Ming-Feng Wei

Aptamer-based tumor-targeted drug delivery for photodynamic therapy.

A specialized G-rich DNA structure, G-quadruplex, has been studied for its special physical characteristics and biological effects. Herein we report a novel strategy of using G-quadruplex as a drug carrier to target cancer cells for photodynamic therapy (PDT). A G-quadruplex forming AS1411 aptamer could be physically conjugated with six molecules of porphyrin derivative, 5,10,15,20-tetrakis(1-methylpyridinium-4-yl)porphyrin (TMPyP4), to fabricate the apt-TMP complex. The TMPyP4 molecules in the complex were identified to bind tightly to the aptamer by intercalation and outside binding. Because the G-quadruplex structure is known to target the overexpressed nucleolin in cancer cells, in this study, the effect of the G-quadruplex structure as a carrier for the delivery of TMPyP4 into cancer cells by nucleolin-mediated internalization was investigated. The results showed that the apt-TMP complex exhibited a higher TMPyP4 accumulation in MCF7 breast cancer cells than in M10 normal epithelium cells. After treated with light for 180 s, the photodamage in MCF7 cells was larger than in M10 cells. These results indicated that the TMPyP4 delivery and uptake were mediated by the specific interaction of the apt-TMP complex with nucleolin on the cellular surface and that the use of the AS1411 aptamer as a drug carrier may be a potential tactic in cancer therapy.

Folic Acid-Conjugated Chitosan Nanoparticles Enhanced Protoporphyrin IX Accumulation in Colorectal Cancer Cells

Folic acid can be covalently conjugated to chitosan molecules via its gamma-carboxyl moiety and thus retain a high affinity for colorectal cancer cells bearing folate receptor overexpression. Colorectal cancer is one of the leading causes of malignant death and often goes undetected with current colonoscopy practices. Improved methods of detecting dysplasia and tumors during colonoscopy will improve mortality. A folic acid conjugated chitosan nanoparticle as a suitable vehicle for carrying 5-aminolaevulinic acid (5-ALA) is developed to enhance the detection of colorectal cancer cells in vivo after a short-term uptake period. Chitosan can be successfully conjugated with folic acid to produce folic acid-chitosan conjugate, which is then loaded with 5-ALA to create nanoparticles (fCNA). The loading efficiency of 5-ALA in fCNA particles and the z-average diameter were in the range 35-40% and 100 nm, respectively. The zeta-potential for fCNA was 20 mV, enough to keep the nanoparticle stable without aggregation. The fCNA is then incubated with HT29 and Caco-2 colorectal cancer cell lines overexpressing folate receptor on the surface of the cell membrane to determine the rate of accumulation of protoporphyrin IX (PpIX). The results show that fCNA can be taken up more easily by HT29 and Caco-2 cell lines after short-term uptake period, most likely via receptor-mediated endocytosis, and the PpIX accumulates in cancer cells as a function of the folate receptor expression and the folic acid modification. Therefore, the folic acid-chitosan conjugate appears to be an ideal vector for colorectal-specific delivery of 5-ALA for fluorescent endoscopic detection.

Autophagy promotes resistance to photodynamic therapy-induced apoptosis selectively in colorectal cancer stem-like cells

Recent studies have indicated that cancer stem-like cells (CSCs) exhibit a high resistance to current therapeutic strategies, including photodynamic therapy (PDT), leading to the recurrence and progression of colorectal cancer (CRC). In cancer, autophagy acts as both a tumor suppressor and a tumor promoter. However, the role of autophagy in the resistance of CSCs to PDT has not been reported. In this study, CSCs were isolated from colorectal cancer cells using PROM1/CD133 (prominin 1) expression, which is a surface marker commonly found on stem cells of various tissues. We demonstrated that PpIX-mediated PDT induced the formation of autophagosomes in PROM1/CD133+ cells, accompanied by the upregulation of autophagy-related proteins ATG3, ATG5, ATG7, and ATG12. The inhibition of PDT-induced autophagy by pharmacological inhibitors and silencing of the ATG5 gene substantially triggered apoptosis of PROM1/CD133+ cells and decreased the ability of colonosphere formation in vitro and tumorigenicity in vivo. In conclusion, our results revealed a protective role played by autophagy against PDT in CSCs and indicated that targeting autophagy could be used to elevate the PDT sensitivity of CSCs. These findings would aid in the development of novel therapeutic approaches for CSC treatment.

