Jian Tan

Self-Assembled Biodegradable Protein–Polymer Vesicle as a Tumor-Targeted Nanocarrier

Self-assembled nanostructures based on amphiphilic protein-polymer conjugates have shown great advantages in the field of nanomedicine such as inherent biocompatibility with biosystems because of their excellent performance. Herein, a novel biodegradable protein-polymer conjugate was prepared by covalently linking the tailor-made hydrophobic maleimide-functionalized poly(ε-caprolactone) (PCL) to hydrophilic bovine serum albumin (BSA) via the maleimide-sulfhydryl coupling reaction. This protein-based conjugate with a biodegradable polyester was reported for the first time, and the obtained biohybrid displayed well-defined structure, excellent biocompatibility and low cytotoxicity, and self-assembly behaviors similar to those of the traditional amphiphilic small molecules and block copolymers. The amphiphilic BSA-PCL conjugate can self-assemble into a nanosized vesicle with a negative surface charge. Furthermore, the self-assembled vesicle based on the BSA-PCL conjugate was functionalized via linking targeting ligand cetuximab to its surface to enhance cell uptake, and the doxorubicin (DOX)-encapsulated cetuximab-functionalized vesicle exhibited enhanced antitumor activity compared with that of free DOX in vitro. These results indicate that the biodegradable protein-polymer conjugate based on BSA and PCL had great potential as a drug delivery vehicle for cancer therapy.

Facile Construction of Near Infrared Fluorescence Nanoprobe with Amphiphilic Protein-Polymer Bioconjugate for Targeted Cell Imaging

A simple, straightforward, and reproducible strategy for the construction of a near-infrared (NIR) fluorescence nanoprobe was developed by coating CuInS2/ZnS quantum dots (CIS/ZnS QDs) with a novel amphiphilic bioconjugate. The amphiphilic bioconjugate with a tailor-designed structure of bovine serum albumin (BSA) as the hydrophilic segment and poly(ε-caprolactone) (PCL) as the hydrophobic part was fabricated by chemical coupling the hydrophobic polymer chain to BSA via the maleimide-sulfhydryl reaction. By incorporating CIS/ZnS QDs into the hydrophobic cores of the self-assembly of BSA-PCL conjugate, the constructed NIR fluorescence nanoprobe exhibited excellent fluorescent properties over a wide pH range (pH 3-10) and a good colloidal stability in PBS buffer (pH = 7.4) with or without 10% fetal bovine serum. The presence of the outer BSA shell effectively reduced the nonspecific cellular binding and imparted high biocompatibility and low-toxicity to the probe. Moreover, the NIR fluorescence nanoprobe could be functionalized by conjugating cyclic Arg-Gly-Asp (cRGD) peptide, and the decorated nanoprobe was shown to be highly selective for targeted integrin αvβ3-overexpressed tumor cell imaging. The feasibility of the constructed NIR fluorescence probe in vivo application was further investigated and the results demonstrated its great potential for in vivo imaging. This developed protocol for phase transfer of the CIS/ZnS QDs was universal and applicable to other nanoparticles stabilized with hydrophobic ligands.

Evaluation of therapeutic effectiveness of (131)I-antiEGFR-BSA-PCL in a mouse model of colorectal cancer.

