Potential Gene Delivery Vectors Research Articles

Novel Multi-Responsive Hyperbranched Polyelectrolyte Polyplexes as Potential Gene Delivery Vectors.

In this work, we investigate the complexation behavior of poly(oligo(ethylene glycol)methyl methacrylate)-co-poly(2-(diisopropylamino)ethyl methacrylate), P(OEGMA-co-DIPAEMA), hyperbranched polyelectrolyte copolymers, synthesized by reversible addition fragmentation chain transfer (RAFT) polymerization, with short-linear DNA molecules. The synthesized hyperbranched copolymers (HBC), having a different chemical composition, are prepared in order to study their ability to bind with a linear nucleic acid at various N/P ratios (amine over phosphate groups). Specifically, the three pH and thermo-responsive P(OEGMA-co-DIPAEMA) hyperbranched copolymers were able to form polyplexes with DNA, with dimensions in the nanoscale. Using several physicochemical methods, such as dynamic and electrophoretic light scattering (DLS, ELS), as well as fluorescence spectroscopy (FS), the complexation process and the properties of formed polyplexes were explored in response to physical and chemical stimuli such as temperature, pH, and ionic strength. The mass and the size of polyplexes are shown to be affected by the hydrophobicity of the copolymer utilized each time, as well as the N/P ratio. Additionally, the stability of polyplexes in the presence of serum proteins is found to be excellent. Finally, the multi-responsive hyperbranched copolymers were evaluated regarding their cytotoxicity via in vitro experiments on HEK 293 non-cancerous cell lines and found to be sufficiently non-toxic. Based on our results, these polyplexes could be useful candidates for gene delivery and related biomedical applications.

Star-shaped poly(l-lysine) with polyester bis-MPA dendritic core as potential degradable nano vectors for gene delivery

Efficient initiation from ammonium trifluoroacetate salts (TFA) dendritic end-groups yields well-defined poly(l-lysine) star polypeptides with 8, 16 and 32 arms. Hydrolytic core degradation and plasmid DNA complexation is demonstrated.

Complexation of Oligo- and Polynucleotides with Methoxyphenyl-Functionalized Imidazolium Surfactants.

Interaction between cationic surfactants and nucleic acids attracts much attention due to the possibility of using such systems for gene delivery. Herein, the lipoplexes based on cationic surfactants with imidazolium head group bearing methoxyphenyl fragment (MPI-n, n = 10, 12, 14, 16) and nucleic acids (oligonucleotide and plasmid DNA) were explored. The complex formation was confirmed by dynamic/electrophoretic light scattering, transmission electron microscopy, fluorescence spectroscopy, circular dichroism, and gel electrophoresis. The nanosized lipoplex formation (of about 100-200 nm), contributed by electrostatic, hydrophobic interactions, and intercalation mechanism, has been shown. Significant effects of the hydrocarbon tail length of surfactant and the type of nucleic acid on their interaction was revealed. The cytotoxic effect and transfection ability of lipoplexes studied were determined using M-HeLa, A549 cancer cell lines, and normal Chang liver cells. A selective reduced cytotoxic effect of the complexes on M-HeLa cancer cells was established, as well as a high ability of the systems to be transfected into cancer cells. MPI-n/DNA complexes showed a pronounced transfection activity equal to the commercial preparation Lipofectamine 3000. Thus, it has been shown that MPI-n surfactants are effective agents for nucleic acid condensation and can be considered as potential non-viral vectors for gene delivery.

Dual-Modified Liposome for Targeted and Enhanced Gene Delivery into Mice Brain.

