A chitosan-graft-PEI-candesartan conjugate for targeted co-delivery of drug and gene in anti-angiogenesis cancer therapy

A multifunctional copolymer–anticancer conjugate chitosan-graft-polyethyleneimine-candesartan (CPC) containing low molecular weight chitosan (CS) backbone and polyethyleneimine (PEI) arms with candesartan (CD) conjugated via an amide bond was fabricated as a targeted co-delivery nanovector of drug and gene for potential cancer therapy. Here, CD was utilized to specifically bind to overexpressed angiotensin II type 1 receptor (AT₁R) of tumor cells, strengthen endosomal buffering capacity of CPC and suppress tumor angiogenesis. The self-assembled CPC/pDNA complexes exhibited desirable and homogenous particle size, moderate positive charges, superior stability, and efficient release of drug and gene in vitro. Flow cytometry and confocal laser scanning microscopy analyses confirmed that CD-targeted function and CD-enhanced buffering capacity induced high transfection, specific cellular uptake and efficient intracellular delivery of CPC/pDNA complexes in AT₁R-overexpressed PANC-1 cells. In addition, CPC/wt-p53 complexes co-delivering CD and wild type p53 (wt-p53) gene achieved synergistic angiogenesis suppression by more effectively downregulating the expression of vascular endothelial growth factor (VEGF) mRNA and protein via different pathways in vitro, as compared to mono-delivery and mixed-delivery systems. In vivo investigation on nude mice bearing PANC-1 tumor xenografts revealed that CPC/wt-p53 complexes possessed high tumor-targeting capacity and strong anti-tumor activity. Additional analysis of microvessel density (MVD) demonstrated that CPC/wt-p53 complexes significantly inhibited tumor-associated angiogenesis. These findings suggested that CPC could be an ideal tumor-targeting nanovector for simultaneous transfer of drug and gene, and a multifunctional CPC/wt-p53 co-delivery system with tumor-specific targetability, enhanced endosomal buffering capacity and synergistic anti-angiogenesis efficacy might be a new promising strategy for effective tumor therapy.     

A silica–polymer composite nano system for tumor-targeted imaging and p53 gene therapy of lung cancer

In our study, a silica–polymer composite nano system (MB-NSi–p53–CS ternary complexes) composed of methylene blue-encapsulated amine-terminated silica nanoparticles (MB-NSi) and chondroitin sulfate (CS) were successfully developed for tumor-targeted imaging and p53 gene therapy of lung cancer. MB was employed as a NIR probe for in vivo imaging, MB-NSi nanoparticles were served as gene vector, while CS was applied to be a coating and targeting polymer. MB-NSi–p53–CS ternary complexes displayed nanosized diameter, effective p53 condensation ability, efficient p53 protection profile, and superior bovine serum albumin stability in vitro. Experiments on A549 cell line further revealed low cytotoxicity, high p53 transfection, and anticancer efficacy of MB-NSi–p53–CS ternary complexes. In vivo imaging and tumor targetability assays demonstrated that MB-NSi–p53–CS ternary complexes were a preferable system with desirable imaging and tumor-targeting properties.     

Self-assembled carboxymethyl poly (L-histidine) coated poly (β-amino ester)/DNA complexes for gene transfection

