Fu-Qiang Hu

Selective redox-responsive drug release in tumor cells mediated by chitosan based glycolipid-like nanocarrier

The redox responsive nanocarriers have made a considerable progress in achieving triggered drug release by responding to the endogenous occurring difference between the extra- and intra- cellular redox environments. Despite the promises, this redox difference exists both in normal and tumor tissue. So a non-selective redox responsive drug delivery system may result in an undesired drug release in normal cells and relevant side-effects. To overcome these limitations, we have developed a chitosan based glycolipid-like nanocarrier (CSO-ss-SA) which selectively responded to the reducing environment in tumor cells. The CSO-ss-SA showed an improved reduction-sensitivity which only fast degraded and released drug in 10mM levels of glutathione (GSH). The CSO-ss-SA could transport the drug fast into the human ovarian cancer SKOV-3 cells and human normal liver L-02 cells by internalization, but only fast release drug in SKOV-3 cells. By regulating the intracellular GSH concentration in SKOV-3 cells, it indicated that the cellular inhibition of the PTX-loaded CSO-ss-SA showed a positive correlation with the GSH concentration. The CSO-ss-SA was mainly located in the liver, spleen and tumor in vivo, which evidenced the passive tumor targeting ability. Despite the high uptake of liver and spleen, drug release was mainly occurred in tumor. PTX-loaded CSO-ss-SA achieved a remarkable tumor growth inhibition effect with rather low dose of PTX. This study demonstrates that a smartly designed glycolipid-like nanocarrier with selective redox sensitivity could serve as an excellent platform to achieve minimal toxicity and rapid intracellular drug release in tumor cells.

Novel micelle formulation of curcumin for enhancing antitumor activity and inhibiting colorectal cancer stem cells

Background and methods: Curcumin has extraordinary anticancer properties but has limited use due to its insolubility in water and instability, which leads to low systemic bioavailability. We have developed a novel nanoparticulate formulation of curcumin encapsulated in stearic acid-g-chitosan oligosaccharide (CSO-SA) polymeric micelles to overcome these hurdles. Results: The synthesized CSO-SA copolymer was able to self-assemble to form nanoscale micelles in aqueous medium. The mean diameter of the curcumin-loaded CSO-SA micelles was 114.7 nm and their mean surface potential was 18.5 mV. Curcumin-loaded CSO-SA micelles showed excellent internalization ability that increased curcumin accumulation in cancer cells. Curcumin-loaded CSO-SA micelles also had potent antiproliferative effects on primary colorectal cancer cells in vitro, resulting in about 6-fold greater inhibition compared with cells treated with a solution containing an equivalent concentration of free curcumin. Intravenous administration of curcumin-loaded CSO-SA micelles marginally suppressed tumor growth but did not increase cytotoxicity to mice, as confirmed by no change in body weight. Most importantly, curcumin-loaded CSO-SA micelles were effective for inhibiting subpopulations of CD44+/CD24+ cells (putative colorectal cancer stem cell markers) both in vitro and in vivo. Conclusion: The present study identifies an effective and safe means of using curcumin-loaded CSO-SA micelles for cancer therapy.

Redox-responsive polymer-drug conjugates based on doxorubicin and chitosan oligosaccharide-g-stearic acid for cancer therapy.

Here, a biodegradable polymer-drug conjugate of doxorubicin (DOX) conjugated with a stearic acid-grafted chitosan oligosaccharide (CSO-SA) was synthesized via disulfide linkers. The obtained polymer-drug conjugate DOX-SS-CSO-SA could self-assemble into nanosized micelles in aqueous medium with a low critical micelle concentration. The size of the micelles was 62.8 nm with a narrow size distribution. In reducing environments, the DOX-SS-CSO-SA could rapidly disassemble result from the cleavage of the disulfide linkers and release the DOX. DOX-SS-CSO-SA had high efficiency for cellular uptake and rapidly released DOX in reductive intracellular environments. In vitro antitumor activity tests showed that the DOX-SS-CSO-SA had higher cytotoxicity against DOX-resistant cells than free DOX, with reversal ability up to 34.8-fold. DOX-SS-CSO-SA altered the drug distribution in vivo, which showed selectively accumulation in tumor and reduced nonspecific accumulation in hearts. In vivo antitumor studies demonstrated that DOX-SS-CSO-SA showed efficient suppression on tumor growth and relieved the DOX-induced cardiac injury. Therefore, DOX-SS-CSO-SA is a potential drug delivery system for safe and effective cancer therapy.