The development of neuropharmaceutical gene delivery systems requires strategies to obtain efficient and effective brain targeting as well as blood-brain barrier (BBB) permeability. A brain-targeted gene delivery system based on a transferrin (Tf) and cell-penetrating peptide (CPP) dual-functionalized liposome, CPP-Tf-liposome, was designed and investigated for crossing BBB and permeating into the brain. We selected three sequences of CPPs [melittin, Kaposi fibroblast growth factor (kFGF), and penetration accelerating sequence-R8] and compared their ability to internalize into the cells and, subsequently, improve the transfection efficiency. Study of intracellular uptake indicated that liposomal penetration into bEnd.3 cells, primary astrocytes, and primary neurons occurred through multiple endocytosis pathways and surface modification with Tf and CPP enhanced the transfection efficiency of the nanoparticles. A coculture in vitro BBB model reproducing the in vivo anatomophysiological complexity of the biologic barrier was developed to characterize the penetrating properties of these designed liposomes. The dual-functionalized liposomes effectively crossed the in vitro barrier model followed by transfecting primary neurons. Liposome tissue distribution in vivo indicated superior ability of kFGF-Tf-liposomes to overcome BBB and reach brain of the mice after single intravenous administration. These findings demonstrate the feasibility of using strategically designed liposomes by combining Tf receptor targeting with enhanced cell penetration as a potential brain gene delivery vector. SIGNIFICANCE STATEMENT: Rational synthesis of efficient brain-targeted gene carrier included modification of liposomes with a target-specific ligand, transferrin, and with cell-penetrating peptide to enhance cellular internalization. Our study used an in vitro triple coculture blood-brain barrier (BBB) model as a tool to characterize the permeability across BBB and functionality of designed liposomes prior to in vivo biodistribution studies. Our study demonstrated that rational design and characterization of BBB permeability are efficient strategies for development of brain-targeted gene carriers.

Development of a novel bivalent baculovirus vectors for complement resistance and sustained transgene expression and its application in anti-angiogenesis gene therapy

Baculovirus (BV) is a potential gene delivery vector but only mediates transient transgene expression and easily inactivated by human complement. To this end, we intend to develop a novel bivalent BV vector for complement resistance and sustained transgene expression, and evaluate its effect in anti-angiogenesis gene therapy. The results showed that the hybrid bivalent BV significantly prolonged the expression of enhanced green fluorescent protein (eGFP) in vitro for at least 90 days at over 109 a.u. total fluorescence intensity, and exhibited significantly higher complement resistance. The control BV-mediated eGFP expression gradually declined within 15 days and showed lower transduction efficiency. In vivo studies confirmed that the hybrid bivalent BV exhibited longer duration of eGFP expression and higher transduction efficacy than the control BVs. Based on these findings, we further constructed a hybrid BV expressing the antiangiogenic fusion protein containing human endostatin and angiostatin (hEA). The hybrid BV-expressed hEA significantly prolonged the expression level of hEA with enhanced anti-angiogenic activities compared to the control groups, as evidenced by ELISA, cell proliferation, migration and tubular formation assays. With the stable expression of hEA, the hybrid BV conferred hEA more significant inhibitory effect on hepatocellular carcinoma tumor growth and significantly extended the life span of mice. These data implicate that the SB-based BV surface display system may have broad prospects as a novel platform for gene therapy of tumors.

The Major Hurdle for Effective Baculovirus Transduction into Mammalian Cells Is Passing Early Endosomes.

Baculoviruses, although they infect insects in nature, can transduce a wide variety of mammalian cells and are therefore promising gene therapy vectors. However, baculovirus transduction into many mammalian cells is very inefficient, and the limiting stages and factors remain unknown. An important finding is that a short-duration trigger with low pH can significantly enhance virus transduction efficiency, but the mechanism is poorly understood. Herein, we performed a detailed comparative study on entry mechanisms of the prototypical baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) into insect and mammalian cells. The results showed that AcMNPV could be internalized into mammalian cells efficiently, but fusion in early endosomes (EEs) appeared to be the major obstacle. Measurement of endosomal pH suggested that virus fusion might be restricted under relatively high-pH conditions in mammalian cells. Interestingly, mutations of the major viral fusion protein GP64 that conferred decreased fusogenicity did not affect virus infection of insect cells, whereas virus transduction into mammalian cells was severely impaired, suggesting a more stringent dependence on GP64 fusogenicity for AcMNPV entry into mammalian cells than into insect cells. An increase in the fusogenicity of GP64 mutants resulting from low pH triggered the rescue of fusion-deficient recombinant virus transduction efficiency. Based on the above-described findings, the pH of EEs was specifically reduced with a Na+/K+-ATPase inhibitor, and the AcMNPV transduction of many mammalian cells indeed became highly efficient. This study not only revealed the roadblocks to mammalian cell entry of baculovirus but also provides a new strategy for improving baculovirus-based gene delivery and therapy.IMPORTANCE Baculoviruses can transduce a wide variety of mammalian cells but do so with low efficiency, which greatly limits their practical application as potential gene delivery vectors. So far, the understanding of baculovirus entry into mammalian cells is obscure, and the limiting stages and factors are unclear. In this study, by comparatively analyzing the mechanisms of baculovirus entry into mammalian and insect cells, virus fusion during the early stage of endocytosis was revealed as the major obstacle for efficient baculovirus transduction into mammalian cells. A higher fusogenicity of the major viral fusion protein GP64 was found to be required for virus entry into mammalian cells than for entry into insect cells. Interestingly, by decreasing the pH of early endosomes with a specific agent, virus transduction of a wide range of mammalian cells was greatly enhanced. This study uncovers the roadblocks to mammalian cell entry of baculoviruses and presents mechanisms to overcome the roadblocks.

