An amphiphilic polymer DLPE-S-S-MPEG was synthesized and employed with PCL to prepare two-component reduction-sensitive lipid–polymer hybrid nanoparticles (SLPNPs) for in vitro and in vivo delivery of a hydrophobic anticancer drug (Doxorubicin, DOX). Insensitive lipid–polymer hybrid nanoparticles (ILPNPs) were prepared as a control. The mean sizes of the LPNPs ranged from 100 nm to 120 nm. The TEM observations showed that the LPNPs have spherical morphologies with homogeneous distribution. The disulfide bond of DLPE-S-S-MPEG was cleaved by dithiothreitol (DTT), which resulted in the disassembly of SLPNPs and triggered the release of encapsulated DOX. The in vitro cytotoxicities of DOX/LPNPs against HeLa cells, HepG2 cells and COS-7 cells were studied. It was demonstrated that DOX/SLPNPs showed higher cytotoxicity against HeLa cells and HepG2 cells than DOX/ILPNPs, but showed a slight difference in the case of COS-7 cells. CLSM observation and FCM measurement further confirmed that the introduction of S–S bonds caused fast intracellular release of DOX from SLPNPs. Moreover, compared with DOX/ILPNPs and free DOX, DOX/SLPNPs exhibited higher antitumor activity. Both DOX/SLPNPs and DOX/ILPNPs showed lower cardiac toxicity and kidney toxicity than free DOX, which were confirmed by histological and immunohistochemical analyses. The tissue distribution of DOX in mice exhibited that two kinds of DOX/LPNPs accumulated extensively in the liver and spleen, while free DOX accumulated mainly in the heart and kidney 12 h after injection. Two-component SLPNPs may be a promising drug delivery carrier for reduction-triggered delivery of DOX.

The importance of mitochondrial delivery of an anticancer drug to cancer cells has been recognized to improve therapeutic efficacy. The introduction of lipophilic cations, such as triphenylphosphonium (TPP), onto the surface of nanocarriers was utilized to target mitochondria via strong electrostatic interactions between positively charged TPP and the negatively charged mitochondrial membrane. However, the highly positive charge nature of TPP leads to rapid clearance from the blood, decrease of circulation lifetime, and nonspecific targeting of mitochondria of cells. Here, we report a strategy for improving the anticancer efficacy of paclitaxel via redox triggered intracellular activation of mitochondria-targeting. The lipid–polymer hybrid nanoparticles (LPNPs) are composed of poly(D,L-lactide-co-glycolide) (PLGA), a TPP-containing amphiphilic polymer (C18-PEG2000-TPP) and a reduction-responsive amphiphilic polymer (DLPE-S-S-mPEG4000). The charges of TPP in LPNPs were almost completely shielded by surface coating of a PEG4000 layer, ensuring high tumor accumulation. After uptake by cancer cells, the surface charges of LPNPs were recovered due to the detachment of PEG4000 under intracellular reductive conditions, resulting in rapid and precise localization in mitochondria. This kind of simple, easy and practicable mitochondria-targeting nanoplatform showed high anticancer activity, and the activatable strategy is valuable for developing a variety of nanocarriers for application in the delivery of other drugs.

