Integration of [12]aneN3 and Acenaphtho[1,2-b]quinoxaline as non-viral gene vectors with two-photon property for enhanced DNA/siRNA delivery and bioimaging.

Two-photon fluorescent Acenaphtho[1,2-b]quinoxaline (ANQ) and the hydrophilic di-(triazole-[12]aneN3) moieties were combined through an alkyl chain (ANQ-A-M) or a ß-hairpin motif with two aromatic γ-amino acid residues (ANQ-H-M) to explore their capabilities for in vitro and in vivo gene delivery and tracing. ANQ-A-M and ANQ-H-M showed the same maximum absorption at 420 nm, and their fluorescent intensities around 650 nm were varied in different solvents and became poor in the protic solvents. Gel electrophoresis assays indicated that both compounds completely retarded the migration of pDNA at 20 µM in the presence of DOPE. However, the DNA condensation with ANQ-H-M was not reversible, and the particle size of the corresponding complexes were larger indicated from the SEM and DLS measurements. In vitro transfections indicated ANQ-A-M/DOPE achieved Luciferase and GFP expressions were to be 7.9- and 5.7-fold of those by Lipo2000 in A549 cells respectively. However, ANQ-H-M showed very poor transfection efficiency in Luciferase expression. With the help of single/two-photon fluorescence imaging it clearly demonstrated that the successful transfection of ANQ-A-M was attributed to its cellular uptake, apparent lysosomal escape, and reversible release of DNA; and the poor transfection of ANQ-H-M was resulted from the aggregation of the DNA complexes which prevented them from the cellular uptake, and also the strong binding ability which is not easy to release DNA. ANQ-A-M/DOPE also exhibited robust gene silencing (83% knockdown of Luciferase) and GFP expression (2.47-fold higher) efficiency compared with Lipo2000 in A549 and zebrafish, respectively. The work demonstrated that the linkage structure between fluorescent and di(triazole-[12]aneN3) played the important role for their gene delivery performance, and that ANQ-A-M represents a vector with the strong transfection efficiency in vitro and in vivo as well as the efficient real time bioimaging properties, which is potential for the development in biomedical research.

Highly efficient RNAi and Cas9-based auto-cloning systems for C. elegans research.

RNA interference (RNAi) technology used for the functional analysis of Caenorhabditis elegans genes frequently leads to phenotypes with low penetrance or even proves completely ineffective. The methods previously developed to solve this problem were built on mutant genetic backgrounds, such as those defective for rrf-3, in which endogenous RNAi pathways are overexpressed. These mutations, however, interferes with many other genetic pathways so that the detected phenotype cannot always be clearly linked to the RNAi-exposed gene. In addition, using RNAi-overexpressing mutant backgrounds requires time-consuming genetic crossing. Here, we present an improved RNAi vector that produces specific double-stranded RNA species only, and thereby significantly stronger phenotypes than the standard gene knockdown vector. The further advantage of the new RNAi vector is that the detected phenotype can be specifically linked to the gene silenced. We also created a new all-in-one C. elegans Cas9 vector whose spacer sequence is much easier to replace. Both new vectors include a novel CRISPR/Cas9-based auto-cloning vector system rendering needless the use of restriction and ligase enzymes in generating DNA constructs. This novel, efficient RNAi and auto-cloning Cas9 systems can be easily adapted to any other genetic model.

Modular and Integrative Vectors for Synthetic Biology Applications in Streptomyces spp.

