N-acetylcysteine functionalized chitosan oligosaccharide-palmitic acid conjugate enhances ophthalmic delivery of flurbiprofen and its mechanisms.

An N-acetylcysteine functionalized chitosan oligosaccharide-palmitic acid conjugate (NAC-COS-PA) with bioadhesive and permeation promoting properties was synthesized to enhance transocular drug delivery. Flurbiprofen (FB) loaded self-assembled NAC-COS-PA nanomicelles (NAC-COS-PA-FB) were prepared and the drug loading was 7.35 ± 0.32%. Human immortalized corneal epithelial (HCE-T) cell cytotoxicity and hen's egg test-chorioallantoic membrane assays confirmed that the conjugate had good biocompatibility. The transportation efficiency of coumarin-6 (C6) loaded nanomicelles in the HCE-T cell monolayer was approximately 1.97 times higher than that of free C6. Decreased intracellular Ca2+ concentration and cell membrane potential, increased cell membrane fluidity, and reversible changes in the F-actin cytoskeleton are presumed to be responsible for the enhanced drug permeation. NAC-COS-PA exhibited strong binding capacity with mucin and rabbit eyeball. In vivo pharmacokinetics indicated that the area under the curve (AUC0-6 h) and the maximum concentration (Cmax) of NAC-COS-PA-FB were approximately 1.92 and 2.44 times that of the FB solution, respectively. NAC-COS-PA-FB demonstrated the best in vivo anti-inflammatory efficacy compared to unfunctionalized nanomicelles (COS-PA-FB) and FB solution. Consequently, NAC-COS-PA appears to be a promising bioadhesive carrier for ophthalmic delivery.

A NAG-Guided Nano-Delivery System for Redox- and pH-Triggered Intracellularly Sequential Drug Release in Cancer Cells.

AIM: Sequential treatment with paclitaxel (PTXL) and gemcitabine (GEM) is considered clinically beneficial for non-small-cell lung cancer. This study aimed to investigate the effectiveness of a nano-system capable of sequential release of PTXL and GEM within cancer cells. METHODS: PTXL-ss-poly(6-O-methacryloyl-d-galactopyranose)-GEM (PTXL-ss-PMAGP-GEM) was designed by conjugating PMAGP with PTXL via disulfide bonds (-ss-), while GEM via succinic anhydride (PTXL:GEM=1:3). An amphiphilic block copolymer N-acetyl-d-glucosamine(NAG)-poly(styrene-alt-maleic anhydride)58-b-polystyrene130 acted as a targeting moiety and emulsifier in formation of nanostructures (NLCs). RESULTS: The PTXL-ss-PMAGP-GEM/NAG NLCs (119.6 nm) provided a sequential in vitro release of, first PTXL (redox-triggered), then GEM (pH-triggered). The redox- and pH-sensitive NLCs readily distributed homogenously in the cytoplasm. NAG augmented the uptake of NLCs by the cancer cells and tumor accumulation. PTXL-ss-PMAGP-GEM/NAG NLCs exhibited synergistic cytotoxicity in vitro and strongest antitumor effects in tumor-bearing mice compared to NLCs lacking pH/redox sensitivities or free drug combination. CONCLUSION: This study demonstrated the abilities of PTXL-ss-PMAGP-GEM/NAG NLCs to achieve synergistic antitumor effect by targeted intracellularly sequential drug release.

Enhanced transdermal delivery of lornoxicam by nanostructured lipid carrier gels modified with polyarginine peptide for treatment of carrageenan-induced rat paw edema.

Background: Nanostructured lipid carriers (NLCs) are emerging as attractive drug carriers in transdermal drug delivery. The surface modification of NLCs with cell-penetrating peptides (CPPs) can enhance the skin permeation of drugs. Purpose: The objective of the current study was to evaluate the ability of the cell-penetrating peptide (CPP) polyarginine to translocate NLCs loaded with lornoxicam (LN) into the skin layers and to evaluate its anti-inflammatory effect. Methods: The NLCs were prepared using an emulsion evaporation and low temperature solidification technique using glyceryl monostearates, triglycerides, DOGS-NTA-Ni lipids and surfactants, and then six histidine-tagged polyarginine containing 11 arginine (R11) peptides was modified on the surface of NLCs. Results: The developed NLCs formulated with LN and R11 (LN-NLC-R11) were incorporated into 2% HPMC gels. NLCs were prepared with a particle size of (121.81±3.61)-(145.72±4.78) nm, and the zeta potential decreased from (-30.30±2.07) to (-14.66±0.74) mV after the modification of R11 peptides. The encapsulation efficiency and drug loading were (74.61±1.13) % and (7.92±0.33) %, respectively, regardless of the surface modification. Cellular uptake assays using HaCaT cells suggested that the NLC modified with R11 (0.02%, w/w) significantly enhanced the cell internalization of nanoparticles relative to unmodified NLCs (P<0.05 or P<0.01). An in vitro skin permeation study showed better permeation-enhancing ability of R11 (0.02%, w/w) than that of other content (0.01% or 0.04%). In carrageenan-induced rat paw edema models, LN-NLC-R11 gels inhibited rat paw edema and the production of inflammatory cytokines compared with LN-NLC gels and LN gels (P<0.01). Conclusion: In our investigation, it was strongly demonstrated that the surface modification of NLC with R11 enhanced the translocation of LN across the skin, thereby alleviating inflammation.

