Yong-Zhong Du

Synthesis and antitumor activity of doxorubicin conjugated stearic acid-g-chitosan oligosaccharide polymeric micelles

Doxorubicin conjugated stearic acid-g-chitosan oligosaccharide polymeric micelles (DOX-CSO-SA) was synthesized via cis-aconityl bond between the anticancer drug doxorubicin (DOX) and stearic acid grafted chitosan oligosaccharide (CSO-SA) in this paper. The CSO-SA micelles had been demonstrated faster internalization ability into tumor cells. Here, the CSO-SA with 6.47% amino substituted degree (SD%) was used to synthesize DOX-CSO-SA. The critical micelle concentration (CMC) was about 0.14 mg mL(-1). The micelles with 1 mg mL(-1) CSO-SA concentration had 32.7 nm number average diameter with a narrow size distribution and 51.5 mV surface potential. After conjugating with doxorubicin, CMC of DOX-CSO-SA descended; the micellar size increased; and the zeta potential decreased. The DOX-CSO-SA micelles indicated pH-dependent DOX release behavior. The release rate of DOX from DOX-CSO-SA micelles increased significantly with the reductions of the pH for release medium from 7.2 to 5.0. In vitro antitumor activity tests of DOX-CSO-SA micelles against human breast carcinoma (MCF-7) cells and their multi-drug resistant (MCF-7/Adr) cells presented the reversal activity against DOX resistance MCF-7 cells (MCF-7/Adr). The in vivo antitumor activity results showed that DOX-CSO-SA micelles treatments effectively suppressed the tumor growth and reduced the toxicity against animal body than commercial doxorubicin hydrochloride injection.

Synthesis and antitumor activity of stearate-g-dextran micelles for intracellular doxorubicin delivery.

Stearate-g-dextran (Dex-SA) was synthesized via an esterification reaction between the carboxyl group of stearic acid (SA) and hydroxyl group of dextran (Dex). Dex-SA could self-assemble to form nanoscaled micelles in aqueous medium. The critical micelle concentration (CMC) depended on the molecular weight of Dex and the graft ratio of SA, which ranged from 0.01 to 0.08 mg mL(-1). Using doxorubicin (DOX) as a model drug, the drug encapsulation efficiency (EE%) using Dex-SA with 10 kDa molecular weight of Dex and 6.33% graft ratio of SA could reach up to 84%. In vitro DOX release from DOX-loaded Dex-SA micelles (Dex-SA/DOX) could be prolonged to 48 h, and adjusted by a different molecular weight of Dex, the graft ratio of SA, or the drug-loading content. Tumor cellular uptake test indicated that Dex-SA micelles had excellent internalization ability, which could deliver DOX into tumor cells. In vitro cytotoxicity tests demonstrated the Dex-SA/DOX micelles could maintain the cytotoxicity of commercial doxorubicin injection against drug-sensitive tumor cells. Moreover, Dex-SA/DOX micelles presented reversal activity against DOX-resistant cells. In vivo antitumor activity results showed that Dex-SA/DOX micelles treatments effectively suppressed the tumor growth and reduced the toxicity against animal body compared with commercial doxorubicin injection.

Reversal activity of nanostructured lipid carriers loading cytotoxic drug in multi-drug resistant cancer cells.

To overcome multi-drug resistance (MDR) of cancer cells, paclitaxel (PTX) and doxorubicin (DOX)-loaded nanostructured lipid carriers (NLC) were prepared by solvent diffusion method using monostearin as solid lipid and oleic acid as liquid lipid matrix. The cytotoxicities and reversal activity of drug-loaded NLC were tested against human breast cancer (MCF-7) cells, human ovarian cancer (SKOV3) cells and their multi-drug resistant (MCF-7/ADR and SKOV3-TR30) cells. The chemical conjugant of folic acid and stearic acid (FA-SA) was further synthesized to prepare folated NLC. Comparing with taxol and doxorubicin solution, the NLC loading PTX exhibited high cytotoxicities in MCF-7 and MCF-7/ADR cells, while the NLC loading DOX only indicated high cytotoxicity in MCF-7/ADR cells. The reversal powers of the NLC loading PTX and DOX were 34.3 and 6.4 folds, respectively. The NLC loading PTX and DOX showed the same trends of enhanced cytotoxicity against SKOV3 and SKOV3-TR30 cells. The reversal powers were 31.3 and 2.2 folds for the NLC loading PTX and DOX, respectively. The modification of NLC with FA-SA could further enhance the cytotoxicities of drug in drug sensitive and drug resistant cells.