Biomaterials coated polymer/DNA complexes are developed as an efficient non-viral gene delivery system. It is able to circumvent the changes of various biophysical properties of the biomaterials and the corresponding polymer/DNA nanoparticles with covalent linkage. In the present study, we introduced pH-sensitive carboxymethyl poly (l-histidine) (CM-PLH) and poly (β-amino ester) (PbAE) as functional biomaterials to form CM-PLH/PbAE/DNA core-shell ternary complexes system based on electrostatically adsorbed coatings for gene efficient delivery and transfection. The preparation of the complexes was performed self-assembly in 25 mm sodium acetate buffer solution at pH 5.2. The complexes kept stable nano-size, behaving good condensation capacity and low toxicity, even provided a higher transfection efficiency than the binary complexes (PbAE/DNA without CM-PLH) and transfected up to (89.6 ± 4.45) % in HEK293 and (57.1 ± 2.10) % in B16-F10 in vitro. The ternary complexes significantly enhanced their cellular uptake and endosomal escape which were proved by the results that the complexes could evade the endosomal lumen and localize in the nucleus of treated cells visualized under Fluorescence Confocal Microscopy (FCM). The aforementioned results indicated that CM-PLH with pH-sensitive imidazole groups played an important role in enhancing the endosomal escape and transfection efficiency. The in vivo gene transfection confirmed that the ternary complexes with pGL3-promoter as led to effectively deposit at the tumor site by the EPR effect and shown 4 fold higher luciferase expression in B16-F10 tumor than the binary complexes. Consequently, CM-PLH/PbAE/DNA ternary complexes system exhibited significant improvements in transfection efficiency in comparison with non-coated PbAE/DNA both in vitro and in vivo, highlighting their functional prospect. Our approach and the gene delivery system fabrication could potentially be useful for effective gene delivery and therapies to targeted cells.     

Self-assembled ternary complexes of plasmid DNA, low molecular weight polyethylenimine and targeting peptide for nonviral gene delivery into neurons

Chemical conjugation of targeting ligands to polycation/plasmid DNA complexes has been widely used to improve the transfection efficiency of nonviral gene delivery vectors. However, conjugation reactions may reduce or even inactivate the biological activities of chemically sensitive moieties, such as proteins and peptides. Here we describe a new method for introducing targeting ligands into nonviral vectors, in which ternary complexes are formed via charge interactions among polyethylenimine (PEI) of 600Da, plasmid DNA and targeting peptides with positively charged DNA-binding sequence. Owing to the nerve growth factor (NGF) loop 4 hairpin motif in the targeting peptide, these ternary complexes are capable of mediating gene delivery efficiently and specifically into cells expressing the NGF receptor TrkA. In in vitro experiments, the complexes improved luciferase reporter gene expression by up to 1000-fold while comparing with that produced by complexes with nontargeting control peptide. In an in vivo experiment, the ternary complexes with the targeting peptide was 59-fold more efficient than the control ternary complexes in transfecting dorsal root ganglia (DRG), the peripheral nervous sites with TrkA-expressing neurons. In a cell viability study, the ternary complexes were remarkably different from DNA complexes by PEI of 25 kDa, the gold standard for nonviral gene carriers, displaying no toxicity in tested neuronal cells. Thus, this study demonstrates an alternative method to construct nonviral delivery system for targeted gene transfer into neurons.     

Gene delivery of PEI incorporating with functional block copolymer via non-covalent assembly strategy

A novel functional diblock polymer P(PEGMA-b-MAH) is prepared and incorporated to improve the gene delivery efficiency of poly(ethyleneimine) PEI via non-covalent assembly strategy. First, P(PEGMA-b-MAH) is prepared from l-methacrylamidohistidine methyl ester (MAH) by reversible addition fragmentation chain transfer polymerization, with poly[poly(ethylene glycol) methyl ether methacrylate] (P(PEGMA)) as the macroinitiator. Then P(PEGMA-b-MAH) is assembled with plasmid DNA (pDNA) and PEI (M_w = 10 kDa) to form PEI/P(PEGMA-b-MAH)/pDNA ternary complexes. The agarose gel retardation assay shows that the presence of P(PEGMA-b-MAH) does not interfere with DNA condensation by the PEI. Dynamic light scattering tests show that PEI/P(PEGMA-b-MAH)/pDNA ternary complexes have excellent serum stability. In vitro transfection indicates that, compared to the P(PEGMA-b-MAH) free PEI-25k/pDNA binary complexes, PEI-10k/P(PEGMA-b-MAH)/pDNA ternary complexes have lower cytotoxicity and higher gene transfection efficiency, especially under serum conditions. The ternary complexes proposed here can inspire a new strategy for the development of gene and drug delivery vectors.     

