Selective attachment of a microtubule interacting peptide to plasmid DNA via a triplex forming oligonucleotide for transfection improvement.

In nonviral gene therapy approaches, the linkage of signal molecules to plasmid DNA (pDNA) is of interest for guiding its delivery to the nucleus. Here, we report its linkage to a peptide (P79-98) mediating migration on microtubules by using a triplex-forming oligonucleotide (TFO). pDNA of 5 kbp and 21 kbp containing 6 and 36 oligopurine • oligopyrimidine sites (TH), respectively, inserted outside the luciferase gene sequence were used. TFO with a dibenzocyclooctyl (DBCO) group in 3' end comprising some Bridged Nucleic Acid bases was conjugated by click chemistry with the peptide carrying an azide function in the C-terminal end. We found the formation of 6 and 18 triplex with pDNA of 5 kbp and 21 kbp, respectively. A twofold increase of the transfection efficiency was observed in the hind-limbs upon Hydrodynamic Limb Vein (HLV) injection in mice of naked P79-98 -pDNA of 21 kbp. This work paves the way for the selective equipping of pDNA with intracellular targeting molecules while preserving the full expression of the encoded gene.

Use of Primary and Secondary Polyvinylamines for Efficient Gene Transfection.

Gene transfection with polymeric carrier remains a challenge; particularly, high transfection levels combined with low toxicity are hard to achieve. We herein revisit polyvinylamines, an old and neglected family of cationic polymers. They can be readily obtained by controlled hydrolysis of polyvinylamides prepared through (controlled) radical polymerization. A series of tailor-made and well-defined polyvinylamines bearing primary amino groups, and poly(N-methylvinylamine) bearing secondary amines, were evaluated for the transfection of cells with pDNA as a function of their molar mass, molar mass distribution, and degree of deacetylation. Unexpected high transfection levels, in combination with low cytotoxicity were recorded for both series. Surprisingly, a great impact of the molar mass was observed for the primary amine polyvinylamine series, whereas the results were mostly independent of molar mass or dispersity for the polymer bearing secondary amine. It was further established that a certain percentage of acetamide groups increased the transfection level, while maintaining low cytotoxicity. These results highlight for the first time the real potential of polyvinylamines as gene carriers, and make these polymers very attractive for further development in gene therapy.

Intracellular Availability of pDNA and mRNA after Transfection: A Comparative Study among Polyplexes, Lipoplexes, and Lipopolyplexes.

Intracellular availability of nucleic acids from synthetic vectors is critical and directly influences the transfection efficiency (TE). Herein, we evaluated the TE of polymer- and lipid-based nanoplexes (polyplexes, lipoplexes and lipopolyplexes) of EGFP-encoding mRNA and pDNA. To determine the translation and transcription efficiency of each nucleic acid nanoplex, in vitro expression was measured in HEK293T7 cells that permit gene expression in the cytoplasmic region. Globally, mRNA transfection profile was well corroborative with cytoplasmic transfection of pT7-pDNA as well as with nuclear transfection of pCMV-DNA. Irrespective of the nucleic acid, high TE was observed with histidinylated l-polyethylenimine (His-lPEI) polyplexes and dioleyl succinyl paromomycin/O,O-dioleyl-N-histamine phosphoramidate (DOPS/MM27) lipoplexes. Moreover, His-lPEI polyplexes yielded higher in vitro expression of EGFP for pDNA than for mRNA. Furthermore, a significant enhancement in the TE in the presence of an excess of His-lPEI was observed indicating that this polymer promotes cytosolic delivery. Compared to other nanoplexes, His-lPEI polyplex showed high intracellular availability of DNA and mRNA along with low cytotoxicity, owing to its rapid (complete or partial) unpacking in the cytosol and/or endosomes. This study gives an insight that, whether with mRNA or pDNA, enhancing nanoplex unpacking in the endosomes and cytosol would improve the delivery of nucleic acid in the cytosol and particularly in the case of pDNA where a sufficient available amount of pDNA in the cytoplasm would definitely improve its transport toward the nucleus.