AIM To investigate the biological effects of internal irradiation, and the therapeutic effectiveness was assessed of (131)I-labeled anti-epidermal growth factor receptor (EGFR) liposomes, derived from cetuximab, when used as a tumor-targeting carrier in a colorectal cancer mouse model. METHODS We described the liposomes and characterized their EGFR-targeted binding and cellular uptake in EGFR-overexpressing LS180 colorectal cancer cells. After intra-tumor injections of 74 MBq (740 MBq/mL) (131)I-antiEGFR-BSA-PCL, we investigated the biological effects of internal irradiation and the therapeutic efficacy of (131)I-antiEGFR-BSA-PCL on colorectal cancer in a male BALB/c mouse model. Tumor size, body weight, histopathology, and SPECT imaging were monitored for 33 d post-therapy. RESULTS The rapid radioiodine uptake of (131)I-antiEGFR-BSA-PCL and (131)I-BSA-PCL reached maximum levels at 4 h after incubation, and the (131)I uptake of (131)I-antiEGFR-BSA-PCL was higher than that of (131)I-BSA-PCL in vitro. The (131)I tissue distribution assay revealed that (131)I-antiEGFR-BSA-PCL was markedly taken up by the tumor. Furthermore, a tissue distribution assay revealed that (131)I-antiEGFR-BSA-PCL was markedly taken up by the tumor and reached its maximal uptake value of 21.0 ± 1.01 %ID/g (%ID/g is the percentage injected dose per gram of tissue) at 72 h following therapy; the drug concentration in the tumor was higher than that in the liver, heart, colon, or spleen. Tumor size measurements showed that tumor development was significantly inhibited by treatments with (131)I-antiEGFR-BSA-PCL and (131)I-BSA-PCL. The volume of tumor increased, and treatment rate with (131)I-antiEGFR-BSA-PCL was 124% ± 7%, lower than that with (131)I-BSA-PCL (127% ± 9%), (131)I (143% ± 7%), and normal saline (146% ± 10%). The percentage losses in original body weights were 39% ± 3%, 41% ± 4%, 49% ± 5%, and 55% ± 13%, respectively. The best survival and cure rates were obtained in the group treated with (131)I-antiEGFR-BSA-PCL. The animals injected with (131)I-antiEGFR-BSA-PCL and (131)I-BSA-PCL showed more uniform focused liposome distribution within the tumor area. CONCLUSION This study demonstrated the potential beneficial application of (131)I-antiEGFR-BSA-PCL for treating colorectal cancer. (131)I-antiEGFR-BSA-PCL suppressed the development of xenografted colorectal cancer in nude mice, thereby providing a novel candidate for receptor-mediated targeted radiotherapy.

Telomerase reverse transcriptase promoter-driven expression of iodine pump genes for targeted radioiodine therapy of malignant glioma cells.

Radioiodine is a routine therapy for differentiated thyroid cancers. Non-thyroid cancers can intake radioiodine after transfection of the human sodium iodide symporter (hNIS) gene. The human telomerase reverse transcriptase (hTERT) promoter, an excellent tumor-specific promoter, has potential value for targeted gene therapy of glioma. We used the hTERT promoter to drive the expression of the hNIS and human thyroid peroxidase (hTPO) gene as a primary step for testing the effects of radioiodine therapy on malignant glioma. The U87 and U251 cells were co-transfected with two adenoviral vectors, in which the hNIS gene had been coupled to the hTERT promoter and the hTPO gene had been coupled to the CMV promoter, respectively. Then, we performed Western blot, 125I intake and efflux assays, and clonogenic assay with cancer cells. We also did 99mTc tumor imaging of nude mice models. After co-transfection with Ad-hTERT-hNIS and Ad-CMV-hTPO, glioma cells showed the 125I intake almost 1.5 times higher than cells transfected with Ad-hTERT-hNIS alone. Western blots revealed bands of approximately 70 kDa and 110 kDa, consistent with the hNIS and hTPO proteins. In clonogenic assay, approximately 90% of cotransfected cells were killed, compared to 50% of control cells after incubated with 37 MBq of 131I. These results demonstrated that radioiodine therapy was effective in treating malignant glioma cell lines following induction of tumor-specific iodide intake by the hTERT promoter-directed hNIS expression in vitro. Cotransfected hNIS and hTPO genes can result in increased intake and longer retention of radioiodine. Nude mice harboring xenografts transfected with Ad-hTERT-NIS can take 99mTc scans.

Antitumor Effect of Nanoparticle 131I-Labeled Arginine-Glycine-Aspartate–Bovine Serum Albumin–Polycaprolactone in Lung Cancer

OBJECTIVE The aim of the present study is to investigate the biologic effects of internal irradiation and the therapeutic effectiveness of 131I-labeled arginine-glycine-aspartate (RGD)-bovine serum albumin (BSA)-polycaprolactone (PCL) (131I-RGD-BSA-PCL) in murine lung cancer models. MATERIALS AND METHODS The target binding and cellular uptake of NCI-H460 lung cancer cells overexpressing integrin αvβ3 were observed by confocal microscopy. Flow cytometry was used to assay apoptosis. The biologic effects of internal irradiation and the therapeutic efficacy of 131I-RGD-BSA-PCL were investigated in murine lung cancer models; tumor size, body weight, histopathologic findings, and SPECT/CT imaging findings were also monitored. RESULTS In vitro uptake studies performed using confocal microscopy showed that, after 1 hour of incubation with RGD-BSA-PCL or BSA-PCL, visible fluorescence was present in the cells, and after 8 hours, the florescent signal did not disappear. The mean (± SE) tumor uptake level (i.e., the percentage of the injected dose per gram of tissue [% ID/g]) of 131I-labeled BSA-PCL (131I-BSA-PCL) at 24 and 72 hours after injection was 11.06% ± 2.15% ID/g and 3.83% ± 0.87% ID/g, respectively, which is significantly higher than the uptake levels noted for other organs (p < 0.05). The level of tumor uptake of 131I-RGD-BSA-PCL at 24 and 72 hours after injection was 39.49% ± 6.06% ID/g and 6.97% ± 1.43% ID/g, respectively, which is significantly higher than that of 131I-labeled liposome (p < 0.05). The decrease in body weight in the group treated with 131I-RGD-BSA-PCL was only 3.5% of the original body weight and was much lower than noted in the group receiving saline (i.e., 21.5% of original body weight). The median survival time for the therapeutic groups was prolonged to 27 days and 23 days after treatment with 131I-RGD-BSA-PCL and 131I-BSA-PCL, respectively. CONCLUSION RGD-BSA-PCL has excellent cellular binding in vitro in a non-small cell lung cancer xenograft model. Furthermore, 131I-RGD-BSA-PCL was evaluated as an imaging agent and is an interesting candidate for targeting therapies in the non-small cell lung cancer xenograft model.