With the development of synthetic biology in the field of (actinobacterial) specialized metabolism, new tools are needed for the design or refactoring of biosynthetic gene clusters. If libraries of synthetic parts (such as promoters or ribosome binding sites) and DNA cloning methods have been developed, to our knowledge, not many vectors designed for the flexible cloning of biosynthetic gene clusters have been constructed. We report here the construction of a set of 12 standardized and modular vectors designed to afford the construction or the refactoring of biosynthetic gene clusters in Streptomyces species, using a large panel of cloning methods. Three different resistance cassettes and four orthogonal integration systems are proposed. In addition, FLP recombination target sites were incorporated to allow the recycling of antibiotic markers and to limit the risks of unwanted homologous recombination in Streptomyces strains when several vectors are used. The functionality and proper integration of the vectors in three commonly used Streptomyces strains, as well as the functionality of the Flp-catalyzed excision, were all confirmed. To illustrate some possible uses of our vectors, we refactored the albonoursin gene cluster from Streptomyces noursei using the BioBrick assembly method. We also used the seamless ligase chain reaction cloning method to assemble a transcription unit in one of the vectors and genetically complement a mutant strain.IMPORTANCE One of the strategies employed today to obtain new bioactive molecules with potential applications for human health (for example, antimicrobial or anticancer agents) is synthetic biology. Synthetic biology is used to biosynthesize new unnatural specialized metabolites or to force the expression of otherwise silent natural biosynthetic gene clusters. To assist the development of synthetic biology in the field of specialized metabolism, we constructed and are offering to the community a set of vectors that were intended to facilitate DNA assembly and integration in actinobacterial chromosomes. These vectors are compatible with various DNA cloning and assembling methods. They are standardized and modular, allowing the easy exchange of a module by another one of the same nature. Although designed for the assembly or the refactoring of specialized metabolite gene clusters, they have a broader potential utility, for example, for protein production or genetic complementation.

Inhibition of human lung adenocarcinoma growth and metastasis by JC polyomavirus-like particles packaged with an SP-B promoter-driven CD59-specific shRNA.

Lung cancer ranks first in both incidence and mortality and is a major health concern worldwide. Upon recognition of specific antigens on tumor cells, complement-dependent cytotoxicity (CDC) is activated, arresting cell growth or inducing apoptosis. However, by overexpressing CD59, a membrane complement regulatory protein (mCRP), lung cancer cells develop resistance to CDC. We previously showed that virus-like particles (VLPs) of human JC polyomavirus (JCPyV) could be used as a gene therapy vector to carry a suicide gene expression plasmid with a lung-specific promoter (SP-B (surfactant protein B)) for lung adenocarcinomas. Herein, we designed a CD59-specific short hairpin RNA (shRNA) expression plasmid driven by SP-B (pSPB-shCD59) to effectively and specifically inhibit CD59 overexpression in lung cancer cells. Treatment of lung cancer cells in vitro with JCPyV VLPs containing pSPB-shCD59 (pSPB-shCD59/VLPs) induces CDC and death of cancer cells. Mice that were subcutaneously injected with human lung cancer cells showed an 87% inhibition in tumor growth after tail vein injection of pSPB-shCD59/VLPs. Moreover, in a mouse model of lung cancer metastasis, a reduction in the lung weight by 39%, compared with the control group, was observed in mice treated with pSPB-shCD59/VLPs after tail vein injection of human lung cancer cells. Furthermore, tissue sectioning showed that the number and size of tumors produced was significantly reduced in the lungs of mice in the treatment group than those of the untreated group, indicating inhibition of metastasis by pSPB-shCD59/VLPs. Together, these results demonstrate the potential of pSPB-shCD59/VLPs as a therapeutic agent for CD59 overexpressed lung cancer.

[Synthesis and cell biological properties of polyaspartic acid drug/gene vector].