The effects of coating pDNA/chitosan complexes with chondroitin sulfate on physicochemical characteristics and cell transfection

pDNA/chitosan complexes have been investigated as promising non-viral vectors for gene delivery. However, an increase in transfection efficiency and enhancement of physicochemical stability are required for their practical use. In this study, chondroitin sulfate (CS) was employed as a coating agent to increase the stability and transfection efficiency of a pDNA/chitosan complex. The pDNA/chitosan/CS ternary complexes formed with six kinds of CSs having different limiting viscosities (0.2-1.6) and sulfation degrees (5.0-7.0%) showed considerable differences in particle size, surface charge, and morphology. Among them, CS having a medium limiting viscosity (0.5-0.6) and a high sulfation degree (6.9%) showed significant enhancements in cell transfection efficiency. Analyses of cellular uptake and intracellular trafficking revealed that increased cellular uptake via macropinocytosis, together with reduced entry into lysosomes, may explain the promotion of transfection efficiency of ternary complexes.     

The effect of adenovirus-conjugated NDRG2 on p53-mediated apoptosis of hepatocarcinoma cells through attenuation of nucleotide excision repair capacity

NDRG2 mRNA and protein levels can be upregulated in a p53-dependent manner. NDRG2 enhances p53-mediated apoptosis, whereas overexpression of NDRG2 suppresses tumor cell growth, regardless of whether p53 is mutated. However, the complicated mechanism by which NDRG2 suppresses tumor cell growth and enhances apoptosis mediated by p53 is not fully understood. Here, we demonstrated that Ad-NDRG2 enhanced the apoptosis of HepG2 cells (wild-type p53). Additionally, Ad-NDRG2 combined with rAd-p53 enhanced the apoptosis of Huh7 cells (mutant p53) after chemotherapy, and the expression of the ERCC6 gene (Cockayne syndrome group B protein gene) was suppressed in this process. Ad-NDRG2 combined with rAd-p53 induced the apoptosis of tumor cells (HepG2 and Huh7 cells); however, apoptosis was attenuated after transfection with ERCC6. Our results indicate that Ad-NDRG2 enhances the p53-mediated apoptosis of hepatocarcinoma cells (HepG2 and Huh7) by attenuating the nucleotide excision repair capacity (i.e., by downregulating ERCC6), and ERCC6 is a NDRG2-inducible target gene that is involved in the p53-mediated apoptosis pathway.     

Synthesis,Self‐Assembly,and Host–Guest Response of Naphthalic Anhydride‐Ended Hyperbranched Polyesters

Self‐assemblies fabricated from hyperbranched polymers have exhibited unique performances and a wide range of applications. However, the direct self‐assembly of such hyperbranched polymers into highly ordered macrostructures remains challenging, due to their high polydispersities, irregular architectures, and poorly defined shapes. Herein, hyperbranched polyesters (HBPE) modified with naphthalic anhydride terminal groups, so called HBPE‐(NA‐COOH)₃ and HBPE‐(NA‐COOH)₆, are synthesized by a convenient one‐pot procedure. Furthermore, highly ordered dendritic morphologies are obtained based on their self‐assembly, wherein the interlayer interactions are enhanced by the multiple hydrogen bonds among the carboxyl groups and the π–π stacking among the aromatic rings. In addition, the host–guest interactions of the naphthalic anhydride‐ended hyperbranched polyesters are investigated. Among them, HBPE‐(NA‐COOH)₃ is a promising fluorescent probe to anthracene, while HBPE‐(NA‐COOH)₆ is a promising probe to pyrene.