The glial fibrillary acidic protein promoter directs sodium/iodide symporter gene expression for radioiodine therapy of malignant glioma

Radioiodine is a routine therapy for differentiated thyroid cancers. Non-thyroid cancers may be treated with radio-iodine following transfection with the human sodium/iodide symporter (hNIS) gene. The glial fibrillary acidic protein (GFAP) promoter is an effective tumor-specific promoter for gene expression and thus may be useful in targeted gene therapy of malignant glioma. The present study used GFAP promoter-modulated expression of the hNIS gene in an experimental model of radioiodine-based treatment for malignant glioma. Cells were transfected using a recombination adeno-virus and evaluated in cells by studying the transfected transgene expression through western blot analysis, 125I uptake and efflux, clonogenicity following 131I treatment and radioiodine therapy using a U87 xenograft nude mouse model. Following transfection with the hNIS gene, the cells showed 95–70-fold higher 125I uptake compared with the control cells transfected with Ad-cytomegalovirus (CMV)-enhanced green fluorescent protein (EGFP). The western blotting revealed bands of ∼70, 49 and 43 kDa, consistent with the hNIS, GFAP and β-actin proteins. The clonogenic assay indicated that, following exposure to 500 μCi of 131I-iodide for 12 h, >90% of cells transfected with the hNIS gene were killed. Ad-GFAP-hNIS-transfected and 2 mCi 131I-injected U87 xenograft nude mice survived the longest of the three groups. The hNIS-expressing tumor tissue accumulated 99mTcO4 rapidly within 30 min of it being intraperitoneally injected. The experiments demonstrated that effective 131I therapy was achieved in the malignant glioma cell lines following the induction of tumor-specific iodide uptake activity by GFAP promoter-directed hNIS gene expression in vitro and in vivo. 131I therapy retarded Ad-GFAP-hNIS transfected-tumor growth following injection with 131I in U87 xenograft-bearing nude mice.

Cotransfected Sodium Iodide Symporter and Human Tyroperoxidase Genes Following Human Telomerase Reverse Transcriptase Promoter for Targeted Radioiodine Therapy of Malignant Glioma Cells

INTRODUCTION Radioiodine is a routine therapy for differentiated thyroid cancers. In principle, undifferentiated thyroid cancers as well as nonthyroid cancers can concentrate and, thus, be treated with radioiodine after transfection with the human sodium iodide symporter (hNIS) gene. The human telomerase reverse transcriptase (hTERT) promoter is an effective tumor-specific promoter of gene expression and, thus, may be useful in targeted gene therapy of cancer. METHODS We used hTERT promoter-modulated expression of the hNIS and human thyroperoxidase (hTPO) genes in an experimental model of radioiodine-based treatment of malignant glioma. Cells were cotransfected by adenovirus in which the hNIS gene had been coupled to the hTERT promoter and the hTPO gene had been coupled to the human cytomegalovirus (CMV) promoter (Ad-hTERT-hNIS and Ad-CMV-hTPO, respectively), and they were evaluated in cells thus transfecting transgene expression by western blots, (125)I uptake and influx, and clonogenecity after (131)I treatment. RESULTS After cotransfection with two adenovirus, cells showed about 31-34 times higher (125)I uptake than the control cells transfected with Ad-CMV-EGFP (enhanced green fluorescent protein) and almost 1.3-1.4 times higher (125)I uptake than cells only transfected with Ad-hTERT-hNIS. Western blots revealed two bands of ∼70 and 110 kDa, respectively. The in vitro clonogenic assay indicated that, after exposure to 100-1000 μCi of (131)I-iodide for 12 hours, 91%-94% of cells cotransfected with the hNIS and hTPO genes, 88%-93% of cells transfected with the hNIS gene, and only 62%-68% of control (nontransfected) cells were killed. CONCLUSIONS The experiments demonstrated that an effective therapy of (131)I was achieved in malignant glioma cell lines after induction of tumor-specific iodide uptake activity by the hTERT promoter-directed NIS expression in vitro. Cotransfection of the hNIS and hTPO genes can lead to longer retention of radioiodide, but did not increase cell killing over that achieved with transfection with the hNIS gene alone.

