Objective:To establish a BGJ398-resistant orthotopic model of intrahepatic cholangiocarcinoma(ICC)in mice that simulates the fibroblast growth factor receptor 2(FGFR2)gene rearrangement/fusion mutations and FGFR2 kinase domain mutations in clinical patients,in order to provide a tool for relavant research.Methods:Modified FGFR2 gene encoding FGFR2 protein that's prone to phosphorylation activation was introduced into the mouse liver using hydrodynamic tail vein injection technology to establish a BGJ398-sensitive orthotopic model of ICC.Plasmids injected include pT3-myr-AKT-HA,pT3-EF1a-NICD1,pCMV(CAT)T7-SB100 and pSB[Exp]-myr-FGFR2-FLAG.Then,the mice were treated with BGJ398 for 4 weeks to induce resistance to BGJ398.Ultrasound imaging was used to monitor bile duct obstruction lesions in the model mice in order to access the extent of resistance to BGJ398.HE staining and oil red O staining were used to analyze liver tissue sections for the histological features of the BGJ398-resistant orthotopic ICC model.Immunofluorescence staining was used to analyze the origin of the ICC cells from BGJ398-resistant mice.Primary tumor cells were extracted from the model mice and CCK-8 assay was performed to analyze their sensitivity to BGJ398 in vitro as well as the median inhibition concentration(IC50)of BGJ398.The model mice were treated with BGJ398 and the survival curves were plotted to verify the resistance of the ICC mouse model to drug treatment in vivo.DNA was extracted from drug-resistant ICC tissues and genomic resequencing was used to verify mutations in the kinase domain of the FGFR2 sequence.Results:The successful rate of the BGJ398-resistant orthotopic ICC mouse model construction was 93.33%,with significant tumor burden observed in the liver.Histologically,ICC tumor tissue was arranged in glandular duct patterns in the liver.Large numbers of lipid droplet vacuoles appeared in HE staining and oil red O staining.Immunofluorescence staining of the liver tissue showed positive results for biliary epithelial cell marker cytokeratin 19(CK19)and negative results for hepatocyte marker albumin(ALB).Primary ICC tumor cells were extracted and cultured,and CCK-8 results showed that the IC50 value for BGJ398 was 95.22 nmol in BGJ398-sensitive cells while the number increased to 348.4 nmol in BGJ398-resistant cells.After treatment with BGJ398 through intraperitoneal injection,the survival rate for BGJ398-resistnat mice was significantly lower than that of BGJ398-sensitive mice(P=0.001 7).Subsequent genomic resequencing revealed significant mutations in the FGFR2 gene,verifying that the mutations in the orthotopic ICC mouse model was consistent with those found in clinical ICC patients.Conclusion:This study has successfully established a BGJ398-resistant orthotopic ICC mouse model,providing valuable tools for further exploration of the molecular mechanisms of resistance to FGFR inhibitors and the development of new treatment methods.