Poly(2-methyl-2-oxazoline)-b-poly(tetrahydrofuran)-b-poly(2-methyl-2-oxazoline) amphiphilic triblock copolymers: synthesis, physicochemical characterizations, and hydrosolubilizing properties.

Block copolymers assembled into micelles have gained a lot of attention to improve drug delivery. The recent drawbacks of the poly(ethylene oxide) blocks (PEO) contained in amphiphilic pluronics derivatives made of a central poly(propylene oxide) block surrounded by two PEO blocks were recently revealed, opening the way to the design of new amphiphilic block copolymers able to self-assemble in water and to entrap molecules of interest. Here, a family of p(methyloxazoline)-b-p(tetrahydrofuran)-b-p(methyloxazoline) triblock copolymers (called TBCP) is synthesized using cationic ring opening polymerization. Studies of micelle formation using dynamic light scattering, isothermal titration calorimetry (ITC), NMR diffusion-ordered spectroscopy (DOSY), and fluorescence experiments lead us to draw a relationship between copolymer structure and the physicochemical properties of the block copolymers (critical micellar concentration (CMC), Nagg, core diameter, shell thickness, etc.). The packing parameter of the block copolymers indicates the formation of a core-corona structure. Hydrosolubilizing properties of TBCPs were exemplified with curcumin selected as a highly insoluble drug model. Curcumin, a natural polyphenolic compound, has shown a large spectrum of biological and pharmacological activity, including anti-inflammatory, antimicrobial, antioxidant, and anticarcinogenic activities. An optimized formulation process reveals that the aggregation number is the parameter affecting drug encapsulation. Patch clamp experiments carried out to study the interaction of TBCP with the cell membrane demonstrate their permeation property suitable to promote the cellular internalization of curcumin.

Improved nuclear delivery of antisense 2'-Ome RNA by conjugation with the histidine-rich peptide H5WYG.

BACKGROUND: Antisense oligonucleotides are promising medicines for treating various diseases, although their efficiency still requires high doses. Their delivery in the cytosol and nucleus to reach their mRNA targets would increase their efficiency at the same time as reducing the dose. METHODS: We conjugated the histidine-rich peptide H5WYG (GLFHAIAHFIHGGWHGLIHGWYG) at the 5'-end of the RNase H-incompetent antisense 2'-O-methyl-phosphodiester oligonucleotide (2'-Ome RNA705) targeting aberrant splicing of luciferase pre-mRNA in HeLa pLuc705 cells. H5WYG was also conjugated with 2'-Ome-RNA705 labelled by fluorescein at the 3'-end. Then, H5WYG-2'-Ome-RNA705 conjugate and 2'-Ome-RNA705 were formulated with lipofectamine to favor their uptake in HeLa pLuc705 cells. RESULTS: Confocal microscopy showed that, after 4 h and overnight incubation, the presence of fluorescein-labelled 2'-Ome-RNA705 in the cytosol and nucleus was enhanced when the oligonucleotide was conjugated with H5WYG. We found that H5WYG-2'-Ome-RNA705 increased the splicing redirection and restoration of a functional luciferase mRNA. Luciferase activity and luciferase mRNA levels in these cells were 6.6- and two-fold higher, respectively, with H5WYG-2'-Ome-RNA705 than with 2'-Ome-RNA705. CONCLUSIONS: The results of the present study show that the conjugation of 2'-Ome antisense RNA to peptide H5WYG is a good strategy for improving its cytosol delivery, accumulation in the nucleus and antisense activity.