Dissecting the Cell Entry Pathway of Baculovirus by Single-Particle Tracking and Quantitative Electron Microscopic Analysis.

The budded virus of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) infects insect cells through mainly clathrin-mediated endocytosis. However, the cell entry pathway of AcMNPV remains unclear. In this study, by using population-based analysis of single-virus tracking and electron microscopy, we investigated the internalization, fusion behavior, and endocytic trafficking of AcMNPV. AcMNPV internalization into host insect cells was facilitated by actin polymerization and dynamin. After incorporation into early endosomes, the AcMNPV envelope fused with the membranes of early endosome, allowing for nucleocapsid release into the cytoplasm. Microtubules were implicated in the bidirectional and long-range transport of virus-containing endosomes. In addition, microtubule depolymerization reduced the motility of virus-bearing early endosomes, impairing the progression of infection beyond enlarged early endosomes. These findings demonstrated that AcMNPV internalization was facilitated by actin polymerization in a dynamin-dependent manner, and nucleocapsid release occurred in early endosomes in a microtubule-dependent manner. This study provides mechanistic and kinetic insights into AcMNPV infection and enhance our understanding of the infection pathway of baculoviruses.IMPORTANCE Baculoviruses are used widely as environmentally benign pesticides, protein expression systems, and potential mammalian gene delivery vectors. Despite the significant application value, little is known about the cell entry and endocytic trafficking pathways of baculoviruses. In this study, we demonstrated that the alphabaculovirus AcMNPV exhibited actin- and microtubule-dependent transport for nucleocapsid release predominantly from within early endosomes. In contrast to AcMNPV transduction in mammalian cells, its infection in host insect cells is facilitated by actin polymerization for internalization and microtubules for endocytic trafficking within early endosomes, implying that AcMNPV exhibits cell type specificity in the requirement of the cytoskeleton network. In addition, experimental depolymerization of microtubules impaired the progression of infection beyond enlarged early endosomes. This is the first study that dissects the cell entry pathway of baculoviruses in host cells at the single-particle level, which advances our understanding of the early steps of baculovirus entry.

Synthesis and characterization of polyester-based dendrimers containing peripheral arginine or mixed amino acids as potential vectors for gene and drug delivery

Macromolecular systems with well-defined sizes, shapes and high controlled architecture like dendrimers are of eminent interest in nanomedical applications such as drug delivery, gene transfection, and imaging. In this paper versatile protocols for the synthesis of polyester-based, hydrolysable, polycationic dendrimers have been setup. Fourth and fifth generation dendrimers equipped with several peripheral hydroxyl groups were prepared from 2, 2-bis(hydroxymethyl)propanoic acid. They were successfully esterified with arginine alone or mixed with lysine or O-methyltyrosine and with dipeptide arginine-glycine and seven polycationic dendrimers were finally obtained as hydrochlorides. Their structures and composition were confirmed by NMR analysis and by experimental molecular weight computed by volumetric titration and their buffer capacity was higher than fourth generation polyamidoamine (G4- PAMAM) derivatives taken as reference. The synthesized dendrimers harmonize a polycationic character and a buffer capacity which presuppose a good transfection efficiency with a degradable scaffold thus appearing as a promising team of new non-toxic vectors for biomedical applications.