OBJECTIVE: Taking polysuccinimide as the main chain, amine side chain and alkyl side chain were grafted to prepare the drug/gene co-delivery vector. The property of the polymers with various side links were investigated to select an optimal vector. METHODS: Poly-D, L-polysuccinimide was synthesized by polymerization reaction of D, L-aspartic acid as monomer. Therefore, N, N-dimethylenedipropyl-triamine and 3, 3'-diaminodipropylamine were grafted with dodecylamine/adecylamine/octadecylamine at different proportions by ring-opening reaction to obtain amphiphilic PEECs. The structure of the material was confirmed by 1H NMR; the particle size and surface potential of the micelles were measured by dynamic light scattering; the critical micelle concentration (CMC) was determined by pyrene fluorescent probe; the RNA blocking ability was characterized by agarose gel electrophoresis; the release behavior of the PEECs was examined and the cytotoxicity, cellular uptake and gene silencing efficiency of the PEECs were studied at the cellular level. RESULTS: A series of PEECs with different grafting rates was successfully synthesized. The particle sizes and surface potential of the PEEC derived micelles were between 250 nm and 350 nm and 27 mV and 45 mV, respectively, with a small CMC value. The RNA binding ratio of PEECs was at a mass ratio of about 0.8:1. MTT assay demonstrated that PEEC micelles had certain cytotoxicity. PEECs had excellent micelle formation, drug-loading and gene binding abilities, particularly, PEEC16-2 showed high gene silencing efficiency at the cellular level. CONCLUSIONS: PEECs are able to co-delivery drug and gene, and PEEC16-2 micelles have the best ability of drug encapsulation and gene delivery.

Self-Assembled and Size-Controllable Oligonucleotide Nanospheres for Effective Antisense Gene Delivery through an Endocytosis-Independent Pathway.

The development of efficient gene delivery vectors has faced two major challenges, namely endo- and lysosomal escape and intracellular release. To address these problems, we developed an oligonucleotide (ON)-template-assisted polymerization approach to create ON nanospheres as gene vectors. Guanidinium-containing disulfide monomers were organized on the ON templates to increase their effective local concentrations. Consequently, ring-opening disulfide-exchange polymerization between monomers was accelerated, further facilitating the self-assembly of ON nanospheres. The size of these nanospheres was controlled by varying the length of the ON templates. Importantly, the nanospheres can be directly delivered into the cytosol through an endocytosis-independent pathway, which is followed by intracellular depolymerization in the reductive cytosolic environment to release the packaged ONs, resulting in efficient gene silencing. The ON nanospheres thus hold great promise as candidates for gene therapy.

Structure-activity relationship of novel low-generation dendrimers for gene delivery.

Structure-activity relationship (SAR) studies are very critical to design ideal gene vectors for gene delivery. However, It is difficult to obtain SAR information of low-generation dendrimers due to the lack of easy structural modification ways. Here, we synthesized a novel family of rigid aromatic backbone-based low-generation polyamidoamine (PAMAM) dendrimers. According to the number of primary amines, they were divided into two types: four-amine-containing PAMAM (DL1-DL5) and eight-amine-containing PAMAM (DL6-DL10). Due to the introduction of a rigid aromatic backbone, the low-generation PAMAM could be modified easier by different hydrophobic aliphatic chains. Several assays were used to study the interactions of the PAMAM dendrimers with plasmid DNA, and the results revealed that they not only had good DNA binding ability but also could efficiently condense DNA into spherical-shaped nanoparticles with suitable sizes and zeta potentials. The SAR studies indicated that the gene-transfection efficiency of the synthesized materials depended on not only the structure of their hydrophobic chains but also the number of primary amines. It was found that four-amine-containing PAMAM prepared from oleylamine (DL5) gave the best transfection efficiency, which was 3 times higher than that of lipofectamine 2000 in HEK293 cells. The cellular uptake mechanism mediated by DL5 was further investigated, and the results indicated that DL5/DNA complexes entered the cells mainly via caveolae and clathrin-mediated endocytosis. In addition, these low-generation PAMAMs modified with a single hydrophobic tail showed lower toxicity than lipofectamine 2000 in MC3T3-E1, MG63, HeLa, and HEK293 cells. These results reveal that such a type of low-generation polyamidoamines might be promising non-viral gene vectors, and also give us clues for the design of safe and high-efficiency gene vectors.

Construction strategies for developing expression vectors for recombinant monoclonal antibody production in CHO cells.