Objective:To establish a BGJ398-resistant orthotopic model of intrahepatic cholangiocarcinoma(ICC)in mice that simulates the fibroblast growth factor receptor 2(FGFR2)gene rearrangement/fusion mutations and FGFR2 kinase domain mutations in clinical patients,in order to provide a tool for relavant research.Methods:Modified FGFR2 gene encoding FGFR2 protein that's prone to phosphorylation activation was introduced into the mouse liver using hydrodynamic tail vein injection technology to establish a BGJ398-sensitive orthotopic model of ICC.Plasmids injected include pT3-myr-AKT-HA,pT3-EF1a-NICD1,pCMV(CAT)T7-SB100 and pSB[Exp]-myr-FGFR2-FLAG.Then,the mice were treated with BGJ398 for 4 weeks to induce resistance to BGJ398.Ultrasound imaging was used to monitor bile duct obstruction lesions in the model mice in order to access the extent of resistance to BGJ398.HE staining and oil red O staining were used to analyze liver tissue sections for the histological features of the BGJ398-resistant orthotopic ICC model.Immunofluorescence staining was used to analyze the origin of the ICC cells from BGJ398-resistant mice.Primary tumor cells were extracted from the model mice and CCK-8 assay was performed to analyze their sensitivity to BGJ398 in vitro as well as the median inhibition concentration(IC50)of BGJ398.The model mice were treated with BGJ398 and the survival curves were plotted to verify the resistance of the ICC mouse model to drug treatment in vivo.DNA was extracted from drug-resistant ICC tissues and genomic resequencing was used to verify mutations in the kinase domain of the FGFR2 sequence.Results:The successful rate of the BGJ398-resistant orthotopic ICC mouse model construction was 93.33%,with significant tumor burden observed in the liver.Histologically,ICC tumor tissue was arranged in glandular duct patterns in the liver.Large numbers of lipid droplet vacuoles appeared in HE staining and oil red O staining.Immunofluorescence staining of the liver tissue showed positive results for biliary epithelial cell marker cytokeratin 19(CK19)and negative results for hepatocyte marker albumin(ALB).Primary ICC tumor cells were extracted and cultured,and CCK-8 results showed that the IC50 value for BGJ398 was 95.22 nmol in BGJ398-sensitive cells while the number increased to 348.4 nmol in BGJ398-resistant cells.After treatment with BGJ398 through intraperitoneal injection,the survival rate for BGJ398-resistnat mice was significantly lower than that of BGJ398-sensitive mice(P=0.001 7).Subsequent genomic resequencing revealed significant mutations in the FGFR2 gene,verifying that the mutations in the orthotopic ICC mouse model was consistent with those found in clinical ICC patients.Conclusion:This study has successfully established a BGJ398-resistant orthotopic ICC mouse model,providing valuable tools for further exploration of the molecular mechanisms of resistance to FGFR inhibitors and the development of new treatment methods.

The gene silence induced by small interfering RNA (siR N A) provides a powerful tool for gene therapy of neoplasm. However, safe and efficient delivery of exogenous siRNA is still the key issue for clinical application. Polyethyenimines ( PEI ) and its copolymers can encapsulate siRNA into complex in the formation of nanoparticle wherein the stability and activity of siRNA are closely related to the structure and molecular weight of PEI, N/P ratio, and the composition of copolymers.The siRNA-encapsulated nanoparticles can effectively inhibit the proliferation of tumor cell not only in vitro, but also in vivo by administration via intratumoral,intraperitoneal or venous injections.

Objective To investigate the intracellular delivery of siRNAs through the applications of ultrasound targeted microbubbles destruction(UTMD)and biodegradable nanoparticles carriers.Methods Preparation of nanoparticles with and without RGD sequences,parameters optimization via L16(45)orthogonal design,control experiments in groups of optimization,RGD targeted nanoparticles,non-RGD nanoparticles and blank control, and determinations by inverted fluorescence microscope and flow cytometry were performed.Results The uptake and fluorescence intensity of PC-3 cells in group of RGD targeted nanoparticle was (93.49±1.37)% and 34.28±2.06 respectively,and that in group of optimization was (88.33±1.24)% and 30.59±3.93 respectively(P＞0.05).Whereas the uptake and fluorescence intensity of PC-3 cells in group of non-RGD nanoparticles was(71.24±2.80)% and 18.39±0.90 respectively,and that in group of optimization was (84.78±2.13)% and 27.18±0.91 respectively(P＜0.05).ConclusionsThe applications of UTMD with RGD targted nanoparticles cannot increase the intracellular delivery of siRNAs.