Ammonium salt modified mesoporous silica nanoparticles for dual intracellular-responsive gene delivery

Effective gene delivery system plays an importmant role in the gene therapy. Mesoporous silica nanoparticle (MSN) has become one potential gene delivery vector because of its high stability, good biodegradability and low cytotoxicity. Herein, MSN-based dual intracellular responsive gene delivery system CMSN-A was designed and fabricated. Short chain ammonium group, which is modified with disulfide bond and amide bond simultaneously, is facilely grafted onto the mesoporous silica nanoparticles. As-synthesized CMSN-A is endowed with small size (80–110nm), large conical pores (15–23nm), and moderate Zeta potential (+25±2mV), which behaves high gene loading capacity, good stability and effectively gene transfection. Moreover, CMSN-A exhibits dual micro-environment responsive (lower pH, more reducing substances) due to the redox-sensitive disulfide bond and pH-sensitive amide bond in the short chain ammonium group. The cellular uptake study indicates that CMSN-A could transfer both plasmid DNA (pDNA) and siRNA into different kinds of tumour cells, which demonstrate the promising potential of CMSN-A as effective and safe gene-delivery vectors.

The synthesis and comparison of chondroitin sulfate-modified PDMAEMA with chondroitin sulfate-modified PEI as a potential gene delivery vector

CS-PDMAEMA obtained by reacting HS-PDMAEMA with CSMA via Michael addition, shows lower cytotoxicity and better transfection efficiency than PDMAEMA. The transfection efficiency is higher in 3T3 cells than in U87 cells owing to higher expression of ASGP-R.

Cytotoxic T lymphocyte antigen 4 decreases humoral and cellular immunity by adenovirus to enhance target GFP gene transfer in C57BL/6 mice

Adenoviruses (Ad) are once potential and promising vectors for gene delivery, but the immunogenicity attenuates its transfer efficiency. Cytotoxic T lymphocyte antigen 4 (CTLA-4) can inhibit T cell immunity. Thus, we aimed to study the effect of CTLA-4 in the process of Ad-mediated gene transfer. The C57BL/6 mice were injected by Ad vectors at twice, and CTLA-4 was administrated after the first Ad injection. Then, the CD3+CD4+ T cells and circulating levels of IL-2, IL-4, and anti-Ad IgG were decreased by CTLA-4, while Ad generated immune responses. The green fluorescence protein (GFP) expressions of tissues were enhanced by CTLA-4 till injection of Ad at twice. Our results indicate that CTLA-4 can inhibit humoral and cellular immunity by adenovirus generation to enhance GFP delivery, and provide a potential way to assist in Ad-mediated gene transfer.

Folic Acid Linked Chondroitin Sulfate-Polyethyleneimine Copolymer Based Gene Delivery System.

In our previous study, chondroitin sulfate-polyethylenimine copolymers (CP) have been synthesized and confirmed as potential gene delivery vectors. Efficient gene transfection is realized by chondroitin sulfate (ChS) that promotes CD44- mediated endocytosis and enhances the cellular uptake of CP/pDNA polyplexes besides clathrin-mediated endocytosis. In this study, the CP was functionalized with a folic acid (FA) molecule. This ancillary ligand allows polyplexes to bind with folate receptors (FR) in addition to the CD44 receptor. We conjugated FA-linked polyethylene glycol (FA-PEG) onto CP (FPCP) for tumor targeting and also synthesized mPEG-CP (MPCP) for comparison. The in vitro cell tests of polymer/pDNA polyplexes were done in FR-expressed U87 and FR-deficient A549 cells. The polymers exhibited less cytotoxicity than PEI-10K as well as PEI-25K against U87 and A549 cells. The transfection efficiency of FPCP/pDNA was higher than those of MPCP/pDNA and CP/pDNA. The cellular uptake pathways of FPCP/pDNA were tested in the cells in the presence of different endocytic chemical inhibitors. The CD44-, folate-, and caveolae-mediated pathways are involved in internalization of FPCP/pDNA. Recognition of FPCP to those receptors on the tumor surface is beneficial for enhanced cellular uptake of FPCP/pDNA, resulting in higher transgene expression than CP/pDNA and MPCP/pDNA.