131I-labeled and DOX-loaded multifunctional nanoliposomes for radiotherapy and chemotherapy in brain gliomas

The codelivery of different therapeutics is a promising option because of its synergetic effects of drugs. In this study, a new combination therapy that used the doxorubicin-loaded and 131I-labeled nanoliposomes (131I-DOX-NL) was proposed to delay tumor growth of gliomas, which are characterized by significant mortality and morbidity. 131I-DOX-NL was constructed based on bovine serum albumin (BSA)-tailor made hydrophobic maleimide-functionalized poly(ε-caprolactone) (PCL) (Fig. 1) and was evaluated by cellular viability in vitro and by U87 xenograft models in vivo. Compared with using 131I-NL or DOX-NL alone, our experimental results show that 131I-DOX-NL exhibits similar high cellular uptake but enhanced efficacy to cure gliomas because of its codelivery of 131I and DOX. In the U87 mouse tumor models, the combination therapy resulted in higher survival rates of mice and smaller tumor sizes than monotherapy did alone. In conclusion, multifunctional nanoliposome 131I-DOX-NL is a good candidate for the codelivery of 131I-mediated radiotherapy and DOX-mediated chemotherapy due to its ability to inhibit U87 cell proliferation and tumor growth. 131I-DOX-NL can be used as a promising effective therapy for malignant gliomas and deserves further investigation.

Effect of alkylglycerone phosphate synthase on the expression profile of circRNAs in the human thyroid cancer cell line FRO

Thyroid cancer is a common primary tumor in China. Therefore, it is important to investigate the underlying molecular mechanism of thyroid cancer in order to achieve effective individualized treatments. In our previous study, a positive correlation between the expression of alkylglycerone phosphate synthase (AGPS) and the malignant phenotype of thyroid cancer cell lines was identified. The inactivation of AGPS was able to decrease the malignancy of cancer, and inhibit tumor growth and invasion. However, the function of AGPS on thyroid cancer was unclear. In the present study, it was revealed that AGPS was able to regulate the expression of circular RNAs (circRNAs), which may be the mechanism of its anticancer activity. Therefore, the effects of AGPS silencing and knockout on circRNA expression in the thyroid cancer cell line FRO were investigated using circRNAs microarray, and Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses were performed in order to investigate the underlying molecular mechanism of AGPS for the regulation of thyroid cancer through circRNAs.

Therapy of cervical cancer using 131I-labeled nanoparticles

Objective To evaluate the effectiveness of two kinds of Arg-Gly-Asp (RGD)-targeted 131I-containing nanoliposomes for the treatment of cervical cancer in vitro and in vivo. Methods The nanoparticle liposomes designated RGD-131I-tyrosine peptide chain (TPC)-L and 131I-RGD-L were prepared. The emulsion solvent evaporation method was used to encapsulate the polypeptide into liposomes. The quantity of entrapped polypeptide was measured using UV spectrophotometry. The labeling rates, radiochemical purities, and total radioactivities were measured using paper chromatography. Cytotoxicity was assessed using the MTS assay and flow cytometry. Therapeutic efficacy was monitored using a mouse xenograft model of cervical cancer. Results The labeling efficiency, radiochemical purity, and specific radioactivity of RGD-131I-TPC-L were greater than those of 131I-RGD-L. The cytotoxicity test indicated that late apoptosis of cells treated with RGD-131I-TPC-L and 131I-RGD-L was higher than that of cells treated with Na131I. The therapeutic effect of RGD-131I-TPC-L was better than that of 31I-RGD-L in the mouse model. Conclusions The specific activity of liposome-encapsulated RGD-131I-TPC-L was higher than that of 131I-RGD-L, which labeled liposomes directly. Moreover, the RGD-131I-TPC-L liposomes were more effective for killing xenografted tumor cells.