Preparation and characteristics of linoleic acid-grafted chitosan oligosaccharide micelles as a carrier for doxorubicin

The linoleic acid (LA)-grafted chitosan oligosaccharide (CSO) (CSO-LA) was synthesized in the presence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), and the effects of molecular weight of CSO and the charged amount of LA on the physicochemical properties of CSO-LA were investigated, such as CMC, graft ratio, size, zeta potential. The results showed that these chitosan derivatives were able to self-assemble and form spherical shape polymeric micelles with the size range of 150.7-213.9nm and the zeta potential range of 57.9-79.9mV, depending on molecular weight of CSO and the charged amount of LA. Using doxorubicin (DOX) as a model drug, the DOX-loaded CSO-LA micelles were prepared by dialysis method. The drug encapsulation efficiencies (EE) of DOX-loaded CSO-LA micelles were as high as about 75%. The sizes of DOX-loaded CSO-LA micelles with 20% charged DOX (relating the mass of CSO-LA) were near 200nm, and the drug loading (DL) capacity could reach up to 15%. The in vitro release studies indicated that the drug release from the DOX-loaded CSO-LA micelles was reduced with increasing the graft ratio of CSO-LA, due to the enhanced hydrophobic interaction between hydrophobic drug and hydrophobic segments of CSO-LA. Moreover, the drug release rate from CSO-LA micelles was faster with the drug loading. These data suggested the possible utilization of the amphiphilic micellar chitosan derivatives as carriers for hydrophobic drugs for improving their delivery and release properties.

Improved Transport and Absorption through Gastrointestinal Tract by PEGylated Solid Lipid Nanoparticles

The aim of the present study was to evaluate the potential of PEGylated solid lipid nanoparticle (pSLN) as mucus penetrating particles (MPP) for oral delivery across gastrointestinal mucus. The SLN was prepared by an aqueous solvent diffusion method, subsequently modified with PEG2000-stearic acid (PEG2000-SA) as hydrophilic groups. Surface properties, cytotoxicity, cellular uptake, and transport across Caco-2/HT29 coculture cell monolayers, intestinal absorption, and pharmacokinetics of pSLN were studied compared with that of SLN. Quantitative cellular uptake showed that the internalization of SLN and pSLN was an active transfer process, which would be restrained by several inhibitors of cell activity. Compared with SLN, the permeation ability of pSLN decreased through Caco-2 cell monolayer while it increased through a mucus-secreting Caco-2/HT29 coculture cell monolayer, which indicated that the mucus layer has a significant impact on determining the efficiency of oral nanoformulations. In addition to increasing permeation ability, the stability of the nanoparticles in simulated intestinal fluids was also increased by the PEGylation. Moreover, in vitro everted gut sac technique and the ligated intestinal loops model in vivo also demonstrated that pSLN can rapidly penetrate mucus secretions, whereas the SLN were strongly trapped by highly viscoelastic mucus barriers. The pharmacokinetic studies presented that pSLN exhibited improved absorption efficiency and prolonged blood circulation times with a 1.99-fold higher relative bioavailability compared with SLN. In conclusion, PEGylated solid lipid nanoparticles had advantages in enhancing the bioavailability of oral administration.

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.

PEGylated chitosan-based polymer micelle as an intracellular delivery carrier for anti-tumor targeting therapy.

Stearic acid-grafted chitosan oligosaccharide (CSO-SA) micelles presented a potential candidate for intracellular drug delivery carrier due to its special spatial structure. In this article, CSO-SA was further modified by polyethylene glycol (PEG). The physicochemical properties of PEGylated CSO-SA (PEG-CSO-SA) micelles were characterized. After PEGylation, the critical micelle concentration (CMC) of PEG-CSO-SA had no significant change; the micelle size increased; and the zeta potential decreased. The cellular uptake of CSO-SA micelles before and after PEGylation in macrophage RAW264.7, immortalized rat liver cells BRL-3A and human liver tumor cells HepG2 was studied. About 58.4+/-0.63% of CSO-SA micelles were uptaked by RAW264.7 in 24h, however, only 17.7+/-0.94% of PEG-CSO-SA micelles were internalized into RAW264.7 after the CSO-SA was modified with PEG in five molar times. Meanwhile, there were no changes in the uptake after PEGylation of CSO-SA in BRL-3A and HepG2. Using mitomycin C as a model drug, the in vitro anti-tumor activities of the drug loaded in the micelles were investigated. The 50% cellular growth inhibition (IC(50)) of the drug decreased from 1.97+/-0.2 to 0.13+/-0.02mug/mL after mitomycin C was loaded into CSO-SA micelles, and the IC(50) value of the drug had no obvious change when the CSO-SA was modified by PEG.