Bioreducible cross-linked polymers based on G1 peptide dendrimer as potential gene delivery vectors

A series of cationic polymers based on low generation (G1) peptide dendrimer were synthesized with disulfide-containing linkages. The DNA binding abilities of the target polymers were studied by gel electrophoresis and fluorescence quenching assay. The bioreducible property of the disulfide-containing polymers P2 and P3 was also investigated in the presence of dithiothreitol (DTT). Results from dynamic light scattering (DLS) and transmission electron microscopy (TEM) assays reveal that these materials may condense DNA into nanoparticles with proper sizes and zeta-potentials. In vitro cell experiments show that compared to branched 25 KDa PEI, P2 and P3 may exhibit much higher gene transfection efficiency and lower cytotoxicity in both HEK293 and U-2OS cells. Additionally, polymer prepared from Michael addition gives better gene transfection ability, while polymer prepared from ring-opening reaction has better serum tolerance. Results indicate that these polymers might be promising non-viral gene vectors for their easy preparation, very low cytotoxicity, and good transfection efficiency.

Assessment of Cholesterol-Derived Ionic Copolymers as Potential Vectors for Gene Delivery

A library of cholesterol-derived ionic copolymers were previously synthesized via reversible addition-fragmentation chain transfer (RAFT) polymerization as 'smart' gene delivery vehicles that hold diverse surface charges. Polyplex systems formed with anionic poly(methacrylic acid-co-cholesteryl methacrylate) (P(MAA-co-CMA)) and cationic poly(dimethylamino ethyl methacrylate-co-cholesteryl methacrylate) (Q-P(DMAEMA-co-CMA)) copolymer series were evaluated for their therapeutic efficiency. Cell viability assays, conducted on SHEP, HepG2, H460, and MRC5 cell lines, revealed that alterations in the copolymer composition (CMA mol %) affected the cytotoxicity profile. Increasing the number of cholesterol moieties in Q-P(DMAEMA-co-CMA) copolymers reduced the overall toxicity (in H460 and HepG2 cells) while P(MAA-co-CMA) series displayed no significant toxicity regardless of the CMA content. Agarose gel electrophoresis was employed to investigate the formation of stable polyplexes and determine their complete conjugation ratios. P(MAA-co-CMA) copolymer series were conjugated to DNA through a cationic linker, oligolysine, while Q-P(DMAEMA-co-CMA)-siRNA complexes were readily formed via electrostatic interactions at conjugation ratios beginning from 6:1:1 (oligolysine-P(MAA-co-CMA)-DNA) and 20:1 (Q-P(DMAEMA-co-CMA)-siRNA), respectively. The hydrodynamic diameter, ζ potential and complex stability of the polyplexes were evaluated in accordance to complexation ratios and copolymer composition by dynamic light scattering (DLS). The therapeutic efficiency of the conjugates was assessed in SHEP cells via transfection and imaging assays using RT-qPCR, Western blotting, flow cytometry, and confocal microscopy. DNA transfection studies revealed P(MAA-co-CMA)-oligolysine-DNA ternary complexes to be ineffective transfection vehicles that mostly adhere to the cell surface as opposed to internalizing and partaking in endosomal disrupting activity. The transfection efficiency of Q-P(DMAEMA-co-CMA)-GFP siRNA complexes were found to be polymer composition and N/P ratio dependent, with Q-2% CMA-GFP siRNA polyplexes at N/P ratio 20:1 showing the highest gene suppression in GFP expressing SHEP cells. Cellular internalization studies suggested that Q-P(DMAEMA-co-CMA)-siRNA conjugates efficiently escaped the endolysosomal pathway and released siRNA into the cytoplasm. The gene delivery profile, reported herein, illuminates the positive and negative attributes of each therapeutic design and strongly suggests Q-P(DMAEMA-co-CMA)-siRNA particles are extremely promising candidates for in vivo applications of siRNA therapy.

