reduction compared with PBS-treated mice (P<0.05). CONCLUSION: A novel targeted ursolic acid compound is synthesized as the folate receptor targeting functional material. It has obvious inhibitory effect on human epidermoid carcinoma xenografts and it might be an effective antitumor drug delivery system.

Interaction between a novel folate receptor targeted rhaponticin conjugate and human serum albumin by molecular spectroscopy / 国际药学研究杂志

Objective To investigate the affinity and inteaction of a folate receptor targeted rhapontion (RHA) conjugate (FRHA) with human serum albumins(HSA). Method The interaction between FRHA and HSA under physiological conditions was investigated by fluorescence spectroscopy, UV- visual (vis) spectroscopy and circular dichroism (CD) spectroscopy. Great attempts were made to investigate their interaction mechanism regarding the quenching mechanism, the specific binding site, the type of interaction force, and the effect of FRHA on the micro-environmental and conformational changes in HSA molecules. Results The formation of the complex of FRHA-HSA would lead to fluorescence quenching. The corresponding values of Ka were 1.4322×105, 1.1793× 105, 0.9334×105 and 0.7896×105 L/mol when the temperature were 298, 302, 306, and 310 K, respectively. The enthalpy change (ΔH) and entropy change (ΔS) were calculated to be -38.772 kJ/mol and -31.39 J/(mol·K), indicating that van der Waals force and hydrogen bonds played major roles in stabilizing the complex. Conclusion The interaction process of the formation of FRHA-HSA is spontaneous. The negative values of enthalpy change (ΔH) and entropy change (ΔS) indicate that van der Waals force and hydrogen bonds play major roles in stabilizing the complex. The conformational investigation reveals the α·-helical structure is decreased and the microenvironment of HSA is changed upon the addition of FRHA. The fluorescence quenching of HSA caused by FRHA is static quenching. Furthermore, the results of site marker competitive experiment suggest that FRHA binds to the sub-domain II A of HSA.

Expression of folate receptor alpha and CA125 and its clinical significance in endometrial carcinoma / 实用医学杂志

Objective To explore the expression and the pathomechanism of folate receptor alpha(FRα) and CA125 in the development and progression of endometrial carcinoma.Methods Sixty samples of endometrial carcinoma tissues,46 samples of endometrial hyperplasia tissues and 10 normal endometrial tissues were collected in the study.Immunohistochemical methods were used to detect the expression of Frαand CA125 in all tissues.The expressions of FRα and CA125 and their correlation with clinicopathological characteristics were analyzed.Results FRα was positively expressed in 93.9％ of the endometrial carcinoma tissues,with a strongly positive rate of 65.0％,which was significantly higher than that in endometrial hyperplasia tissues and normal endometrial tissues (P ＜ 0.05).The highly expressed FRαin endometrial carcinoma tissues was associated with age,FIGO stage and histologic types (P ＜ 0.05),while no statistical significance was found between the high expression of FRαand myometrial invasion.The expression of FRα in endometrial atypical hyperplasia was higher than that in other hyperplasia subgroups.The expression of CA125 in endometrial carcinoma tissues and endometrial hyperplasia tissues were both higher than that in normal endometrial tissues (P ＜ 0.05).Conclusion FRα may play an important role in the carcinogenesis and progression of endometrial carcinoma,and act as a target of therapy and a kind of assessment for prognosis in endometrial carcinoma.CA125 may be involved in the development of endometrial lesions and further researches are needed to confirm a physiological mechanism between FRA and CA125 in carcinogenesis of endometrial carcinoma.

Interaction between a novel folate receptor targeted rhaponticin conjugate and human serum albumin by molecular spectroscopy / 国际药学研究杂志

Objective To investigate the affinity and interaction of a folate receptor targeted rhapontion(RHA)conjugate (FRHA)with human serum albumins(HSA). Method The interaction between FRHA and HSA under physiological conditions was investigated by fluorescence spectroscopy,UV-visual(vis)spectroscopy and circular dichroism(CD)spectroscopy. Great attempts were made to investigate their interaction mechanism regarding the quenching mechanism,the specific binding site,the type of inter-action force,and the effect of FRHA on the micro-environmental and conformational changes in HSA molecules. Results The forma-tion of the complex of FRHA-HSA would lead to fluorescence quenching. The corresponding values of Ka were 1.4322 × 105,1.1793 × 105,0.9334 × 105 and 0.7896 × 105 L/mol when the temperature were 298,302,306,and 310 K,respectively. The enthalpy change (ΔH)and entropy change(ΔS)were calculated to be-38.772 kJ/mol and-31.39 J/(mol·K),indicating that van der Waals force and hydrogen bonds played major roles in stabilizing the complex. Conclusion The interaction process of the formation of FRHA-HSA is spontaneous. The negative values of enthalpy change(ΔH)and entropy change(ΔS)indicate that van der Waals force and hydrogen bonds play major roles in stabilizing the complex. The conformational investigation reveals theα-helical structure is decreased and the microenvironment of HSA is changed upon the addition of FRHA. The fluorescence quenching of HSA caused by FRHA is static quenching. Furthermore,the results of site marker competitive experiment suggest that FRHA binds to the sub-domainⅡA of HSA.