Redox-sensitive micelles based on retinoic acid modified chitosan conjugate for intracellular drug delivery and smart drug release in cancer therapy.

Novel chitosan-cystamine-retinoic acid conjugate (CS-SS-RA) was synthesized and could self-assemble into redox-sensitive micelles in aqueous environment with low critical micelle concentration value. CS-SS-RA micelles were characterized with spherical shape, desirable particle size, negative zeta potential, high paclitaxel (PTX) loading and encapsulation efficiency and redox-sensitivity. Hemolysis and cytotoxicity studies proved the safety of CS-SS-RA micelles for intravenous administration. Cytotoxicity study against HepG2 cells and the growth inhibition study on three-dimensional multicellular tumor spheroids (MCTSs) revealed that PTX-loaded CS-SS-RA micelles exhibited higher antitumor activity than free PTX. The in vitro cellular uptake profiles of FITC-labeled CS-SS-RA micelles evaluated via confocal laser scanning microscopy and flow cytometry indicated that CS-SS-RA micelles could enhance cellular uptake efficiency of PTX, and their internalization by HepG2 cells were mediated by clathrin-mediated endocytosis and macropinocytosis. These results demonstrated that CS-SS-RA micelles could be developed as a promising platform for intracellular delivery of hydrophobic antitumor agents.

Tumor-targeted polymeric nanostructured lipid carriers with precise ratiometric control over dual-drug loading for combination therapy in non-small-cell lung cancer.

Gemcitabine (GEM) and paclitaxel (PTX) are effective combination anticancer agents against non-small-cell lung cancer (NSCLC). At the present time, a main challenge of combination treatment is the precision of control that will maximize the combined effects. Here, we report a novel method to load GEM (hydrophilic) and PTX (hydrophobic) into simplex tumor-targeted nanostructured lipid carriers (NLCs) for accurate control of the ratio of the two drugs. We covalently preconjugated the dual drugs through a hydrolyzable ester linker to form drug conjugates. N-acetyl-d-glucosamine (NAG) is a glucose receptor-targeting ligand. We added NAG to the formation of NAG-NLCs. In general, synthesis of poly(6-O-methacryloyl-d-galactopyranose)-GEM/PTX (PMAGP-GEM/PTX) conjugates was demonstrated, and NAG-NLCs were prepared using emulsification and solvent evaporation. NAG-NLCs displayed sphericity with an average diameter of 120.3±1.3 nm, a low polydispersity index of 0.233±0.04, and accurate ratiometric control over the two drugs. A cytotoxicity assay showed that the NAG-NLCs had better antitumor activity on NSCLC cells than normal cells. There was an optimal ratio of the two drugs, exhibiting the best cytotoxicity and combinatorial effects among all the formulations we tested. In comparison with both the free-drug combinations and separately nanopackaged drug conjugates, PMAGP-GEM/PTX NAG-NLCs (3:1) exhibited superior synergism. Flow cytometry and confocal laser scanning microscopy showed that NAG-NLCs exhibited higher uptake efficiency in A549 cells via glucose receptor-mediated endocytosis. This combinatorial delivery system settles problems with ratiometric coloading of hydrophilic and hydrophobic drugs for tumor-targeted combination therapy to achieve maximal anticancer efficacy in NSCLC.

A shell-crosslinked polymeric micelle system for pH/redox dual stimuli-triggered DOX on-demand release and enhanced antitumor activity.