Cyclodextrins for Non-Viral Gene and siRNA Delivery

Considerable research is focused on the development of non-viral vectors for gene and RNA interference therapies, with significant advancements in this field over the past number of years. Cationic lipids and polymers have been extensively investigated for these purposes, but there still remains a need for alternative vectors. Cyclodextrins (CDs) are cyclic oligosaccharides derived from starch and are well characterised pharmaceutical excipients. They offer many advantages as potential non-viral vectors for gene and siRNA delivery, in particular the ease with which they can be chemically modified and their lack of toxicity. In recent years, there has been a surge in the number of publications concerning CDs in this field. In this paper, we will review the two main approaches to the use of CDs for gene and siRNA delivery. In the first instance, CDs are used as a scaffold, to which various chemical groups can be grafted, yielding monodisperse functionalised CDs which can self-assemble in the presence of oligonucleotides. CDs are particularly amenable to chemical modification and this approach enables specific and precise design of CD vectors for targeting to various cell and tissue types. In the second approach, CDs can be included as a component of a delivery system, for example, as part of a polymer backbone, appended to a dendrimeric vector, or in polyrotaxane systems. Here, the inclusion of CDs facilitates postmodification of the vector through the formation of inclusion complexes with adamantane and, in some instances, reduces toxicity of the vector. Lastly, we will consider the development of in vivo CD vectors for therapeutic use and other novel applications including siRNA delivery in neurons and the CNS.

Cyclodextrins for Non-Viral Gene and siRNA Delivery

Considerable research is focused on the development of non-viral vectors for gene and RNA interference therapies, with significant advancements in this field over the past number of years. Cationic lipids and polymers have been extensively investigated for these purposes, but there still remains a need for alternative vectors. Cyclodextrins (CDs) are cyclic oligosaccharides derived from starch and are well characterised pharmaceutical excipients. They offer many advantages as potential non-viral vectors for gene and siRNA delivery, in particular the ease with which they can be chemically modified and their lack of toxicity. In recent years, there has been a surge in the number of publications concerning CDs in this field. In this paper, we will review the two main approaches to the use of CDs for gene and siRNA delivery. In the first instance, CDs are used as a scaffold, to which various chemical groups can be grafted, yielding monodisperse functionalised CDs which can self-assemble in the presence of oligonucleotides. CDs are particularly amenable to chemical modification and this approach enables specific and precise design of CD vectors for targeting to various cell and tissue types. In the second approach, CDs can be included as a component of a delivery system, for example, as part of a polymer backbone, appended to a dendrimeric vector, or in polyrotaxane systems. Here, the inclusion of CDs facilitates postmodification of the vector through the formation of inclusion complexes with adamantane and, in some instances, reduces toxicity of the vector. Lastly, we will consider the development of in vivo CD vectors for therapeutic use and other novel applications including siRNA delivery in neurons and the CNS. Keywords: Click chemistry, cyclodextrins, genes, non-viral delivery, pre-clinical, siRNA.

Exploring Quaternized Hydroxyethylcellulose as Potential Gene Carriers

AbstractCationic polysaccharides have been receiving more attentions and used as nonviral gene delivery vectors. In this paper, quaternized hydroxyethylcellulose (QHEC) derivatives were studied as gene carriers for their efficient DNA binding abilities. All QHECs could form stable QHEC/DNA complexes and resist the degradation of DNase I. And the dynamic light scatter (DLS) results showed that all QHEC/DNA complexes could form compact particles. These QHEC/DNA complexes exhibited effective transfection abilities in comparison to the naked DNA. The cytotoxicities of QHEC and QHEC/DNA complexes were also evaluated in four cell lines which were relatively low compared with 25 kDa bPEI. All results indicated that these quaternized hydroxyethylcelluloses could be used as potential gene delivery vectors.