Recent years have seen the use of recombinant proteins in the treatment of different diseases. Among them, monoclonal antibodies (mAbs) are currently the fastest growing class of bio-therapeutic recombinant proteins. Chinese hamster ovary (CHO) cells are the most commonly used host cells for production of these recombinant mAbs. Expression vectors determine the expression level and quality of recombinant mAbs. Currently, few construction strategies for recombinant mAbs expression vectors in CHO cells have been developed, including monocistronic vector, multiple-promoter expression vector, and tricistronic vector mediated by internal ribosome entry site (IRES) or Furin-2A element. Among them, Furin-2A-mediated vector is an effective approach due to advantages of high "self-cleavage" efficiency, and equal expression of light and heavy chains from a single open reading frame. Here, we have reviewed the progress in development of different strategies for constructing recombinant mAb expression vectors in CHO cells and its potential advantages and disadvantages.

Polymer functionalized gold nanoparticles as nonviral gene delivery reagents.

BACKGROUND: In the present study, we investigated the ability of polyethylene glycol (PEG) functionalized gold nanoparticles to function as nonviral vectors in the transfection of different cell lines, comparing them with commercial lipoplexes. METHODS: Positively-charged gold nanoparticles were synthesized using polyethylenimine (PEI) as a reducing and stabilizer agent and its cytotoxicity was reduced by its functionalization with PEG. We bound the nanoparticles to three plasmids with different sizes (4-40 kpb). Vector internalization was evaluated by confocal and electronic microscopy. Its transfection efficacy was studied by fluorescence microscopy and flow cytometry. The application of the resulting vector in gene therapy was evaluated indirectly using ganciclovir in HeLa cells transfected to express the herpes virus thymidine kinase. RESULTS: An appropriate ratio between the nitrogen from the PEI and the phosphorous from the phosphate groups of the DNA, together with a reduced size and an elevated electrokinetic potential, are responsible for an increased nanoparticle internalization and enhanced protein expression when carrying plasmids of up to 40 kbp (plasmid size close to the limit of the DNA-carrying capacity of viral vectors). Compared to a commercial transfection reagent, an equal or even higher expression of reporter genes (on HeLa and Hek293t) and a suicide effect on HeLa cells transfected with the herpes virus thymidine kinase gene were observed when using this novel nanoparticulated vector. CONCLUSIONS: Nonviral vectors based on gold nanoparticles covalently coupled with PEG and PEI can be used as efficient transfection reagents showing expression levels that are the same or greater than those obtained with commercially available lipoplexes.

Organometallic DNA-B12 Conjugates as Potential Oligonucleotide Vectors: Synthesis and Structural and Binding Studies with Human Cobalamin-Transport Proteins.

The synthesis and structural characterization of Co-(dN)25 -Cbl (Cbl: cobalamin; dN: deoxynucleotide) and Co-(dN)39 -Cbl, which are organometallic DNA-B12 conjugates with single DNA strands consisting of 25 and 39 deoxynucleotides, respectively, and binding studies of these two DNA-Cbl conjugates to three homologous human Cbl transporting proteins, transcobalamin (TC), intrinsic factor (IF), and haptocorrin (HC), are reported. This investigation tests the suitability of such DNA-Cbls for the task of eventual in vivo oligonucleotide delivery. The binding of DNA-Cbl to TC, IF, and HC was investigated in competition with either a fluorescent Cbl derivative and Co-(dN)25 -Cbl, or radiolabeled vitamin B12 (57 Co-CNCbl) and Co-(dN)25 -Cbl or Co-(dN)39 -Cbl. Binding of the new DNA-Cbl conjugates was fast and tight with TC, but poorer with HC and IF, which extends a similar original finding with the simpler DNA-Cbl, Co-(dN)18 -Cbl. The contrasting affinities of TC versus IF and HC for the DNA-Cbl conjugates are rationalized herein by a stepwise mechanism of Cbl binding. Critical contributions to overall affinity result from gradual conformational adaptations of the Cbl-binding proteins to the DNA-Cbl, which is first bound to the respective ß domains. This transition is fast with TC, but slow with IF and HC, with which weaker binding results. The invariably tight interaction of the DNA-Cbl conjugates with TC makes the Cbl moiety a potential natural vector for the specific delivery of oligonucleotide loads from the blood into cells.