The important characteristics of a nanoparticle-based anticancer drug delivery system at least include high stability in blood, highly efficient tumor cellular uptake and stimuli-responsive release of the anticancer drug inside the tumor cells. We here report a nanomicellar drug delivery system via self-assembly of PLA-SS-PAEMA/DMMA, an amphiphilic block copolymer composed of a poly(lactide) block and a poly(PAEMA/DMMA) block with disulfide linkage. Doxorubicin (DOX) was loaded into the micelles with a high drug-loading efficiency (10.6%, LE) and entrapment efficiency (59.5%, EE). In a neutral environment, PLA-SS-PAEMA/DMMA micelles are negatively charged, which ensures low adsorption of protein in the blood and excellent hemocompatibility. The surface charges of the micelles convert to positive under slightly acidic conditions at the tumor site, which improves the uptake of the micelles by the tumor cells. Once the drug-loaded micelles enter into the tumor cells, rapid release of DOX is triggered by reductive agents inside the tumor cells, such as GSH. In comparison to single responsive DOX-loaded PLA-CC-PAEMA/DMMA micelles and DOX-loaded PLA-SS-PAEMA/SA micelles, DOX-loaded PLA-SS-PAEMA/DMMA micelles showed higher cytotoxicity against HeLa cells due to the combinational effect of charge-reversal on cellular uptake and reduction-sensitivity on intracellular DOX release. The synergetic enhancement of antitumor efficacy by charge-reversal and reduction-sensitivity in DOX-loaded PLA-SS-PAEMA/DMMA micelles was further revealed by CLSM and flow cytometry analysis.

A salicylaldazine-based amphiphilic polymer (AIE-1) with AIE characteristics was synthesized and self-assembled to form sub-20 nm micelles (AIE-M). The green fluorescence of AIE-M was completely quenched by Cu2+ and then recovered with Na2S, which was utilized for the specific and sensitive detection of S2− in solution and mitochondrial imaging of H2S in HeLa cells.

The development and evaluation of folate-targeted and reduction-triggered biodegradable nanoparticles are introduced to the research on targeted delivery of doxorubicin (DOX). This type of folate-targeted lipid–polymer hybrid nanoparticles (FLPNPs) is comprised of a poly(D,L-lactide-co-glycolide) (PLGA) core, a soybean lecithin monolayer, a monomethoxy-poly(ethylene glycol)-S-S-hexadecyl (mPEG-S-S-C16) reduction-sensitive shell, and a folic acid-targeted ligand. FLPNPs exhibited high size stability but fast disassembly in a simulated cancer cell reductive environment. The experiments on the release process in vitro revealed that as a reduction-sensitive drug delivery system, FLPNPs released DOX faster in the presence of 10 mM dithiothreitol (DTT). Results from flow cytometry, confocal image and in vitro cytotoxicity assays revealed that FLPNPs further enhanced cell uptake and generated higher cytotoxicity against human epidermoid carcinoma in the oral cavity than non-targeted redox-sensitive and targeted redox-insensitive controls. Furthermore, in vivo animal experiments demonstrated that systemic administration of DOX-loaded FLPNPs remarkably reduced tumor growth. Experiments on biodistribution of DOX-loaded FLPNPs showed that an increasing amount of DOX accumulated in the tumor. Therefore, FLPNPs formulations have proved to be a stable, controllable and targeted anticancer drug delivery system.

•A Y-shaped amphiphilic reduction-sensitive polymer [mPEG-S-S-(PCL)2] was developed.•The polymer could self-assemble into micelles and encapsulate DOX.•The PEG shell of these micelles could be shed in the presence of reducing agent.•The DOX-loaded micelles exhibited faster DOX release in GSH-OEt-pretreated cells.A new type of shell-sheddable micelles with disulfide linkages between the hydrophobic polyester core and hydrophilic poly(ethylene glycol) (PEG) shell was developed based on Y-shaped amphiphilic polymers mPEG-S-S-(PCL)2. The micelles were then used for the glutathione-mediated intracellular delivery of the anticancer drug doxorubicin (DOX) into tumor cells. The polymer could self-assemble into micelles with an average diameter of 135 nm in aqueous solution and load DOX at a total content of 3.6%. The hydrophilic PEG shell of these micelles could be shed in the presence of reducing agent dithiothreitol (DTT), which resulted in size change of the micelles. In vitro release studies revealed that DOX-loaded mPEG-S-S-(PCL)2 micelles exhibited faster DOX release in the presence of DTT. MTT assay demonstrated that DOX-loaded mPEG-S-S-(PCL)2 micelles showed higher cytotoxicity against 10 mM of glutathione monoester (GSH-OEt) pretreated HeLa cells than that of the non-pretreated ones. Confocal laser scanning microscopy and flow cytometry analyses indicated that DOX-loaded mPEG-S-S-(PCL)2 micelles were efficiently internalized into HeLa cells and exhibited faster DOX release in GSH-OEt-pretreated cells than in cells with no pretreatment. Endocytosis inhibition results proved that mPEG-S-S-(PCL)2 micelles entered the cells mainly through the clathrin-mediated endocytosis pathway, and caveolae-mediated endocytosis was involved to a small extent. These results indicate the great potential of the proposed Y-shaped reduction-sensitive polymer for application in effective intracellular anticancer drug delivery.