Brain-targeting study of stearic acid–grafted chitosan micelle drug-delivery system

Purpose Therapy for central nervous system disease is mainly restricted by the blood–brain barrier. A drug-delivery system is an effective approach to overcome this barrier. In this research, the potential of polymeric micelles for brain-targeting drug delivery was studied. Methods Stearic acid–grafted chitosan (CS-SA) was synthesized by hydrophobic modification of chitosan with stearic acid. The physicochemical characteristics of CS-SA micelles were investigated. bEnd.3 cells were chosen as model cells to evaluate the internalization ability and cytotoxicity of CS-SA micelles in vitro. Doxorubicin (DOX), as a model drug, was physically encapsulated in CS-SA micelles. The in vivo brain-targeting ability of CS-SA micelles was qualitatively and quantitatively studied by in vivo imaging and high-performance liquid chromatography analysis, respectively. The therapeutic effect of DOX-loaded micelles in vitro was performed on glioma C6 cells. Results The critical micelle concentration of CS-SA micelles with 26.9% ± 1.08% amino substitute degree was 65 μg/mL. The diameter and surface potential of synthesized CS-SA micelles in aqueous solution was 22 ± 0.98 nm and 36.4 ± 0.71 mV, respectively. CS-SA micelles presented excellent cellular uptake ability on bEnd.3 cells, the IC50 of which was 237.6 ± 6.61 μg/mL. DOX-loaded micelles exhibited slow drug-release behavior, with a cumulative release up to 72% within 48 hours in vitro. The cytotoxicity of DOX-loaded CS-SA micelles against C6 was 2.664 ± 0.036 μg/mL, compared with 0.181 ± 0.066 μg/mL of DOX · HCl. In vivo imaging results indicated that CS-SA was able to transport rapidly across the blood–brain barrier and into the brain. A maximum DOX distribution in brain of 1.01%/g was observed 15 minutes after administration and maintained above 0.45%/g within 1 hour. Meanwhile, free DOX · HCl was not detected in brain. In other major tissues, DOX-loaded micelles were mainly distributed into lung, liver, and spleen, with a reduction of DOX accumulation in heart. Conclusion The CS-SA micelles were able to be used as a promising carrier for a braintargeting drug delivery system.

Preparation and pharmacodynamics of stearic acid and poly (lactic-co-glycolic acid) grafted chitosan oligosaccharide micelles for 10-hydroxycamptothecin.

Stearic acid (SA) and poly (lactic-co-glycolic acid) (PLGA) grafted chitosan oligosaccharide (SA-CSO-PLGA SCP) tripolymer was synthesized via the reaction between the carboxyl group of SA or PLGA with carboxylic side group, and the amine group of CSO in the presence of 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC). The degrees of amino-substitution for SA and PLGA were assayed through 2, 4, 6-trinitrobenzene sulfonic acid (TNBS) test and (13)C NMR spectrum, which were 8.15% and 5.82%, respectively; the critical micelle concentrations of SCP in PBS (pH 7.4) and deionized water (DI water) were about 34.9 and 14.5 microg/ml, respectively. Using 10-hydroxycamptothecin (HCPT) as a model drug, the drug-loaded micelles showed above 86% encapsulation efficiency, which not only enhanced the solubility of HCPT in aqueous medium markedly, but also protected the lactone ring of HCPT. Cellular uptakes of SCP micelles against A549, MCF-7 and HepG-2 tumor cells showed a faster cellular internalization. Comparing to the commercial HCPT injection, HCPT-loaded micelles showed higher cytotoxicities against A549, MCF-7 and HepG-2 cells. The increased folds were 22, 18 and 15, respectively. These results suggested the SCP could be applied as a carrier for hydrophobic drugs.