     

Binary and ternary complexes based on polycaprolactone-graft-poly (N, N-dimethylaminoethyl methacrylate) for targeted siRNA delivery

Small interfering RNA (siRNA) is a powerful gene silencing tool and has promising prospects in basic research and the development of therapeutic reagents. However, the lack of an effective and safe tool for siRNA delivery hampers its application. Here, we introduced binary and ternary complexes that effectively mediated siRNA-targeted gene silencing. Both complexes showed excellent siRNA loading even at the low N/P/C ratio of 3:1:0. FACS and confocal microscopy demonstrated that nearly all cells robustly internalized siRNAs into the cytoplasm, where RNA interference (RNAi) occurred. Luciferase assay and Western blot verified that silencing efficacy reached >80%, and introducing folate onto the ternary complexes further enhanced silencing efficacy by about 10% over those without folate at the same N/P/C ratio. In addition, the coating of PGA-g-mPEG decreased the zeta potential almost to electroneutrality, and the MTT assay showed decreased cytotoxicity. In vivo distribution measurement and histochemical analysis executed in C57BL/6 and Hela tumor-bearing BALB/c nude mice showed that complexes accumulated in the liver, lungs, pancreas and tumors and were released slowly for a long time after intravenous injection. Furthermore, ternary complexes showed higher siRNA fluorescence intensity than binary complexes at the same N/P ratio in tumor tissues, those with folate delivered more siRNAs to tumors than those without folate, and more folate induced more siRNA transport to tumors. In addition, in vivo functional study showed that both binary and ternary complexes mediated down-regulation of ApoB in liver efficiently and consequently blocked the secretion of fatty acids into the blood, resulted in lipid accumulation in liver, liver steatosis and hepatic dysfunction. In conclusion, these complexes provided a powerful means of administration for siRNA-mediated treatment of liver-related diseases and various cancers, especial for pancreatic and cervical cancer.     

Ternary complexes of pDNA,polyethylenimine, and γ-polyglutamic acid for gene delivery systems

We discovered a vector coated by γ-polyglutamic acid (γ-PGA) for effective and safe gene delivery. In order to develop a useful non-viral vector, we prepared several ternary complexes constructed with pDNA, polyethylenimine (PEI), and various polyanions, such as polyadenylic acid, polyinosinic–polycytidylic acid, α-polyaspartic acid, α-polyglutamic acid, and γ-PGA. The pDNA/PEI complex had a strong cationic surface charge and showed extremely high transgene efficiency although it agglutinated with erythrocytes and had extremely high cytotoxicity. Those polyanions changed the positive ζ-potential of pDNA/PEI complex to negative although they did not affect the size. They had no agglutination activities and lower cytotoxicities but most of the ternary complexes did not show any uptake and gene expression; however, the pDNA/PEI/γ-PGA complex showed high uptake and gene expression. Most of the pDNA/PEI/γ-PGA complexes were located in the cytoplasm without dissociation and a few complexes were observed in the nuclei. Hypothermia and the addition of γ-PGA significantly inhibited the uptake of pDNA/PEI/γ-PGA by the cells, although l-glutamic acid had no effect. These results strongly indicate that the pDNA/PEI/γ-PGA complex was taken up by γ-PGA-specific receptor-mediated energy-dependent process. Thus, the pDNA/PEI/γ-PGA complex is useful as a gene delivery system with high transfection efficiency and low toxicity.     

Self-assembled oligopeptide nanostructures for co-delivery of drug and gene with synergistic therapeutic effect

In this study, oligopeptide amphiphile containing three blocks of amino acids, Ac-(AF)₆-H₅-K₁₅-NH₂ (FA32), were synthesized and evaluated as carriers for co-delivery of drug and gene. Doxorubicin (DOX), luciferase reporter gene, and p53 gene were used as a model drug and genes. The peptide amphiphile self-assembled into cationic core–shell nanostructures (i.e. micelles), with a CMC value of around 0.042 mg/mL, estimated by fluorescent spectroscopy technique. FA32 nanostructures had an average size of 102 ± 19 nm, and a zeta potential of 22.8 ± 0.2 mV. These nanostructures had a high capacity for DOX encapsulation, with a DOX loading level of up to 22%. In addition, DOX release from the micelles was sustained without obvious initial burst. DOX-loaded micelles were effectively taken up by HepG2 cells, with an IC₅₀ of 1.8 mg/L for DOX-loaded FA32, which was higher than that of free DOX (0.25 mg/L). In addition, FA32 micelles condensed DNA efficiently to form small complexes with net positive charge on the surface. In vitro gene transfection studies showed that FA32 induced comparable gene expression level to polyethylenimine. Co-delivery of drug and gene using FA32 micelles was demonstrated via confocal imaging, luciferase expression in the presence of DOX, and synergy in cytotoxic effect between p53 gene and DOX. It was shown that through simultaneous delivery of both p53 gene and DOX using FA32 micelles, an increase in p53 mRNA expression level as well as end point cytotoxicity towards HepG2 cells was achieved. FA32 micelles, therefore, have a great potential in delivering hydrophobic anticancer drug and gene simultaneously for improved cancer therapy.     