Porous hematite nanorods (PHNRs) are potential candidates as drug delivery carriers because of their pores which are available for the encapsulation of drugs and genes, large surface area for loading biomacromolecules, biocompatibility, storage stability and inexpensive large-scale preparation. We report a facile synthesis of PHNRs loaded with a poorly water-soluble drug, paclitaxel (PTX). PHNRs were used as templates for the precipitation of PTX in a dimethyl sulfoxide (DMSO)–water mixture. The precipitation of amorphous PTX in the pores of PHNRs was characterized by nitrogen adsorption–desorption analysis, differential thermal analysis (DTA) and fourier transform infrared spectroscopy (FT-IR). Thermogravimetric analysis (TGA) and ultraviolet-visible (UV-vis) measurements determined that the loading of paclitaxel in PHNRs was high (17.6 wt%) and that the loading efficiency was up to 96%. We further studied the effect of free PTX or PTX-loaded PHNRs on the growth inhibition of HeLa cells by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and live/dead staining. PTX-loaded PHNRs showed significant cytotoxicity to HeLa cells. However, the ability of PTX-loaded PHNRs to inhibit cell growth was slightly lower or similar to that of free PTX after 48 h incubation. Confocal laser scanning microscopy (CLSM) and flow cytometric analysis suggested that HeLa cell death induced by free PTX or PTX-loaded PHNRs occurred via apoptosis, which was detected by Annexin V antibody and propidium iodide staining.

As a powerful technology to enhance the efficiency of gene delivery, magnetofection has attracted considerable attention in the past decade. In this work, we introduced 6 generation of PAMAM dendrimer modified superparamagnetic nanoparticles (DMSPION-G6) to PEI/DNA polyplexes by a two-step process and enhanced the transfection efficiency of PEI with the help of a magnetic field. We prepared DMSPION-G6/DNA/PEI ternary magnetoplexes by precondensing DMSPION-G6 with DNA at a low mass ratio to yield DMSPION-G6/DNA magnetoplexes with negative surface charge, followed by further coating with branched PEI (25 kDa) via electrostatic interactions. We measured the transfection efficiencies of DMSPION-G6/DNA/PEI ternary magnetoplexes in COS-7, 293T and HeLa cells in the presence or absence of a magnetic field. Compared with PEI/DNA polyplexes, DMSPION-G6/DNA/PEI ternary magnetoplexes exhibited enhanced transfection efficiencies in all the three cell lines when a magnetic field was applied, especially in the presence of 10% FBS. Time-resolved and dose-resolved transfection indicated that high-level transgene expression was achievable with a relatively short incubation time and low DNA dose when magnetofection was employed. Further evidence from Prussian blue staining, quantification of cellular iron concentration and cellular uptake of Cy-3 labelled DNA demonstrated that the magnetic field could quickly gather the magnetoplexes to the surface of target cells and consequently enhanced the uptake of magnetoplexes by the cells. This represents a novel strategy for polycation-based in vitrogene delivery enhanced by a magnetic field.