Colorectal cancer cell detection by 5-aminolaevulinic acid-loaded chitosan nano-particles

Colorectal cancer is one of the leading causes of malignant death in Taiwan because it often remains undetected until later stages of the disease. In this study, we designed an oral form nano-particle to encapsulate 5-aminolaevulinic acid (5-ALA) to improve the detection of colorectal cancer cells in vivo. The nano-particle should escape from bacteria uptake in the gastrointestinal tract which seriously interferes the results of endoscopic observation. In this study, chitosan was mixed with sodium tripolyphosphate (STPP) and 5-ALA to prepare chitosan nano-particles (CN) and 5-ALA loaded chitosan nano-particles (CNA) by adding different pH values and concentrations of 5-ALA solution. The average particle size and zeta-potential of CN and CNA were measured by the Zetasizer-3000. The results revealed that particle size with different zeta-potential could be manipulated just by 5-ALA concentrations and pH values. CNA particles prepared at pH 7.4 and pH 9 of 5-ALA solutions with a concentration higher than 0.5 mg/ml showed a promising loading efficiency of up to 75% and an optimum average particle size of 100 nm. The zeta-potential for CNA was over 30 mV that kept the nano-particle stable without aggregation when stored in suspension solution. Fluorescence microscope examination showed that CNA could be engulfed by Caco-2 colon cancer cells but showed no evidence of being taken up by Escherichia coli. This result implies that CNA could exclude the influence of normal flora inside the gut and serves as an adequate tool for fluorescent endoscopic detection of colorectal cancer cells in vivo.

The use of polyethylenimine–DNA to topically deliver hTERT to promote hair growth

The present study investigates the efficacy of polyethylenimine (PEI)–DNA complex that expressed human telomerase reverse transcriptase (hTERT) to transfect hair follicle stem cells and produce sufficient hTERT to stimulate hair growth. Transfection with pLC–hTERT–DNA–PEI complex (D+P group) in vitro induced expression of proliferating cell nuclear antigen in 35.8% of the purified stem cell population, suggesting enhanced cell proliferation. In vivo transfection efficiency of rat dorsal skin was determined by staining for β-gal activity. Cells positive for β-gal were located in the bulge region and dermal sheath of hair follicles. The follicles in the hTERT-transfected region entered anagenon day 15 after transfection, whereas non-transfected (Neg) controls remained in telogen. The similar effect was observed in 50-day-old rat dorsal skin. D+P group displayed a specific expression of hTERT and sufficient to initiate a transition to the anagen phase and promote new hair synthesis 18 days after the transfection. hTERT promoted follicle neogenesis following wounding. In all, 60 days after wounding, tissues of the D+P group showed more newly regenerating hair follicles (83±52 regenerated follicles per rat) in contrast to control group tissues (15±15 regenerated follicles per rat). These studies provide a potential approach for gene therapy of skin disease.

Targeting Colorectal Cancer Stem-Like Cells with Anti-CD133 Antibody-Conjugated SN-38 Nanoparticles

Cancer stem-like cells play a key role in tumor development, and these cells are relevant to the failure of conventional chemotherapy. To achieve favorable therapy for colorectal cancer, PEG-PCL-based nanoparticles, which possess good biological compatibility, were fabricated as nanocarriers for the topoisomerase inhibitor, SN-38. For cancer stem cell therapy, CD133 (prominin-1) is a theoretical cancer stem-like cell (CSLC) marker for colorectal cancer and is a proposed therapeutic target. Cells with CD133 overexpression have demonstrated enhanced tumor-initiating ability and tumor relapse probability. To resolve the problem of chemotherapy failure, SN-38-loaded nanoparticles were conjugated with anti-CD133 antibody to target CD133-positive (CD133(+)) cells. In this study, anti-CD133 antibody-conjugated SN-38-loaded nanoparticles (CD133Ab-NPs-SN-38) efficiently bound to HCT116 cells, which overexpress CD133 glycoprotein. The cytotoxic effect of CD133Ab-NPs-SN-38 was greater than that of nontargeted nanoparticles (NPs-SN-38) in HCT116 cells. Furthermore, CD133Ab-NPs-SN-38 could target CD133(+) cells and inhibit colony formation compared with NPs-SN-38. In vivo studies in an HCT116 xenograft model revealed that CD133Ab-NPs-SN-38 suppressed tumor growth and retarded recurrence. A reduction in CD133 expression in HCT116 cells treated with CD133Ab-NPs-SN-38 was also observed in immunohistochemistry results. Therefore, this CD133-targeting nanoparticle delivery system could eliminate CD133-positive cells and is a potential cancer stem cell targeted therapy.