Gene Transfection Mediated by Catiomers Requires Free Highly Charged Polymer Chains To Overcome Intracellular Barriers.

The prospective use of the block copolymers poly(ethylene oxide)113-b-poly[2-(diethylamino)ethyl methacrylate]50 (PEO113-b-PDEA50) and poly[oligo(ethylene glycol)methyl ether methacrylate]70-b-poly[oligo(ethylene glycol)methyl ether methacrylate10-co-2-(diethylamino)ethyl methacrylate47-co-2-(diisopropylamino)ethyl methacrylate47] (POEGMA70-b-P(OEGMA10-co-DEA47-co-DPA47)) as nonviral gene vectors was evaluated. The polymers are able to properly condense DNA into nanosized particles (RH ≈ 75 nm), which are marginally cytotoxic and can be uptaken by cells. However, the green fluorescent protein (GFP) expression assays evidenced that DNA delivery is essentially negligible meaning that intracellular trafficking hampers efficient gene release. Subsequently, we demonstrate that cellular uptake and particularly the quantity of GFP-positive cells are substantially enhanced when the block copolymer polyplexes are produced and further supplemented by BPEI chains (branched polyethylenimine). The dynamic light scattering/electrophoretic light scattering/isothermal titration calorimetry data suggest that such a strategy allows the adsorption of BPEI onto the surface of the polyplexes, and this phenomenon is responsible for increasing the size and surface charge of the assemblies. Nevertheless, most of the BPEI chains remain freely diffusing in the systems. The biological assays confirmed that cellular uptake is enhanced in the presence of BPEI and principally, the free highly charged polymer chains play the central role in intracellular trafficking and gene transfection. These investigations pointed out that the transfection efficiency versus cytotoxicity issue can be balanced by a mixture of BPEI and less cytotoxic agents such as for instance the proposed block copolymers.

Oncolytic Adenovirus Coated with Multidegradable Bioreducible Core-Cross-Linked Polyethylenimine for Cancer Gene Therapy.

Recently, adenovirus (Ad) has been utilized as a viral vector for efficient gene delivery. However, substantial immunogenicity and toxicity have obstructed oncolytic Ad's transition into clinical studies. The goal of this study is to generate an adenoviral vector complexed with multidegradable bioreducible core-cross-linked polyethylenimine (rPEI) polymer that has low immunogenicity and toxicity while having higher transduction efficacy and stability. We have synthesized different molecular weight rPEIs and complexed with Ad at varying molar ratios to optimize delivery of the Ad/polymer complex. The size and surface charge of Ad/rPEIs were characterized. Of note, Ad/rPEIs showed significantly enhanced transduction efficiency compared to either naked Ad or Ad/25 kDa PEI in both coxsackievirus and adenovirus receptor (CAR) positive and negative cancer cells. The cellular uptake result demonstrated that the relatively small size of Ad/16 kDa rPEIs (below 200 nm) was more critical to the complex's internalization than its surface charge. Cancer cell killing effect and viral production were significantly increased when oncolytic Ad (RdB/shMet, or oAd) was complexed with 16 kDa rPEI in comparison to naked oAd-, oAd/25 kDa PEI-, or oAd/32 kDa rPEI-treated cells. This increased anticancer cytotoxicity was more readily apparent in CAR-negative MCF7 cells, implying that it can be used to treat a broad range of cancer cells. Furthermore, A549 and HT1080 cancer cells treated with oAd/16 kDa rPEI had significantly decreased Met and VEGF expression compared to either naked oAd or oAd/25 kDa PEI. Overall, these results demonstrate that shMet expressing oncolytic Ad complexed with multidegradable bioreducible core-cross-linked PEI could be used as efficient and safe cancer gene therapy.

Engineered glycated amino dendritic polymers as specific nonviral gene delivery vectors targeting the receptor for advanced glycation end products.