Organic-inorganic hybrid silica nanoparticles (PM-1, PM-2 and PM-3) with positive surface charge and size below 200 nm were one-pot prepared viaMichael addition between 3-methacryloxypropyl- trimethoxysilane (MPTMS) and polyethylenimine (PEI), followed by hydrolysis and polycondensation of siloxanes. The nanoparticles were characterized by FT-IR, element analysis and particle size analysis. The average sizes of the nanoparticles were 130–180 nm and the surface charge was about 40 mV. The acid–base titration showed that the nanoparticles had higher buffer capacity than PEI 25 kDa. The positively charged nanoparticles can condense negatively charged DNA to form complexes and completely retard the DNA mobility in agarose gel at a weight ratio of 5. The average sizes of PM-1/DNA and PM-2/DNA complexes were below 250 nm and the surface charge of the complexes was in the range of 30–40 mV at the weight ratio of 100. An in vitro transfection assay demonstrated that the transfection efficiencies of the nanoparticles were dependent on the PEI content, and PM-1 showed improved transfection efficiency compared with PEI 25 kDa in the presence of 10% serum. The intracellular trafficking assay of PM-1 nanoparticle/Cy3-labelled DNA complexes in COS-7 cells in the presence of 10% serum indicated that a large amount of complexes crossed the cell membrane and located in the cytoplasm and only a small amount of complexes entered into the cell nucleus after 24 h incubation. The uptake of PM-1 nanoparticle/DNA complexes by COS-7 cells in the presence of serum was higher than that of PEI/DNA complexes. In addition, the cytotoxicity of PM nanoparticles was significantly lower than that of PEI 25 kDa. The results indicate that the synthesized nanoparticles will show potential in nonviral gene delivery.

Porous films were fabricated from nonporous layer-by-layer multilayers composed of a blend of positively charged disulfide-containing polyamidoamine and poly(allylamine hydrochoride), and negatively charged poly(acrylic acid), followed by removal of cleavable disulfide-containing polycation after incubation in 1 mM DTT solution. The thickness of original multilayered films decreased with the increase of incubation time in DTT solution. Atomic force microscopy (AFM) measurements and electrochemical analysis demonstrated the formation of nanopores with sizes ranging from 50 to 120 nm. The formed porous films were stable in buffer solution at pHs ranging from 7.4 to 1.6, whereas they showed slight changes in pore number and pore size when incubated in PBS buffer at a pH of 10.0. This research might provide a universal method for the fabrication of noncrosslinked porous multilayered films.

An amphiphilic disulfide-containing polyamidoamine was synthesized by Michael-type polyaddition reaction of piperazine to equimolar N, N′-bis(acryloyl)cystamine with 90% yield. The polycationic micelles (198 nm, 32.5 mV), prepared from the amphiphilic polyamidoamine by dialysis method, can condense foreign plasmid DNA to form nanosized polycationic micelles/DNA polyelectrolyte complexes with positive charges, which transfected 293T cells with high efficiency. Under optimized conditions, the transfection efficiencies of polycationic micelles/DNA complexes are comparable to, or even higher than that of commercially available branched PEI (Mw 25 kDa).

Three hydrolytically degradable poly(β-aminoester)s containing ester bonds in the main chain and primary amines in the side chain, synthesized by Michael polyaddition, were applied to deliver foreign DNA into cells in vitro. These linear polycations can condense DNA into small-sized particles with positive surface charge at high N/P ratios. Their high buffer capacity at pH 5−7 facilitated the escape of DNA from the endosome and resulted in efficient gene expression. Under the optimal conditions, poly(β-aminoester)s with a pendant aminoethyl group (1a) showed higher transfection efficiencies than branched poly(ethylenimine) (PEI) 25KDa in 293T cells. The effect of side chain structure of the poly(β-aminoester) on transfection efficiency has been investigated, which indicated that the poly(β-aminoester) containing the pendant aminoethyl group was the most efficient carrier for both of 293T cells and COS-7 cells. The combination of hydrolytical degradation, high buffer capacity, relatively low cytotoxicity, and high transfection efficiency suggested that this kind of poly(β-aminoester)s are novel promising nonviral gene carriers.