Folate Receptor-Targeted Diagnostics and Therapeutics for Inflammatory Diseases

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-α, FR-β, and FR-γ, 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.

Tissue distribution and tumor uptake of folate receptor-targeted epothilone folate conjugate, BMS-753493, in CD2F1 mice after systemic administration

To assess targeting of an epothilone folate conjugate (BMS-753493) to the folate receptor (FR)-overexpressed tumor in mice bearing both FR+ and FR- tumors, a series of experiments were conducted by quantitative whole-body autoradiography (QWBA) and LC-MS/MS following i.v. administration of BMS-753493 or its active moiety, BMS-748285 in mice bearing FR+ (98M109) and FR- (M109) tumors. QWBA showed [H]BMS-753493-derived radioactivity was extensively distributed to various tissues. The FR over-expressing 98M109 tumors showed consistently higher level of radioactivity than FR-negative tumors (., M109 tumors) up to 48 h post dose of [H]BMS-753493, despite the magnitude of difference between the tumors is relatively small (generally 3~5-fold). The radioactivity level in 98M109 tumors was 2~12-fold of normal tissues except intestine/content at 48 h post dose. No selective radioactivity uptake into 98M109 tumors over M109 or normal tissues was observed after i.v. administration of the active epothilone, [H]BMS-748285. LC-MS/MS measurements demonstrated that the concentrations of BMS-748285, presumably from hydrolysis of the folate conjugate, in 98M109 tumors were greater than those in M109 tumors after i.v. administration of BMS-753493 (2-3-fold) whereas no differential uptake in the tumors following BMS-748285 administration. Those data were consistent with radioactivity determinations. Those results demonstrated that the folate conjugation in BMS-753493 enabled moderately preferential distribution of the active epothilone to FR over-expressing 98M109 tumors, thereby supporting targeted delivery of cytotoxics through the folate receptor.

Folate-conjugated polyspermine for lung cancer-targeted gene therapy

Biodegradable polyamines have long been studied as potential recombinant viral gene vectors. Spermine (SPE) is an endogenous tetra-amine with excellent biocompatibility yet poor gene condensation capacity. We have previously synthesized a polyspermine based on SPE and poly(ethylene glycol) (PEG) diacrylate (SPE-alt-PEG) for enhanced transfection performance, but the synthesized SPE-alt-PEG still lacked specificity towards cancer cells. In this study, folic acid (FA) was incorporated into SPE-alt-PEG to fabricate a targeted gene delivery vector (FA-SPE-PEG) via an acylation reaction. FA-SPE-PEG exhibited mild cytotoxicity in both cancer cells and normal cells. FA-SPE-PEG possessed higher transfection efficiency than PEI 25 K and Lipofectamine(®) 2000 in two tested cancer cell lines at functional weight ratios, and its superiority over untargeted SPE-alt-PEG was prominent in cells with overexpressed folate receptors (FRs). Moreover, in vivo delivery of green fluorescent protein (GFP) with FA-SPE-PEG resulted in highest fluorescent signal intensity of all investigated groups. FA-SPE-PEG showed remarkably enhanced specificity towards cancer cells both in vivo and in vitro due to the interaction between FA and FRs. Taken together, FA-SPE-PEG was demonstrated to be a prospective targeted gene delivery vector with high transfection capacity and excellent biocompatibility.