The receptor for advanced glycation end products (RAGE) is involved in diabetes or angiogenesis in tumors. Under pathological conditions, RAGE is overexpressed and upon ligand binding and internalization stimulates signaling pathways that promote cell proliferation. In this work, amino dendritic polymers PEI 25 kDa and alkylated derivatives of PAMAM-G2 were engineered by the nonenzymatic Maillard glycation reaction to generate novel AGE-containing gene delivery vectors targeting the RAGE. The glycated dendritic polymers were easily prepared and retained the capability to bind and protect DNA from endonucleases. Furthermore, while glycation decreased the transfection efficiency of the dendriplexes in CHO-k1 cells which do not express RAGE, glycated dendriplexes acted as efficient transfection reagents in CHO-k1 cells which stably express recombinant RAGE. In addition, preincubation with BSA-AGEs, a natural ligand of the RAGE, or dansyl cadaverine, an inhibitor of the RAGE internalization, blocked transfection, confirming their specificity toward RAGE. The results were confirmed in NRK and RAW264.7 cell lines, which naturally express the receptor. The glycated compounds retain their transfection efficiency in the presence of serum and promote in vivo transfection in a mouse model. Accordingly, RAGE is a suitable molecular target for the development of site-directed engineered glycated nonviral gene vectors.

Hepatic-targeted gene delivery using cationic mannan vehicle.

The incidence of hepatic diseases continuously increases worldwide and causes significant mortality because of inefficient pharmacotherapy. Gene therapy is a new strategy in the treatment of hepatic diseases, but the disadvantages of insufficient retention in the liver and undesirable side effects hinder its application. In this study, we developed a novel nonviral vehicle targeted to liver. Mannan was cationized with spermine at varying grafted ratios to deliver the gene and was optimized in cytotoxicity and transfection in vitro. A spermine-mannan (SM)-based delivery system was proven to be hepatic targeted and was capable of prolonging the gene retention period in the liver. Moreover, SM at N/P of 20 was confirmed to be less interfered with by the serum. Optimized SM vehicle has relatively high hepatic transfection with almost no toxicity induction in the liver, which highlighted its potential in the treatment of hepatic diseases.

Amphiphilic polyethylenimine (PEI) as highly efficient non-viral gene carrier.

Efficient and safe gene vectors are important for gene therapy. Here, a novel family of amphiphilic polyethylenimine (PEI) LD1-PEI bearing a polar group of branched PEI 25K and four dodecyl chains was developed. Agarose gel electrophoresis was used to confirm the formation of complexes. The transfection activity of the amphiphilic carrier was evaluated in different cell lines. The in vitro study showed that LD1-PEI showed a higher transfection efficiency with improved biocompatibility than PEI 25K. Serum showed almost no or only a slight effect on LD1-PEI/DNA transfection efficiency. In summary, LD1-PEI is a promising nonviral gene carrier.

Properties and evaluation of quaternized chitosan/lipid cation polymeric liposomes for cancer-targeted gene delivery.

Development of high-stability and efficient nonviral vectors with low cytoxicity is important for targeted tumor gene therapy. In this study, cationic polymeric liposomes (CPLs), with similar lipid bilayer structure and high thermal stability, were prepared from polymeric surfactants of quaternized (carboxymethyl)chitosan with different carbon chains (dodecyl, tetradecyl, hexadecyl, and octadecyl). By comparing different factors that influence gene delivery, tetradecyl-quaternized (carboxymethy)chitosan (TQCMC) CPLs, with suitable size (184.4 ± 17.1 nm), ζ potentials (27.5 ± 4.9 mV), and productivity for synthesis TQCMC (weight yield 13.1%), were selected for gene transfection evaluation in various cancer cell lines. Although TQCMC CPLs have lower gene transfection efficiency compared with cationic liposomes (Lipofectamine 2000) in vitro, they displayed higher reporter gene delivery ability for cancer tissues (bearing U87 and SMMC-7721 tumors) in vivo after intravenous injection. TQCMC CPLs also have lower cell cytotoxicity and lower cytokine production or liver injury for BALB/c mice. We conclude that the CPLs are promising gene delivery systems that may be used to target various cancers.