Porous hematite nanorods (PHNRs) are potential candidates as drug delivery carriers because of their pores which are available for the encapsulation of drugs and genes, large surface area for loading biomacromolecules, biocompatibility, storage stability and inexpensive large-scale preparation. We report a facile synthesis of PHNRs loaded with a poorly water-soluble drug, paclitaxel (PTX). PHNRs were used as templates for the precipitation of PTX in a dimethyl sulfoxide (DMSO)–water mixture. The precipitation of amorphous PTX in the pores of PHNRs was characterized by nitrogen adsorption–desorption analysis, differential thermal analysis (DTA) and fourier transform infrared spectroscopy (FT-IR). Thermogravimetric analysis (TGA) and ultraviolet-visible (UV-vis) measurements determined that the loading of paclitaxel in PHNRs was high (17.6 wt%) and that the loading efficiency was up to 96%. We further studied the effect of free PTX or PTX-loaded PHNRs on the growth inhibition of HeLa cells by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and live/dead staining. PTX-loaded PHNRs showed significant cytotoxicity to HeLa cells. However, the ability of PTX-loaded PHNRs to inhibit cell growth was slightly lower or similar to that of free PTX after 48 h incubation. Confocal laser scanning microscopy (CLSM) and flow cytometric analysis suggested that HeLa cell death induced by free PTX or PTX-loaded PHNRs occurred via apoptosis, which was detected by Annexin V antibody and propidium iodide staining.

Organic-inorganic hybrid silica nanoparticles (PM-1, PM-2 and PM-3) with positive surface charge and size below 200 nm were one-pot prepared viaMichael addition between 3-methacryloxypropyl- trimethoxysilane (MPTMS) and polyethylenimine (PEI), followed by hydrolysis and polycondensation of siloxanes. The nanoparticles were characterized by FT-IR, element analysis and particle size analysis. The average sizes of the nanoparticles were 130–180 nm and the surface charge was about 40 mV. The acid–base titration showed that the nanoparticles had higher buffer capacity than PEI 25 kDa. The positively charged nanoparticles can condense negatively charged DNA to form complexes and completely retard the DNA mobility in agarose gel at a weight ratio of 5. The average sizes of PM-1/DNA and PM-2/DNA complexes were below 250 nm and the surface charge of the complexes was in the range of 30–40 mV at the weight ratio of 100. An in vitro transfection assay demonstrated that the transfection efficiencies of the nanoparticles were dependent on the PEI content, and PM-1 showed improved transfection efficiency compared with PEI 25 kDa in the presence of 10% serum. The intracellular trafficking assay of PM-1 nanoparticle/Cy3-labelled DNA complexes in COS-7 cells in the presence of 10% serum indicated that a large amount of complexes crossed the cell membrane and located in the cytoplasm and only a small amount of complexes entered into the cell nucleus after 24 h incubation. The uptake of PM-1 nanoparticle/DNA complexes by COS-7 cells in the presence of serum was higher than that of PEI/DNA complexes. In addition, the cytotoxicity of PM nanoparticles was significantly lower than that of PEI 25 kDa. The results indicate that the synthesized nanoparticles will show potential in nonviral gene delivery.

As a powerful technology to enhance the efficiency of gene delivery, magnetofection has attracted considerable attention in the past decade. In this work, we introduced 6 generation of PAMAM dendrimer modified superparamagnetic nanoparticles (DMSPION-G6) to PEI/DNA polyplexes by a two-step process and enhanced the transfection efficiency of PEI with the help of a magnetic field. We prepared DMSPION-G6/DNA/PEI ternary magnetoplexes by precondensing DMSPION-G6 with DNA at a low mass ratio to yield DMSPION-G6/DNA magnetoplexes with negative surface charge, followed by further coating with branched PEI (25 kDa) via electrostatic interactions. We measured the transfection efficiencies of DMSPION-G6/DNA/PEI ternary magnetoplexes in COS-7, 293T and HeLa cells in the presence or absence of a magnetic field. Compared with PEI/DNA polyplexes, DMSPION-G6/DNA/PEI ternary magnetoplexes exhibited enhanced transfection efficiencies in all the three cell lines when a magnetic field was applied, especially in the presence of 10% FBS. Time-resolved and dose-resolved transfection indicated that high-level transgene expression was achievable with a relatively short incubation time and low DNA dose when magnetofection was employed. Further evidence from Prussian blue staining, quantification of cellular iron concentration and cellular uptake of Cy-3 labelled DNA demonstrated that the magnetic field could quickly gather the magnetoplexes to the surface of target cells and consequently enhanced the uptake of magnetoplexes by the cells. This represents a novel strategy for polycation-based in vitrogene delivery enhanced by a magnetic field.