Based on targeted amphiphilic block copolymer N-acetyl glucosamine-poly (styrene-alt-maleic anhydride)58-b-polystyrene130 (NAG-P(St-alt-MA)58-b-PSt130), a pH/redox dual-triggered shell-crosslinked polymeric micelle system was constructed. The shell-crosslinked micelles (CLM) were prepared by post-crosslinking method to regulate drug release kinetics using cystamine as linkers between carboxy groups of the shell. Compared with non-crosslinked micelles (NCLM), CLM showed spherical shapes with little increased mean diameter of 102.40±0.54nm, low polydispersity index (PDI) of 0.19±0.36, enlarged zeta potential value from -41.46±0.99 to -9.31±0.50mV, indicating the successful modification of disulfide bonds in shell. In vitro drug release study clearly exhibited a pH and redox dual-sensitive drug release profile with significantly accelerated drug release under pH 5.0 and 10mM GSH conditions (46.84% in 96h) without burst release. Both CLM and NCLM showed quite different release profiles between physiological (pH 7.4) and tumoral microenvironment (pH 5.0), effectively avoiding the premature drug leakage and realizing on-demand drug release. The MTT assay implied that CLM presented a time- and concentration-dependent manner to inhibit proliferation of A549 and MCF-7 cells and much lower IC50 values in comparison with that of NCLM after 72h incubation. Both FCM and CLSM results showed that CLM displayed much higher cellular uptake efficiency and anti-tumor activities than NCLM and free DOX. CLM and NCLM could be internalized by energy-dependent endocytosis mechanism due to similar surface properties. Overall, this dual-stimuli triggered micelle system provided a promising tumor-responsive platform for cancer therapy.

Galactose-Containing Polymer-DOX Conjugates for Targeting Drug Delivery.

A novel multifunctional drug delivery system was fabricated by conjugating galactose-based polymer, methoxy-poly(ethylene glycol)-block-poly(6-O-methacryloyl-D-galactopyranose) (mPEG-b-PMAGP) with doxorubicin (DOX) via an acid-labile carbamate linkage. The mPEG-b-PMAGP-co-DOX nanoparticles were spherical in shape, and the diameter determined by dynamic light scattering (DLS) was 54.84 ± 0.58 nm, larger than that characterized by transmission electron microscopy (TEM). The in vitro drug release profiles were studied, and the release of DOX from the nanoparticles was pH-responsive. The cellular uptake behavior of free-DOX and mPEG-b-PMAGP-co-DOX nanoparticles by asialoglycoprotein (ASGP) receptor-positive cancer cell line (HepG2) and ASGP receptor-negative cancer cell lines (MCF-7 and A549 cells) was evaluated by confocal laser scanning microscopy (CLSM) and flow cytometry (FCM), respectively. The mPEG-b-PMAGP-co-DOX nanoparticles which contain galactose functional groups exhibited higher cellular uptake behavior via ASGP receptor-mediated endocytosis in HepG2 cells than in other two cancer cells. The in vitro cytotoxicity assay manifested that the mPEG-b-PMAGP-co-DOX nanoparticles exhibited higher anticancer efficacy against HepG2 cells than MCF-7 cells. These results indicated that the multifunctional mPEG-b-PMAGP-co-DOX nanoparticles possessing pH-responsible and hepatoma-targeting function have great potential to be used as a targeting drug delivery system for hepatoma therapy.

Multifunctional mesoporous silica nanoparticles modified with tumor-shedable hyaluronic acid as carriers for doxorubicin.

In this paper, a CD44-targeted and redox-responsive drug delivery system based on mesoporous silica nanoparticles (MSNs) was synthesized by conjugating tumor-shedable hyaluronic acid (HA) on the surface of MSNs via disulfide bonds. Doxorubicin hydrochloride (DOX·HCl) was physically encapsulated into HA modified MSNs (MSNs/SS/HA@DOX) as a model drug. MSNs/SS/HA@DOX (40nm) had a high drug loading (14.1%) and redox-responsive drug release property. The cellular uptake behaviors of MSNs/SS/HA@DOX by HeLa and LO2 cells were evaluated by confocal laser scanning microscopy (CLSM) and flow cytometry (FCM). MSNs/SS/HA@DOX exhibited higher cellular uptake efficacy via CD44-mediated endocytosis by HeLa cells (CD44 over-expressed cells) than by LO2 cells (CD44 deficient cells). The in vitro cytotoxicity assay demonstrated that MSNs/SS/HA@DOX exhibited higher cytotoxicity to HeLa cells than to LO2 cells. These results indicated that MSNs/SS/HA@DOX might be promising as a multifunctional drug delivery system to improve the anti-tumor efficacy of chemotherapeutic drugs.