Physical exercise of moderate intensity is becoming readily accepted as an adjunct therapy to enhance curative effects of chemotherapy in patients with breast cancer. In this study, we investigated the putative effect of physical exercise on inhibition of breast cancer and the possible mechanism therein involoved. Balb/c female mice were transplanted with BCAP-37 breast xenografts and randomly assigned to four groups: (a) saline control, (b) exercise-only, (c) DHAQ-loaded NPs, (d) exercise + DHAQ-loaded NPs. The mice in exercise groups performed progressive wheel running up to 15 m/min for 30 minutes, 6 d/wk for 4 weeks. Tumor growth delay was significantly longer in the DHAQ-loaded NPs group and the exercise + DHAQ-loaded NPs groups compared with that in the control group (P < 0.05; P < 0.01, respectively). Tumor volume and the value of hemoglobin (HGB) showed significant difference between the DHAQ-loaded NPs and exercise + DHAQ-loaded NPs groups (P < 0.05), suggesting that physical exercise of moderate intensity can significantly induce an influence of DHAQ-loaded NPs on delay in tumor growth, and can enhance the anti-tumor efficacy of DHAQ-loaded PLA-PLL-RGD NPs. It is a contributor to adjuvant therapy for breast cancer.
Animals ; Antineoplastic Agents ; administration & dosage ; Drug Carriers ; administration & dosage ; Drug Delivery Systems ; Exercise Therapy ; Female ; Mammary Neoplasms, Experimental ; therapy ; Mice ; Mice, Inbred BALB C ; Mitoxantrone ; administration & dosage ; Nanoparticles ; administration & dosage ; Random Allocation

Background and purpose:A pressing obstacle in clinical chemotherapy is drug resistance in breast cancer.A nano-delivery system,which has many advantages as a drug carrier,such as carrying anticancer drugs,can be used effectively to overcome drug resistance in tumors.This paper examined a new nano-delivery system,called calcium phosphate and glycerophosphocholine-mPEG(CAP/GPC-MPEG)composite nanoparticle and its influence on the cellular drug uptake of BCRP-over expressing mitoxantrone(MIT)-resistant breast cancer cell MCF-7/MIT.This paper will also examine its effect on overcoming drug resistance in the MCF-7/MIT cells.Methods:After the calcium phosphate and GPC-mPEG composite nanoparticles were designed and prepared,the entrapment efficiency and in vitro drug release of mitoxantrone-loaded nanoparticles were investigated.Quantitative comparisons were made between cellular uptake of drug-loaded nanoparticles and free drugs.Finally,a confocal laser scanning microscopy Was used to compare the subcellular distribution of drug-loaded nanoparticles and the free drugs.Results:Calcium phosphate and GPC-mPEG composite nanoparticles were nanoporous spherical particles with diameters between 50-100 mn.The MIT-loaded nanoparticles have an entrapment efficiency of(89.45±0.05)%.Although the drug-loaded nanoparticles showed an initial burst of drug release,it was followed by a more sustained release.The concentration of mitoxantrone was 1.89 times treated with MIT-loaded nanoparticles for 1 h compared to that treated with free mitoxantrone for 1 h in MCF-7/MIT cells.and which was 2.33 times in MCF-7 cells.Fluorescent red mitoxantrone appeared in the cytoplasm and nucleus of the MCF-7 and MCF-7,MIT cells treated with MlT-loaded nanoparticles whereas it is almost undetected in both cells treated with free mitoxantrone.Conclusion:Calcium phosphate and GPC-mPEG composite nanoparticles Can promote the cellular uptake and entering of mitoxantrone to the nucleus in MCF-7 and its corresponding BCRP-over expressing MIT-resistant MCF-7/MIT breast cancer cell lines.This nanoparticle is a potential nano-carrier for overcoming drug resistance in tumors.

To identify the advantages of modification treated poly (D, L-lactic and glycolic acid) with poly (ethylene glycol) (PLGA/mPEG, PELGA) block copolymer, blood compatibility was evaluated in vitro. Three different proportional block copolymers (LA/GA80: 20, 70: 30, 50: 50) with 15% mPEG were synthesized. According to the international standard organization (ISO10993) and National Standards of P. R. China GB/T 16886, siliconized glass tube was used as a negative control sample, while non-siliconized glass tube as a positive one. Blood compatibility of PELGA was evaluated by hemolytic ratio analysis, platelet adhesion investigation, dynamic clotting time, plasma recalcification time (PRT) measurements, plasma prothrombin time (PT) and clotting time (CT) test. The results revealed that blood compatibility of the synthesized materials was good. Nanoparticles made by this kind of materials might be promising for intravenous use.
Biocompatible Materials ; chemistry ; Drug Carriers ; chemistry ; Drug Delivery Systems ; Lactic Acid ; chemistry ; Materials Testing ; Platelet Adhesiveness ; drug effects ; Polyethylene Glycols ; chemistry ; Polyglycolic Acid ; chemistry