The development and evaluation of folate-targeted and reduction-triggered biodegradable nanoparticles are introduced to the research on targeted delivery of doxorubicin (DOX). This type of folate-targeted lipid–polymer hybrid nanoparticles (FLPNPs) is comprised of a poly(D,L-lactide-co-glycolide) (PLGA) core, a soybean lecithin monolayer, a monomethoxy-poly(ethylene glycol)-S-S-hexadecyl (mPEG-S-S-C16) reduction-sensitive shell, and a folic acid-targeted ligand. FLPNPs exhibited high size stability but fast disassembly in a simulated cancer cell reductive environment. The experiments on the release process in vitro revealed that as a reduction-sensitive drug delivery system, FLPNPs released DOX faster in the presence of 10 mM dithiothreitol (DTT). Results from flow cytometry, confocal image and in vitro cytotoxicity assays revealed that FLPNPs further enhanced cell uptake and generated higher cytotoxicity against human epidermoid carcinoma in the oral cavity than non-targeted redox-sensitive and targeted redox-insensitive controls. Furthermore, in vivo animal experiments demonstrated that systemic administration of DOX-loaded FLPNPs remarkably reduced tumor growth. Experiments on biodistribution of DOX-loaded FLPNPs showed that an increasing amount of DOX accumulated in the tumor. Therefore, FLPNPs formulations have proved to be a stable, controllable and targeted anticancer drug delivery system.

An amphiphilic polymer DLPE-S-S-MPEG was synthesized and employed with PCL to prepare two-component reduction-sensitive lipid–polymer hybrid nanoparticles (SLPNPs) for in vitro and in vivo delivery of a hydrophobic anticancer drug (Doxorubicin, DOX). Insensitive lipid–polymer hybrid nanoparticles (ILPNPs) were prepared as a control. The mean sizes of the LPNPs ranged from 100 nm to 120 nm. The TEM observations showed that the LPNPs have spherical morphologies with homogeneous distribution. The disulfide bond of DLPE-S-S-MPEG was cleaved by dithiothreitol (DTT), which resulted in the disassembly of SLPNPs and triggered the release of encapsulated DOX. The in vitro cytotoxicities of DOX/LPNPs against HeLa cells, HepG2 cells and COS-7 cells were studied. It was demonstrated that DOX/SLPNPs showed higher cytotoxicity against HeLa cells and HepG2 cells than DOX/ILPNPs, but showed a slight difference in the case of COS-7 cells. CLSM observation and FCM measurement further confirmed that the introduction of S–S bonds caused fast intracellular release of DOX from SLPNPs. Moreover, compared with DOX/ILPNPs and free DOX, DOX/SLPNPs exhibited higher antitumor activity. Both DOX/SLPNPs and DOX/ILPNPs showed lower cardiac toxicity and kidney toxicity than free DOX, which were confirmed by histological and immunohistochemical analyses. The tissue distribution of DOX in mice exhibited that two kinds of DOX/LPNPs accumulated extensively in the liver and spleen, while free DOX accumulated mainly in the heart and kidney 12 h after injection. Two-component SLPNPs may be a promising drug delivery carrier for reduction-triggered delivery of DOX.