[Genetic, immunological, and pharmacological strategies to generate improved oncolytic viruses]. / Stratégies génétiques, immunologiques et pharmacologiques au service d'une nouvelle génération de virus anticancéreux.

Since over a century, medical literature has reported cases of viral infections leading to tumour regression. This phenomenon, now understood, can be exploited for cancer therapy. It involves viruses defined as "oncolytic". These viruses, either wild-type or genetically engineered, replicate preferentially in malignant cells. They induce tumour regression through various mechanisms including direct cell lysis and stimulation of an anti-tumour immune response. Several oncolytic viruses have reached late-stage clinical investigation and could be approved soon for treating certain neoplasms. While already promising, there is still room for improvement and various genetic, immunological, and pharmacological strategies are currently under development to increase their therapeutic efficacy.

Purification and characterization of the human γ-secretase activating protein.

Alzheimer's disease is a fatal neurological disorder that is a leading cause of death, with its prevalence increasing as the average life expectancy increases worldwide. There is an urgent need to develop new therapeutics for this disease. A newly described protein, the γ-secretase activating protein (GSAP), has been proposed to promote elevated levels of amyloid-ß production, an activity that seems to be inhibited using the well-establish cancer drug, imatinib (Gleevec). Despite much interest in this protein, there has been little biochemical characterization of GSAP. Here we report protocols for the recombinant bacterial expression and purification of this potentially important protein. GSAP is expressed in inclusion bodies, which can be solubilized using harsh detergents or urea; however, traditional methods of refolding were not successful in generating soluble forms of the protein that contained well-ordered and homogeneous tertiary structure. However, GSAP could be solubilized in detergent micelle solutions, where it was seen to be largely α-helical but to adopt only heterogeneous tertiary structure. Under these same conditions, GSAP did not associate with either imatinib or the 99-residue transmembrane C-terminal domain of the amyloid precursor protein. These results highlight the challenges that will be faced in attempts to manipulate and characterize this protein.

Synthesis and gene transfer activities of novel serum compatible reducible tocopherol-based cationic lipids.

The molecular structure of the cationic lipids greatly influences their transfection efficiency. High transfection efficiencies of tocopherol-based simple monocationic transfection lipids with hydroxylethyl headgroups were recently reported by us (Kedika, B., et al. J. Med. Chem.2011, 54 (2), 548-561). Toward enhancing the transfection efficiency of tocopherol-based lipids, we have synthesized two tocopherol-based dicationic lipids (1 and 2) using simple cystine in the headgroup region. The efficiency of tocopherol-based lipids (1 and 2) were compared with nontocopherol-based lipids (3 and 4) with cystine in the headgroup region. We report also a comprehensive structure-activity relationship study that identified tocopherol-based gemini cationic lipid 1 is a better transfecting agent than its monomeric lipid counterpart 2 and two other nontocopherol-based gemini cationic lipids (3 and 4). The transfection efficiency of lipid 1 was also greater than that of commercial formulation in HepG2 cell lines. A major characteristic feature of this investigation is that serum does not inhibit the transfection activity of tocopherol-based lipids (1 and 2) in general and in particular lipid 1 which is found to be highly serum-compatible even at higher concentrations of serum when compared to its monomeric counterpart lipid 2 and the other two control lipid analogues 3 and 4.

Cyclam-based polymeric copper chelators for gene delivery and potential PET imaging.

A series of reducible polycationic copper chelators (RPCs) based on 1,4,8,11-tetraazacyclotetradecane (cyclam) were synthesized by Michael addition. Molecular weight of the polycations was controlled by reaction stoichiometry and reaction conditions, resulting in polymers with molecular weights ranging from 4400 to 13 800. The cyclam moieties in the polycations retained their ability to form complexes with Cu(II). The presence of disulfide bonds in the polycations resulted in substantially lower cytotoxicity than control 25 kDa poly(ethyleneimine). RPC as well as their complexes with Cu(II) exhibited high transfection activity in vitro. The reported polycationic Cu(II) chelates represent promising nucleic acid delivery vectors with potential for future theranostic applications.