Biodegradable Self-Assembled Nanoparticles of Galactose-Containing Amphiphilic Triblock Copolymers for Targeted Delivery of Paclitaxel to HepG2 Cells.

Biodegradable self-assembled polymeric nanoparticles (NPs) composed of poly(6-O-methacryloyl-D-galactopyranose)-b-poly(L-lactide)-b-poly(6-O-methacryloyl-D-galactopyranose) (PMAGP-b-PLA-b-PMAGP) are prepared as carriers for the hydrophobic anticancer drug paclitaxel (PTX), to achieve target delivery to hepatoma cells. PTX can be encapsulated by the NPs with various molar ratios of L-lactide (LA) and 6-O-methacryloyl-D-galactopyranose (MAGP) during the process of self-assembly, and the resulting NPs exhibit high drug loading efficacy and substantial stability in aqueous solution. The size, size distribution, and morphology of the NPs are characterized using a Zetasizer Nano ZS and transmission electron microscopy. The hemolysis assay and cell cytotoxicity assay indicate that the polymeric NPs are biocompatible and non-toxic. The cellular uptake assay demonstrates that the galactose-containing NPs can be selectively recognized and subsequently accumulate in HepG2 cells. All of these results demonstrate that galactose-containing polymeric NPs are potential carriers for hepatoma-targeted drug delivery and liver cancer therapy in clinical medicine.

Coordinated pH/redox dual-sensitive and hepatoma-targeted multifunctional polymeric micelle system for stimuli-triggered doxorubicin release: Synthesis, characterization and in vitro evaluation.

Multifunctional polymeric micelles self-assembled from a DOX-conjugated methoxypolyethylene glycols-b-poly (6-O-methacryloyl-D-galactopyranose)-disulfide bond-DOX (mPEG-b-PMAGP-SS-DOX) copolymer were prepared as an antitumor carrier for doxorubicin delivery, of which the chemical modification with disulfide bonds and hydrazone bonds allowed micelles to release doxorubicin (DOX) selectively at acidic pH and high redox conditions. The resulting micelles exhibited coordinated pH/redox dual-sensitive and hepatoma-targeted multifunction with sustaining stability in aqueous media. The multifunctional micelles showed spherical shapes with a mean diameter of 93 ± 2.08 nm, a low polydispersity index (PDI) of 0.21, a low CMC value of 0.095 mg/mL, a high drug grafting degree of 56.9% and a drug content of 39.0%. Remarkably, in vitro drug release studies clearly exhibited a pH and redox dual-sensitive drug release profile with significantly accelerated drug release treated with pH 5.0 and 10mM GSH (88.4% in 72 h) without drug burst release. The tumor proliferation assays indicated that DOX-grafted micelles, along with low cytotoxicity and well biocompatibility to normal cells up to a concentration of 10 µg/mL, inhibited the proliferation of HepG2 cells in a formulation-, time- and concentration-dependent manner in comparison with MCF-7 cells which was similar to free DOX. Anticancer activity releaved that the disulfide-modified micelles possessed much higher anti-hepatoma activity with a low IC50 value of 1.1 µg/mL following a 72 h incubation. Furthermore, the intracellular uptake tested by CLSM and FCM demonstrated that multifunctional polymeric micelles could be more efficiently taken up by HepG2 cells compared with MCF-7 cells, agreed well with MTT assays, suggesting these well-defined micelles provide a potential drug delivery system for dual-responsive controlled drug release and enhanced anti-hepatoma therapy.

Polymeric nanoparticles based on chitooligosaccharide as drug carriers for co-delivery of all-trans-retinoic acid and paclitaxel.

An amphiphilic all-trans-retinoic acid (ATRA)-chitooligosaccharide (RCOS) conjugate was synthesized to form self-assembled polymeric nanoparticles to facilitate the co-delivery of ATRA and paclitaxel (PTX). The blank RCOS nanoparticles possessed low hemolytic activity and cytotoxicity, and could efficiently load PTX with a drug loading of 22.2% and a high encapsulation efficiency of 71.3%. PTX-loaded RCOS nanoparticles displayed a higher cytotoxicity to HepG2 cells compared to PTX plus ATRA solution when corrected by the accumulated drug release. Cellular uptake profiles of RCOS nanoparticles were evaluated via confocal laser scanning microscope and flow cytometry with FITC as a fluorescent mark. The RCOS nanoparticles could be rapidly and continuously taken up by HepG2 cells via endocytosis and transported into the nucleus, and the uptake rates increased with particle concentration. These results revealed the promising potential of RCOS nanoparticles as drug carriers for co-delivery of ATRA and PTX or other hydrophobic therapeutic agents.