Influence of the polyanion on the physico-chemical properties and biological activities of polyanion/DNA/polycation ternary polyplexes

It was recently reported that polyanion/DNA/polycation ternary polyplexes markedly improve gene transfection activity in comparison with the original DNA/polycation binary polyplexes. In this study to explore the influence of the polyanion on the physico-chemical properties and biological activity of polyanion/pDNA/polycation ternary polyplexes four types of biocompatible polyanions were selected, mainly based on the acid strength of the anionic functional groups and the molecular rigidity on forming ternary polyplexes with 25 kDa polyethyleneimine and DNA. Polyanion loosening of the DNA polyplex, weakening of the adsorption of serum proteins and improving of cellular uptake, which are thought to be important factors leading to a high transfection efficiency of DNA ternary polyplexes, were specifically investigated. Electrophoresis retardation analysis indicated that the loosening capacity of polyanions depended on the pK(a) value of the functional anion groups as well as the flexibility of the polyanion. The low pK(a) and flexible structure of the polyanions tended to loosen the compact DNA polyplexes. Thermodynamic analysis by isothermal titration calorimetry provided direct evidence about the serum protein-DNA ternary polyplex interactions. The polyanion/pDNA/polycation ternary polyplexes exhibited obviously lower binding affinities and less adsorption to serum proteins compared with the original DNA/polycation binary polyplexes. These relatively stable DNA ternary polyplexes maintained high levels of cellular uptake and intracellular accumulation in serum-containing medium that correlated with their high transfection efficiency. In contrast, the original pDNA/polycation binary polyplexes became clustered by strong adsorption of large amounts of serum proteins, leading to a sharp reduction in cellular uptake and intracellular accumulation, and thus low gene transfer efficiency. These results provide a basis for the development of polyanion/DNA/polycation ternary polyplexes for polyfection.     

Gene transfer by chemical vectors, and endocytosis routes of polyplexes, lipoplexes and lipopolyplexes in a myoblast cell line

Chemical vectors are widely developed for providing safe DNA delivery systems. It is well admitted that their endocytosis and intracellular trafficking are critical for the transfection efficiency. Here, we have compared the endocytic pathways of lipoplexes, polyplexes and lipopolyplexes formed with carriers of various chemical compositions. Engineered C2C12 mouse myoblast cells expressing Rab5-EGFP, Rab7-EGFP or Cav1-GFP were used to monitor the location of the plasmid DNA into the endocytic compartments by real time fluorescence confocal microscopy. We observed that (i) DNA complexes made with dioleyl succinyl paromomycin:O,O-dioleyl-N-histamine phosphoramidate (DOSP/MM27) liposomes or histidinylated lPEI (His-lPEI) allowing the highest transfection efficiency displayed a positive ζ potential and were internalized by clathrin-mediated endocytosis, (ii) DOSP/MM27 lipoplexes were 6-times more internalized than His-lPEI polyplexes, (iii) all negatively charged DNA complexes lead to less efficient transfection and entered the cells via caveolae and (iv) lipopolyplexes allowing high transfection efficiency were weakly internalized via caveolae. Our results indicate that the transfection efficiency is better correlated with the nature of the endocytic pathway than with the uptake efficacy. This study shows also that engineered cells expressing specific fluorescent compartments are convenient tools to monitor endocytosis of a fluorescent plasmid DNA by real time fluorescence confocal microscopy.     