A series of poly (lacticacid-co-glycolicacid)-poly(ethylene glycol) (PLGA-PEG, PELGA) block copolymers and poly (ethylene glycol)-poly (lacticacid-co-glycolicacid)-poly (ethylene-glycol) (PELGE) was synthesized by ring-opening polymerization. PELGA nanoparticles and PELGE nanoparticles were prepared using the emulsion-solvent evaporation technique (O/W). To study the behavior and mechanism of the degradation of PELGA-NP and PELGA-NP, we determined the lactic acids by UV spectrophotometry. The method confirmed that degradation was much faster for polymers with a decrease in the LA content of the polymers or an increase in the PEG content of the polymers.
Biocompatible Materials ; chemistry ; Biodegradation, Environmental ; Drug Carriers ; Drug Delivery Systems ; Humans ; Lactic Acid ; chemical synthesis ; chemistry ; Microspheres ; Nanostructures ; Nanotechnology ; Polyesters ; chemical synthesis ; chemistry ; Polyethylene Glycols ; chemical synthesis ; chemistry ; Polyglactin 910 ; chemistry ; Polyglycolic Acid ; chemical synthesis ; chemistry ; Polymers ; chemical synthesis ; chemistry

Bone-like apatite formation on the surface of calcium phosphate ceramics has been believed to be necessary for new bone to grow on the ceramics and to be related to the osteoinductivity of the material. The research of bone-like apatite formation is a great help to understanding the mechanism of osteoinduction. Synthetic porous calcium phosphate ceramics (HA/TCP = 70/30) were implanted intramuscularly in pigs, dogs, rabbits and rats to make a comparative study of the bone-like apatite formation onto the porous HA/TCP ceramics in different animals. Specimens were harvested at 14 days after implantation. Samples were detected for the surface morphology with SEM. The chemical composition of the sample surface after implantation was analyzed with reflection infrared (R-IR). Obvious bone-like apatite formation could be detected in the sections of porous specimens harvested from all animals after 14 days intramuscular implantation. Crystal deposition could be only observed on the surface of the concave regions of the samples collected from dogs, rabbits and rat. On the contrary, evenly distributed flake-shaped crystal could be found on the pore surface and also on the outer surface of the materials implanted in pigs. The morphology of bone-like apatite in pigs was different from that in the others animals. Bone-like apatite was not observed in dense specimen implanted intramuscularly. Bone-like apatite formed faster on specimens implanted in rabbit than that in other animals. This formation sequence is different from the sequence of osteoinductivity of biphasic calcium phosphate ceramics implanted in these animals. The results demonstrated that the formation of bone-like apatite on materials is a prerequisite condition to their osteoinduction but other factors also play important roles in osteoinduction.
Animals ; Apatites ; chemical synthesis ; Body Fluids ; chemistry ; Bone Substitutes ; chemical synthesis ; chemistry ; Calcium Phosphates ; chemistry ; Ceramics ; chemistry ; Dogs ; Male ; Materials Testing ; Prostheses and Implants ; Rabbits ; Rats ; Swine

Bone-like apatite formation on the surface of calcium phosphate ceramics has been believed to be the prerequisite of new bone growth on ceramics and to be related to the osteoinductivity of the material. The research of the factors effecting bone-like apatite formation is a great help in understanding the mechanism of osteoinduction. This paper is aimed to a comparative study of in vitro formation of bone-like apatite on the surface of dense and rough calcium phosphate ceramics with SBF flowing at different rates. The results showed that the rough surface was beneficial to the formation of bone-like apatite, and the apatite formed faster in 1.5 SBF than in SBF. Rough surface, namely, larger surface area, increased the dissolution of Ca2+ and HPO4(2-) and higher concentration of Ca2+ and HPO4(2-) ions of SBF and was in turn advantageous to the accumulation of Ca2+, HPO4(2-), PO4(3-) near the ceramic surface. Local supersaturating concentration of Ca2+, HPO4(2-), PO4(3-) near sample surface was essential to nucleation of apatite on the surface of sample.
Apatites ; Biocompatible Materials ; Calcium Phosphates ; Ceramics ; Materials Testing ; Surface Properties