A variable gene delivery carrier—biotinylated chitosan/polyethyleneimine

A variable gene delivery system has been developed based on conjugating chitosan to biotin through a functionalized poly(ethylene glycol) (PEG) spacer, which can be used to further bind different molecules on the outer layer of a polymer/DNA complex by streptavidin (SA)-biotin linkage. In this study, TAT-conjugated SA was used as the model molecule to prove the conjugation function of the prepared complex. In addition, low-molecular-weight poly(ethyleneimine) (PEI) was added into the polymer/DNA complex to increase the transfection efficiency. The results of the luciferase assay show that the transfection efficiency of the prepared complex was significantly correlated with the amount of PEI and was further enhanced when TAT was conjugated to the complex by SA-biotin linkage. Considered to have negligible cytotoxic effects, the variable gene delivery complex prepared in this study would be of considerable potential as carriers for in vitro applications.

Imidazole/poly(Ethylene glycol)-substituted poly(n-(8-aminooctyl)acrylamide) as biocompatible and efficient gene carrier.

A novel cationic polymer was developed by conjugating imidazole and poly(ethylene glycol) (PEG) on poly(N-(8-aminooctyl)acrylamide) (P8Am) for complexing with pDNA to exhibit high gene expression with low cytotoxicity and the resistance against erythrocyte agglutination and serum inhibition. Cytotoxicity results indicated that these P8Am derivatives with varying substitutions were more biocompatible than unmodified P8Am and PEI control. Moreover, the particle size and zeta potential experiment demonstrated that they were capable of complexing pDNA into sub-micrometer (135–625 nm) and positively charged (+10 to +43 mV) particles, while the high degree of substitution might impede their pDNA complexation ability that formed less positive and larger polyplexes. Flow cytometry analysis demonstrated that the cellular uptake efficiency was dependent on the degree of substitution; low degree of substitution would mediate high uptake efficiency. The gene-transfection ability, evaluated by luciferase assay, revealed low substitution in P8Am-IM11 (substituted with 11 mol% imidazole moieties) and P8Am-PG7 (substituted with 7 mol% PEG moieties) in transfected cells more efficient than unmodified P8Am. Therefore, a multi-functional P8Am derivative, P8Am-IM11-PG7, containing both imidazole and PEG, was developed according to the optimized contents. In the presence of serum, P8Am-IM11-PG7 polyplexes significantly enhanced the gene-transfection efficiency relative to unmodified P8Am polyplexes. Moreover, they exhibited minimal cytotoxicity and the erythrocyte aggregation assay showed that P8Am-IM11-PG7 polyplexes had good blood compatibility as compared to P8Am and PEI polyplexes. This indicated that, by chemical modification, P8Am-IM11-PG7 could possess the required abilities to overcome the difficulties encountered in gene transfection and be a promising alternative of a gene carrier.

PEGylated Guanidinylated Polyallylamine as Gene-Delivery Carrier

A novel cationic co-polymer was developed by grafting poly(ethylene glycol) (PEG) on guanidinylated polyallylamine (PAA) for gene delivery. Characterization of PEG-g-guanidinylated PAA/DNA complexes demonstrated that particle size increased and surface charge decreased with increasing the amount of PEG. The results of cytotoxicity assay proved that grafted PEG could effectively decrease the cytotoxicity of the complexes. In transfection efficiency assay, HeLa cells treated with PEG(2)-g-guanidinylated PAA (formed with 17.5 μmol guanidinylated PAA and 2 μmol PEG)/DNA (0.2 μg EGFP plasmid) complexes showed a very high level of EGFP expression. In conclusion, combination of guanidinylation and PEGylation could effectively decrease the cytotoxicity and significantly increase the transfection efficiency of PAA.