Cellular uptake of Tumor-targeted nanoparticles derived from pullulan acetate conjugate / 医学研究生学报

Objective Folate receptors ( FRs) , overexpressed on the surface of a variety of tumor cells , are potential targets for tumor targeting therapy .This study aimed to prepare an FR-mediated drug nanocarrier with folate conjugated pullulan acetate ( FPA) to target chemotherapeutic agents to FR-overexpressed tumor cells and investigate its tumor-suppressing effect in vitro. Methods The cytotoxicity of epirubicin-loaded FPA nanoparticles ( FPA/EPI NP) against HepG2 and Hela cells was evaluated by MTS assay.The HepG2 and Hela cells were divided into five groups to be treated with NPs (NP control), chlorpromazine, chloro-quine, amiloride, and folate, respectively, followed by detection of the fluorescence intensity by flow cytometry . Results FPA/EPI NP was successfully formulated into NPs , with the mean particle size of (268.5 ±12.0) nm, by dialysis with an almost spherical shape . The drug-loading rate and entrapment rate of FPA/EPI NP were (6.45 ±1.04) and (72.45 ±11.50) %, respectively.The survival rates of the HepG2 and Hela cells were both >95%after 24 hours of incubation with FPA NP at 5, 40, 200, 400 and 1000μg/mL and 90.0%after 72 hours.The survival rates of the HepG2 cells treated with 5, 40, 200, 400 and 1000μg/mL FPA/EPI NP for 24 hours were (92.3 ±5.2), (70.4 ±4.6), (50.0 ±4.0), (41.1 ±4.1) and (27.0 ±3.6) %, respectively.Compared with the NP control group, the Hela cells of the chlorpromazine , amiloride and folate groups showed a significantly lower rate of NP uptake (P<0.05), and so did the HepG2 cells pretreated with chlorpromazine or amilo-ride (P<0.05).At 72 hours, the half maximal inhibitory concentrations (IC50) of FPA/EPI NP against HepG2 and Hela cells were 168 and 105μg/mL, respectively . Conclusion Clathrin-mediated endocytosis and macropinocytosis are involved in the internaliza-tion of FPA/EPI NP in HepG2 cells, while clathrin-and FR-mediated endocytosis in that of Hela cells .FPA NP may serve as a new drug carrier for tumor-targeted therapy .

The intake of folate-conjugated silica-coated gold nanorods in rabbits with VX-2 liver cancer：an ;experimental study / 介入放射学杂志

Objective To establish the rabbit model with hepatic VX-2 tumor and to investigate the intake of folate-conjugated silica-coated gold nanorods (GNRs@SiO2-FA) in experimental rabbits. Methods Under CT-guidance, animal model with VX-2 liver cancer was established in 27 rabbits by using puncture inoculation method. CT scanning and sonography were employed to observe the tumor growth. After two weeks, the rabbits were randomly and equally divided into blank control group (n=9, injection of saline), portal vein injection group (n=9, injection of GNRs@SiO2-FA) and intra-tumoral injection group (n=9, injection of GNRs@SiO2-FA). Every three rabbits from each group were sacrificed each time at 24 h, 48 h and 72 h after the treatment. The tumor tissue and the major organs were collected and sent for pathological examination. The cellular uptake of GNRs@SiO2-FA was studied by confocal laser scanning microscopy. Results The rabbit model of VX-2 liver cancer was successfully established. CT and sonography examination indicated that the tumor was rich in blood supply. Confocal laser scanning microscopy revealed that GNRs@SiO2-FA could specifically bind with tumor cells within 24 hours after injection, then the GNRs@SiO2-FA entered into the tumor cells and gathered in the tumor cytoplasm. Conclusion GNRs@SiO2-FA has highly targeted effect on the liver cancer cells in experimental animals, which has very important application prospect in targeting hyperthermia therapy and in 125I seed implantation therapy.

Expression and significance of folate receptor alpha in serum of patients with endometrial carcinoma / 实用医学杂志

Objective To detect serum concentration of folate receptor alpha and to investigate its significance in the clinical application of patients with endometrial carcinoma. Methods Thirty-seven patients with endometrial carcinoma, 33 patients with endometrial hyperplasia and 10 healthy women were enrolled in this study. Sera were used to detect the the folate receptor alpha using an Enzyme-linked Immunosorbent Assay (ELISA) technique.The expression level of serum folate receptor alpha in different groups was analyzed. The correlation between the expression level of serum folate receptor alpha and age of patients, menopause, tumor morphology, myometrial invasion and clinical stage was was also analyzed in patients with endometrial carcinoma. Results Level of folate receptor alpha was successfully detected in serum of healthy women and patients with endometrial diseases. Level of folate receptor alpha in patients with endometrial carcinoma was much higher than that in patients with endometrial hyperplasia. Level of folate receptor alpha in patients with endometrial hyperplasia was also higher than that in the healthy controls, with significant difference (P 0.05). Conclusion The Serum level of folate receptor alpha can be detected, and its expression will contribute to the diagnosis, treatment and predicting the prognosis of patients with endometrial carcinoma.