N-Acetyl-D-glucosamine decorated polymeric nanoparticles for targeted delivery of doxorubicin: Synthesis, characterization and in vitro evaluation.

A novel targeting drug delivery system containing poly(styrene-alt-maleic anhydride)58-b-polystyrene130 (P(St-alt-MA)58-b-PSt130) as a copolymer backbone, N-acetyl glucosamine (NAG) as a targeting moiety was designed and synthesized. The NAG grafted copolymer (NAG-P(St-alt-MA)58-b-PSt130) was characterized by FTIR and (1)H NMR. The NAG-P(St-alt-MA)58-b-PSt130 nanoparticles exhibited spherical shapes with an average diameter about 56.27±0.43 nm, low critical micelle concentration of 0.028 mg/mL, negative zeta potential -41.46±0.99 mV, high drug loading 25.83±1.09% and encapsulation efficiency 69.69±3.98%. In vitro cell cytotoxicity was conducted to confirm the safety of the NAG-P(St-alt-MA)58-b-PSt130 nanoparticles. Confocal laser scanning microscopy (CLSM) and flow cytometry (FCM) results showed that the NAG targeting moiety enhanced the internalization and targeting ability of NAG-P(St-alt-MA)58-b-PSt130 nanoparticles. Anticancer activity toward MCF-7 cells and HT29 cells showed that DOX-loaded NAG-P(St-alt-MA)58-b-PSt130 nanoparticles exhibited a higher antitumor activity compared to DOX-loaded P(St-alt-MA)58-b-PSt130 nanoparticles, which could attribute to NAG receptor-mediated endocytosis. These results suggest that the biocompatible and non-toxic NAG-P(St-alt-MA)58-b-PSt130 nanoparticles may be used as an effective targeting drug delivery system for cancer therapy.

Hepatoma-targeting and pH-sensitive nanocarriers based on a novel D-galactopyranose copolymer for efficient drug delivery.

Smart nanoparticles based on the mechanisms of asialoglycoprotein (ASGP)-mediated endocytosis and pH-induced drug release were developed for the efficient treatment of hepatoma using a newly developed copolymer, methoxy-polyethylene glycols (PEG)-b-poly (d-galactopyranose) (MPEG-b-PMaIPG). The particles exhibited spherical shapes, uniform particle size distribution (100 ± 4.43 nm), negative zeta potential (-32.8 ± 0.23 mV), high drug loading (24.77 ± 2.68%) and encapsulation efficiency (66.12 ± 9.44%). The in vitro drug release was also investigated, resulting that the release of drug from particles depended on different pH value. In vitro cell cytotoxicity and hemolysis assays were conducted to confirm the safety of the MPEG-b-PMaIPG nanoparticles. Anticancer activity showed that DOX-loaded MPEG-b-PMaIPG nanoparticles exhibited a high antitumor activity toward HepG2 cells, which was similar to free DOX, while blank MPEG-b-PMaIPG nanoparticles were non-toxic up to a tested concentration of 1.0mg/mL. Confocal laser scanning microscopy (CLSM) and flow cytometry (FCM) were used to verify the targeting efficiency of d-galactopyranose-modified nanoparticles. The results clearly demonstrated that d-galactopyranose-modified nanoparticles were taken up quickly by the HepG2 cells, which suggests that MPEG-b-PMaIPG nanoparticles with good biocompatibility and non-toxic for normal cells may be used as an effective cancer-targeting drug delivery system for chemotherapy.

A new fluorescence "off-on" chemodosimeter for L-cysteine based on water-soluble polythiophene.

A novel water-soluble cationic conjugated polymer, denoted as poly 2,5-[3-(1,1-dimethyl-4-piperidine methylene)thiophene] chloride（PDPMT-Cl was a novel fluorescent material. Fluorescence can be quenched by [AuCl4](-) effectively. On addition of l-cysteine (l-Cys) in [AuCl4](-)-PDPMT-Cl, fluorescence recovered. A new method to detect l-Cys was established successfully by designing a fluorescent "off-on" probe. The method showed good sensitivity and selectivity. Under optimized condition, the fluorescence intensity was linear to l-Cys concentration varying from 1.0 × 10(-8)M to 6.0 × 10(-5)M (γ=0.9982). The detection limit (3σ) was 1.39 × 10(-10)M. The method was successfully used for the determination of l-Cys in human serum and compound amino acid injection.