Photo-activatable ternary complex based on a multifunctional shielding material for targeted shRNA delivery in cancer treatment

A photo-activatable ternary complex (PTC) consisting of multifunctional shielding material (MSM) with photosensitizer (PS)-conjugated chondroitin sulfate and polyethyleneimine based binary complexes containing epidermal growth factor receptor (EGFR)-shRNA delivery for CD44 targeted cancer therapy has been developed. The PTC has a negative surface charge of −37.9 mV, and a size of approximately 90 nm, and the ternary complexes were found to be stable against plasma proteins. The stable nanostructure of PTC especially could enable CD44-receptor mediated tumor-targeted delivery and PS-mediated endosomal disruption for efficient gene silencing, and an enhanced rate of cancer cell death was achieved both in vitro and in vivo. This suggests that PTC could represent a promising strategy for the delivery of other therapeutic genes for cancer therapy.     

Uptake and transfection efficiency of PEGylated cationic liposome–DNA complexes with and without RGD-tagging

Steric stabilization of cationic liposome–DNA (CL–DNA) complexes is required for in vivo applications such as gene therapy. PEGylation (PEG: poly(ethylene glycol)) of CL–DNA complexes by addition of PEG2000-lipids yields sterically stabilized nanoparticles but strongly reduces their gene delivery efficacy. PEGylation-induced weakening of the electrostatic binding of CL–DNA nanoparticles to cells (leading to reduced uptake) has been considered as a possible cause, but experimental results have been ambiguous. Using quantitative live-cell imaging in vitro, we have investigated cell attachment and uptake of PEGylated CL–DNA nanoparticles with and without a custom synthesized RGD-peptide grafted to the distal ends of PEG2000-lipids. The RGD-tagged nanoparticles exhibit strongly increased cellular attachment as well as uptake compared to nanoparticles without grafted peptide. Transfection efficiency of RGD-tagged PEGylated CL–DNA NPs increases by about an order of magnitude between NPs with low and high membrane charge density (σ_M; the average charge per unit area of the membrane; controlled by the molar ratio of cationic to neutral lipid), even though imaging data show that uptake of RGD-tagged particles is only slightly enhanced by high σ_M. This suggests that endosomal escape and, as a result, transfection efficiency of RGD-tagged NPs is facilitated by high σ_M. We present a model describing the interactions between PEGylated CL–DNA nanoparticles and the anionic cell membrane which shows how the PEG grafting density and membrane charge density affect adhesion of nanoparticles to the cell surface.     

Suppression of p53-activated gene,PAG608, attenuates methamphetamine-induced neurotoxicity

The p53-activated gene 608 (PAG608) is a proapoptotic gene activated and regulated by p53 expression in oxidative stress-induced apoptosis of neuronal cells. In this study, we determined the role of PAG608 in methamphetamine-induced neurotoxicity. Treatment of mouse dopaminergic CATH.a cells with 2 mM methamphetamine increased PAG608 expression at 3 h followed by increase in phosphorylated p53 expression. Transient transfection of PAG608 antisense cDNA or RNA interference using PAG608 small interfering RNA significantly attenuated the dose-dependent decrease in cell viability of CATH.a cells by methamphetamine (1–4 mM) exposure. In monoaminergic neuronal B65 cells, which contain serotonin rather than dopamine, methamphetamine-induced cell death was also significantly but partially protected by transient transfection of PAG608 antisense cDNA. Furthermore, cell death of PC12 cells produced by methamphetamine (1–5 mM) was almost completely prevented by stable expression of PAG608 antisense cDNA, compared with significant reduction of cell viability in control PC12 cells. Our results showed that suppression of PAG608 using transient and stable transfection with PAG608 antisense cDNA or small interfering RNA attenuates methamphetamine-induced death of various monoaminergic neuronal cells, suggesting that methamphetamine neurotoxicity in monoaminergic cells is related, at least in part, to induction of PAG608 expression.     