Studies on PEG-modified SLNs loading vinorelbine bitartrate (I): Preparation and evaluation in vitro

In this study, the conjugate of PEG2000-stearic acid (PEG2000-SA) was used to prepare PEGylated solid lipid nanoparticles loading vinorelbine bitartrate (VB-pSLNs) by cold homogenization technique. The particle size and zeta potential of resulted VB-pSLNs ranged 180-250nm and 0-10mV, which were determined using a Zetasizer, respectively. Although the drug entrapment efficiency (EE) slightly decreased after the PEG modification of VB-SLNs, above 60 % EE could be reached. The drug release tests in vitro indicated the faster drug release from VB-pSLNs than that from VB-SLNs without PEG modification. To investigate the cellular uptake of VB-pSLNs, the chemical conjugate of octadecylamine-fluorescein isothiocynate (FITC-ODA) was synthesized, and was used as a fluorescence marker to incorporate into nanoparticles. The results from cellular uptake indicated that the phagocytosis of VB-pSLNs by RAW264.7 cells was inhibited effectively by the PEG modification of SLNs, while the uptake by cancer cells (MCF-7 and A549) could be improved significantly. The assay of anticancer activity in vitro demonstrated that the anticancer activity of VB was significantly enhanced by the encapsulation of SLNs and pSLNs due to the increased cellular internalization of drug. The results suggested that SLNs and pSLNs could be excellent carrier candidates to entrap VB for tumor chemotherapeutics.

pH triggered doxorubicin delivery of PEGylated glycolipid conjugate micelles for tumor targeting therapy.

The main objective of this study was aimed at tumor microenvironment-responsive vesicle for targeting delivery of the anticancer drug, doxorubicin (DOX). A glucolipid-like conjugate (CS) was synthesized by the chemical reaction between chitosan and stearic acid, and polyethylene glycol (PEG) was then conjugated with CS via a pH-responsive cis-aconityl linkage to produce acid-sensitive PEGylated CS conjugates (PCCS). The conjugates with a critical micelle concentration (CMC) of 181.8 μg/mL could form micelles in aqueous phase, and presented excellent DOX loading capacity with a drug encapsulation efficiency up to 87.6%. Moreover, the PCCS micelles showed a weakly acid-triggered PEG cleavage manner. In vitro drug release from DOX-loaded PCCS micelles indicated a relatively faster DOX release in weakly acidic environments (pH 5.0 and 6.5). The CS micelles had excellent cellular uptake ability, which could be significantly reduced by the PEGylation. However, the cellular uptake ability of PCCS was enhanced comparing with insensitive PEGylated CS (PCS) micelles in weakly acidic condition imitating tumor tissue. Taking PCS micelles as a comparative group, the PCCS drug delivery system was demonstrated to show much more accumulation in tumor tissue, followed by a relatively better performance in antitumor activity together with a security benefit on xenograft tumor model.

RGD peptide-mediated chitosan-based polymeric micelles targeting delivery for integrin-overexpressing tumor cells

Background Solid tumors need new blood vessels to feed and nourish them as well as to allow tumor cells to escape into the circulation and lodge in other organs, which is termed “angiogenesis.” Some tumor cells within solid tumors can overexpress integrins αvβ3 and αvβ5, which can specifically recognize the peptide motif Arg-Gly-Asp (RGD). Thus, the targeting of RGD-modified micelles to tumor vasculature is a promising strategy for tumor-targeting treatment. Methods RGD peptide (GSSSGRGDSPA) was coupled to poly(ethylene glycol)-modified stearic acid-grafted chitosan (PEG-CS-SA) micelles via chemical reaction in the presence of N,N′-Disuccinimidyl carbonate. The critical micelle concentration of the polymeric micelles was determined by measuring the fluorescence intensity of pyrene as a fluorescent probe. The micelle size, size distribution, and zeta potential were measured by light scattering and electrophoretic mobility. Doxorubicin (DOX) was chosen as a model anticancer drug to investigate the drug entrapment efficiency, in vitro drug-release profile, and in vitro antitumor activities of drug-loaded RGD-PEG-CS-SA micelles in cells that overexpress integrins (ανβ3 and ανβ5) and integrin-deficient cells. Results Using DOX as a model drug, the drug encapsulation efficiency could reach 90%, and the in vitro drug-release profiles suggested that the micelles could be used as a controlled-release carrier for the hydrophobic drug. Qualitative and quantitative analysis of cellular uptake indicated that RGD-modified micelles could significantly increase the DOX concentration in integrin-overexpressing human hepatocellular carcinoma cell line (BEL-7402), but not in human epithelial carcinoma cell line (Hela). The competitive cellular-uptake test showed that the cellular uptake of RGD-modified micelles in BEL-7402 cells was significantly inhibited in the presence of excess free RGD peptides. In vitro cytotoxicity tests demonstrated DOX-loaded RGD-modified micelles could specifically enhance the cytotoxicity against BEL-7402 compared with DOX-loaded PEG-CS-SA and doxorubicin hydrochlorate. Conclusion This study suggests that RGD-modified PEG-CS-SA micelles are promising drug carriers for integrin-overexpressing tumor active targeting therapy.