Effect of Thiol Pendant Conjugates on Plasmid DNA Binding, Release, and Stability of Polymeric Delivery Vectors

Polymers have attracted much attention as potential gene delivery vectors due to their chemical and structural versatility. However, several challenges associated with polymeric carriers, including low transfection efficiencies, insufficient cargo release, and high cytotoxicity levels have prevented clinical implementation. Strong electrostatic interactions between polymeric carriers and DNA cargo can prohibit complete cargo release within the cell. As a result, cargo DNA never reaches the cell's nucleus where gene expression takes place. In addition, highly charged cationic polymers have been correlated with high cytotoxicity levels, making them unsuitable carriers in vivo. Using poly(allylamine) (PAA) as a model, we investigated how pH-sensitive disulfide cross-linked polymer networks can improve the delivery potential of cationic polymer carriers. To accomplish this, we conjugated thiol-terminated pendant chains onto the primary amines of PAA using 2-iminothiolane, developing three new polymer vectors with 5, 13, or 20% thiol modification. Unmodified PAA and thiol-conjugated polymers were tested for their ability to bind and release plasmid DNA, their capacity to protect genetic cargo from enzymatic degradation, and their potential for endolysosomal escape. Our results demonstrate that polymer-plasmid complexes (polyplexes) formed by the 13% thiolated polymer demonstrate the greatest delivery potential. At high N/P ratios, all thiolated polymers (but not unmodified counterparts) were able to resist decomplexation in the presence of heparin, a negatively charged polysaccharide used to mimic in vivo polyplex-protein interactions. Further, all thiolated polymers exhibited higher buffering capacities than unmodified PAA and, therefore, have a greater potential for endolysosomal escape. However, 5 and 20% thiolated polymers exhibited poor DNA binding-release kinetics, making them unsuitable carriers for gene delivery. The 13% thiolated polymers, on the other hand, displayed high DNA binding efficiency and pH-sensitive release.

Lower Rim Guanidinocalix[4]arenes: Macrocyclic Nonviral Vectors for Cell Transfection

Guanidinium groups were introduced through a spacer at the lower rim of calix[4]arenes in the cone conformation to give new potential nonviral vectors for gene delivery. Several structural modifications were explored, such as the presence or absence of a macrocyclic scaffold, lipophilicity of the backbone, length of the spacer, and nature of the charged groups, in order to better understand the factors which affect the DNA condensation ability and transfection efficiency of these derivatives. The most interesting compound was a calix[4]arene unsubstituted at the upper rim and having four guanidinium groups linked at the lower rim through a three carbon atom spacer. This compound, when formulated with DOPE, showed low toxicity and transfection efficiency higher than the commercially available lipofectamine LTX in the treatment of human Rhabdomiosarcoma and Vero cells. Most of the investigated compounds showed a tendency to self-aggregate in pure water or in the presence of salts, as evidenced by NMR and AFM studies, and it was found that the ability to condense DNA plasmids in nanometric globules is a necessary but not sufficient condition for transfection. The superiority of macrocyclic vectors over linear Gemini-type analogues and of guanidinium compared to other ammonium head groups in determining the biological activity of the vectors was also ascertained.

Effects of chirality on the intracellular localization of binuclear ruthenium(II) polypyridyl complexes

Interest in binuclear ruthenium(II) polypyridyl complexes as luminescent cellular imaging agents and for biomedical applications is increasing rapidly. We have investigated the cellular localization, uptake, and biomolecular interactions of the pure enantiomers of two structural isomers of [μ-bipb(phen)4Ru2]4+ (bipb is bis(imidazo[4,5-f]-1,10-phenanthrolin-2-yl)benzene and phen is 1,10-phenanthroline) using confocal laser scanning microscopy, emission spectroscopy, and linear dichroism. Both complexes display distinct enantiomeric differences in the staining pattern of fixed cells, which are concluded to arise from chiral discrimination in the binding to intracellular components. Uptake of complexes in live cells is efficient and nontoxic at 5 μM, and occurs through an energy-dependent mechanism. No differences in uptake are observed between the structural isomers or the enantiomers, suggesting that the interactions triggering uptake are rather insensitive to structural variations. Altogether, these findings show that the complexes investigated are promising for future applications as cellular imaging probes. In addition, linear dichroism shows that the complexes exhibit DNA-condensing properties, making them interesting as potential gene delivery vectors.Electronic supplementary materialThe online version of this article (doi:10.1007/s00775-012-0877-0) contains supplementary material, which is available to authorized users.