Receptor-mediated gene delivery by folic acid-modified stearic acid-grafted chitosan micelles

Background Cationic polymers have been accepted as effective nonviral vectors for gene delivery with low immunogenicity unlike viral vectors. However, the lack of organ or cell specificity sometimes hampers their application and the modification of polymeric vectors has also shown successful improvements in achieving cell-specific targeting delivery and in promoting intracellular gene transfer efficiency. Methods A folic acid-conjugated stearic acid-grafted chitosan (FA-CS-SA) micelle, synthesized by a 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide-coupling reaction, was designed for specific receptor-mediated gene delivery. Results Due to the cationic properties of chitosan, the micelles could compact the plasmid DNA (pDNA) to form micelle/pDNA complexes nanoparticles. The particle size and zeta potential of the FA-CS-SA/pDNA complexes with different N/P ratios were 100–200 nm and −20 to −10 mV, respectively. The DNase I protection assay indicated that the complexes can efficiently protect condensed DNA from enzymatic degradation by DNase I. A cytotoxicity study indicated that the micelles exhibited less toxicity in comparison with LipofectamineTM 2000. Using SKOV3 and A549 as model tumor cells, the cellular uptake of micelles was investigated. Conclusion It was found that cellular uptake of FA-CS-SA in SKOV3 cells with higher folate receptor expression was faster than that in A549 cells with a short incubation time. Luciferase assay and green fluorescent protein detection were used to confirm that FA-CS-SA could be an effective gene vector. Transfection efficiency of the FA-CS-SA/pDNA complexes in SKOV3 cells was enhanced up to 2.3-fold compared with that of the CS-SA/pDNA complexes. However, there was no significant difference between the transfection efficiencies of the two complexes in A549 cells. Importantly, the transfection efficiency of FA-CS-SA/pDNA decreased with free FA pretreatment in SKOV3 cells. It was concluded that the increase in transfection efficiency of the FA-CS-SA/pDNA complexes was attributed to folate receptor-mediated endocytosis.

Oxaliplatin-incorporated micelles eliminate both cancer stem-like and bulk cell populations in colorectal cancer

Purpose The failure of cancer treatments is partly due to the enrichment of cancer stem-like cells (CSLCs) that are resistant to conventional chemotherapy. A novel micelle formulation of oxaliplatin (OXA) encapsulated in chitosan vesicle was developed. The authors postulate that micelle encapsulation of OXA would eliminate both CSLCs and bulk cancer cells in colorectal cancer (CRC). Experimental design In this study, using stearic acid-g-chitosan oligosaccharide (CSO-SA) polymeric micelles as a drug-delivery system, OXA-loaded CSO-SA micelles (CSO-SA/OXA) were prepared. Intracellular uptake of CSO-SA/OXA micelles was assessed by confocal microscope. The effects of free OXA, the empty carrier, and CSO-SA/OXA micelles were tested using human CRC cell lines in vitro and in vivo. Results The micelles showed excellent internalization ability that increased OXA accumulation both in CRC cells and tissues. Furthermore, CSO-SA/OXA micelles could either increase the cytotoxicity of OXA against the bulk cancer cells or reverse chemoresistance of CSLC subpopulations in vitro. Intravenous administration of CSO-SA/OXA micelles effectively suppressed the tumor growth and reduced CD133+/CD24+ cell (putative CRC CSLC markers) compared with free OXA treatment, which caused CSLC enrichment in xenograft tumors (P < 0.05). Conclusion The results of this study indicate that CSO-SA micelle as a drug-delivery carrier is effective for eradicating CSLCs and may act as a new option for CRC therapy.

Improved cytotoxicity and multidrug resistance reversal of chitosan based polymeric micelles encapsulating oxaliplatin.

To overcome the side effects and drug resistance in cancer chemotherapy, oxaliplatin (OXA) was encapsulated in chitosan based polymeric micelles with glycolipid-like structure, which were formed by stearic acid-grafted chitosan oligosaccharide (CSO-SA). CSO-SA with 6.89% amino substituted degree was synthesized in this paper. The critical micelle concentration was about 0.12 mg/mL. CSO-SA micelles with the concentration of 1.0 mg/mL had 34.8 nm number average diameter and +50.8 mV surface potential in the aqueous medium. Thin-film dispersed method mediated by lecithin was chosen to prepare OXA-loaded CSO-SA micelles (CSO-SA/OXA), encapsulation efficiency of which could reach up to about 47%. In vitro anti-tumor activity of CSO-SA/OXA micelles against drug sensitive tumor cells and drug resistant cells was then examined. Using SGC-7901, SKOV3, BEL-7402, K562, and MCF-7 as model drug sensitive tumor cells, the 50% inhibition of cellular growth (IC50) of CSO-SA/OXA micelles could be lowered about 3-6 folds compared to that of free OXA solution. Furthermore, cytotoxicity test of CSO-SA/OXA micelles against MCF-7 and multidrug resistant MCF-7 (MCF-7/Adr) cells presented the reversal activity against MCF-7/Adr cells. The present micelles are a promising carrier candidate for platinum drug to improve the anti-tumor activity.