Ternary complexes of amphiphilic polycaprolactone-graft-poly (N,N-dimethylaminoethyl methacrylate), DNA and polyglutamic acid-graft-poly(ethylene glycol) for gene delivery

Binary complexes of cationic polymers and DNA were used commonly for DNA delivery, whereas, the excess cationic charge of the binary complexes mainly leads to high toxicity and unstability in vivo. In this paper, ternary complexes by coating polyglutamic acid-graft-poly(ethylene glycol)(PGA-g-mPEG) onto binary complexes of polycaprolactone-graft-poly(N,N-dimethylaminoethyl methacrylate) (PCL-g-PDMAEMA) nanoparticles (NPs)/DNA were firstly developed for effective and targeted gene delivery. The coating of PGA-g-mPEG was able to decrease the zeta potential of the nano-sized DNA complexes nearly to electroneutrality without interferring with DNA condensation ability. As a result, the stability, the escape ability from endosomes and the transfection efficiency of the complexes were enhanced. The ternary complexes of PCL-g-PDMAEMA NPs/DNA/PGA-g-mPEG demonstrated lower cytotoxicity in CCK-8 measurements and higher gene transfection efficiency than the binary complexes in vitro. In addition, Lactate dehydrogenase (LDH) assay was performed to quantify the membrane-damaging effects of the complexes, which is consistent with the conclusion of CCK-8 measurement for cytotoxicity assay. The in vivo imaging measurement and histochemical analysis of tumor sessions confirmed that the intravenous administration of the ternary complexes with red fluorescent protein (RFP) as payload led to protein expression in tumor, which was further enhanced by the targeted coating of PGA-g-PEG-folate.     

A facile preparation of novel multifunctional vectors by non-covalent bonds for co-delivery of doxorubicin and gene

In this study, novel multifunctional ternary complexes of biotinylated transferrin-avidin-biotin-poly(ethylene glycol)-poly(L-glutamate acid)/poly(2-(2-aminoethylamino) ethyl methacrylate)/doxorubicin-poly(L-aspartic acid)/pDNA (TAB/PIC-D/pDNA complexes) were prepared based on polyion complex micelles (PIC) and the avidin-biotin system, which aimed to target co-delivery of anti-cancer doxorubicin and gene. Cytotoxicity studies revealed that PIC-D could have anti-tumor effect on HeLa cells and HepG2 cells; TAB coating could increase the biocompatibility of PIC-D/pDNA complexes and the targeting delivery efficiency of doxorubicin. TAB/PIC-D/pDNA complexes possessed higher transfection efficiency than the unmodified complexes in serum, and transferrin could enhance luciferase expression in HeLa cells and HepG2 cells. Furthermore, confocal laser scanning microscopy showed that doxorubicin and gene could be delivered into HepG2 cells simultaneously by TAB/PIC-D/pDNA complexes. The formation of the ternary complexes provides a facile approach to constructing a multifunctional delivery system for targeted co-delivery of anticancer drugs and gene.     

Phosphorescent iridium(III) complexes as multicolor probes for specific mitochondrial imaging and tracking

In the present study, four phosphorescent iridium(III) complexes [Ir(C–N)₂(PhenSe)]⁺ (Ir1–Ir4, in which C–N = 2-(2,4-difluorophenyl)pyridine (dfppy), dibenzo[f,h]quinoxaline (dbq), 2-phenylquinoline (2-pq) and 2-phenylpyridine (ppy), PhenSe = 1,10-phenanthrolineselenazole) with tunable emission colors were developed to image mitochondria and track the dynamics of the mitochondrial morphology. In comparison with commercially available mitochondrial trackers, Ir1–Ir4 possess high specificity to mitochondria in live and fixed cells without requiring prior membrane permeabilization or the replacement of the culture medium. Due to the high resistance of Ir1–Ir4 to the loss of mitochondrial membrane potential as well as the appreciable tolerance to environmental changes, these complexes are applicable for the imaging and tracking of the mitochondrial morphological changes over long periods of time. In addition, Ir2–Ir4 exhibited superior photostability compared to the commercially available mitochondrial trackers. These colorful iridium(III) complexes may contribute to the future development of staining agents for organelle-selective imaging in living cells.