Preparation and evaluation of SiO2-deposited stearic acid-g-chitosan nanoparticles for doxorubicin delivery

Purpose: Both polymer micelles and mesoporous silica nanoparticles have been widely researched as vectors for small molecular insoluble drugs. To combine the advantages of copolymers and silica, studies on the preparation of copolymer-silica composites and cellular evaluation were carried out. Methods: First, a stearic acid-g-chitosan (CS-SA) copolymer was synthesized through a coupling reaction, and then silicone oxide (SiO2)-deposited doxorubicin (DOX)-loaded stearic acid-g-chitosan (CS-SA/SiO2/DOX) nanoparticles were prepared through the sol-gel reaction. Physical and chemical properties such as particle size, zeta potential, and morphologies were examined, and small-angle X-ray scattering (SAXS) analysis was employed to identify the mesoporous structures of the generated nanoparticles. Cellular uptake and cytotoxicity studies were also conducted. Results: CS-SA/SiO2/DOX nanoparticles with different amounts of SiO2 deposited were obtained, and SAXS studies showed that mesoporous structures existed in the CS-SA/SiO2/DOX nanoparticles. The mesoporous size of middle-ratio and high-ratio deposited CS-SA/SiO2/DOX nanoparticles were 4–5 nm and 8–10 nm, respectively. Based on transmission electron microscopy images of CS-SA/SiO2/DOX nanoparticles, dark rings around the nanoparticles could be observed in contrast with CS-SA/DOX micelles. Furthermore, CS-SA/SiO2/DOX nanoparticles exhibited faster release behavior in vitro than CS-SA/DOX micelles; cellular uptake research in A549 indicated that the CS-SA/SiO2/DOX nanoparticles were taken up by A549 cells more rapidly, and that CS-SA/SiO2/DOX nanoparticles entered the cell more easily when the amount of SiO2 was higher. IC50 values of CS-SA/DOX micelles, CS-SA/SiO2/DOX-4, CS-SA/SiO2/DOX-8, and CS-SA/SiO2/DOX-16 nanoparticles against A549 cells measured using the MTT assay were 1.69, 0.93, 0.32, and 0.12 μg/mL, respectively. Conclusion: SiO2-deposited stearic acid-g-chitosan organic–inorganic composites show promise as nanocarriers for hydrophobic drugs such as DOX.

Efficient gene delivery system mediated by cis-aconitate-modified chitosan-g-stearic acid micelles.

Cis-aconitate-modified chitosan-g-stearic acid (CA-CSO-SA) micelles were synthesized in this study to improve the gene transfection efficiency of chitosan-g-stearic acid (CSO-SA). The CA-CSO-SA micelles had a similar size, critical micelle concentration, and morphology, but their zeta potential and cytotoxicity were reduced compared with CSO-SA micelles. After modification with cis-aconitate, the CA-CSO-SA micelles could also compact plasmid DNA (pDNA) to form nanocomplexes. However, the DNA binding ability of CA-CSO-SA was slightly reduced compared with that of CSO-SA. The transfection efficiency mediated by CA-CSO-SA/pDNA against HEK-293 cells reached up to 37%, and was much higher than that of CSO-SA/pDNA (16%). Although the cis-aconitate modification reduced cellular uptake kinetics in the initial stages, the total amount of cellular uptake tended to be the same after 24 hours of incubation. An endocytosis inhibition experiment showed that the internalization mechanism of CA-CSO-SA/pDNA in HEK-293 cells was mainly via clathrin-mediated endocytosis, as well as caveolae-mediated endocytosis and macropinocytosis. Observation of intracellular trafficking indicated that the CSO-SA/pDNA complexes were trapped in endolysosomes, but CA-CSO-SA/pDNA was more widely distributed in the cytosol. This study suggests that modification with cis-aconitate improves the transfection efficiency of CSO-SA/pDNA.