Enhancing natural killer cells proliferation and cytotoxicity using imidazole-based lipid nanoparticles encapsulating interleukin-2 mRNA.

mRNA applications have undergone unprecedented applications-from vaccination to cell therapy. Natural killer (NK) cells are recognized to have a significant potential in immunotherapy. NK-based cell therapy has drawn attention as allogenic graft with a minimal graft-versus-host risk leading to easier off-the-shelf production. NK cells can be engineered with either viral vectors or electroporation, involving high costs, risks, and toxicity, emphasizing the need for alternative way as mRNA technology. We successfully developed, screened, and optimized novel lipid-based platforms based on imidazole lipids. Formulations are produced by microfluidic mixing and exhibit a size of approximately 100 nm with a polydispersity index of less than 0.2. They are able to transfect NK-92 cells, KHYG-1 cells, and primary NK cells with high efficiency without cytotoxicity, while Lipofectamine Messenger Max and D-Lin-MC3 lipid nanoparticle-based formulations do not. Moreover, the translation of non-modified mRNA was higher and more stable in time compared with a modified one. Remarkably, the delivery of therapeutically relevant interleukin 2 mRNA resulted in extended viability together with preserved activation markers and cytotoxic ability of both NK cell lines and primary NK cells. Altogether, our platforms feature all prerequisites needed for the successful deployment of NK-based therapeutic strategies.

An E3-14.7K peptide that promotes microtubules-mediated transport of plasmid DNA increases polyplexes transfection efficiency.

Chemical vectors as cationic polymers and cationic lipids are promising alternatives to viral vectors for gene therapy. Beside endosome escape and nuclear import, plasmid DNA (pDNA) migration in the cytosol toward the nuclear envelope is also regarded as a limiting step for efficient DNA transfection with non-viral vectors. Here, the interaction between E3-14.7K and FIP-1 to favor migration of pDNA along microtubules is exploited. E3-14.7K is an early protein of human adenoviruses that interacts via FIP-1 (Fourteen.7K Interacting Protein 1) protein with the light-chain components of the human microtubule motor protein dynein (TCTEL1). This peptide is conjugated with pDNA and mediates interaction of pDNA in vitro with isolated microtubules as well as with microtubules in cellulo. Videomicroscopy and tracking treatment of images clearly demonstrate that P79-98/pDNA conjugate exhibits a linear transport with large amplitude along microtubules upon 2 h transfection with polyplexes whereas control pDNA conjugate exhibits small non-directional movements in the cytoplasm. Remarkably, P79-98/peGFP polyplexes enhance by a factor 2.5 (up to 76%) the number of transfected cells. The results demonstrate, for the first time, that the transfection efficiency of polyplexes can be drastically increased when the microtubules migration of pDNA is facilitated by a peptide allowing pDNA docking to TCTEL1. This is a real breakthrough in the non viral gene delivery field that opens hope to build artificial viruses.

Impact of net charge, targeting ligand amount and mRNA modification on the uptake, intracellular routing and the transfection efficiency of mRNA lipopolyplexes in dendritic cells.

Targeting mRNA formulations to achieve cell specificity is one of the challenges that must be tackled to mettle their therapeutic potential. Here, lipopolyplexes (LPR) bearing tri-mannose-lipid (TM) are used to target mannose receptor on dendritic cells. We investigated the impact of the net charge and percentage of TM units on the binding, uptake, transfection efficiency (TE) and RNA sensors activation. Binding and uptake capacities of naked and targeted LPR increase with the percent of cationic lipid, but the latter are 2-fold more up taken by the cells. Cationic LPR bearing 5 % and 10 % TM were localized in acidic compartments in contrast to naked LPR and 2.5 % TM-LPR. The drawback is the dramatic decrease of TE as the number of TM-units increases. Cationic LPR bearing 5 % and 10 % TM strongly induced NF-κB and PKR phosphorylation at 6 h. Conversely, mTOR is less activated in line with their low TE. Those side effects are overcome by using 5-methoxyuridine mRNA resulting in an improved TE due to non-phosphorylation of NF-κB and PKR and mTOR activation. Our results point out that targeting DC via mannose receptor triggers a higher uptake of cationic LPRs and fast routing to acidic compartments, and that efficient TE requires low number of TM units use or modified mRNA to escape RNA sensors activation to enhance the translation.

CEBA: A new heterobifunctional reagent for plasmid DNA functionalization by click chemistry.

Here, we report the synthesis of 3,6,9-trioxaundecan-1-{4-[(2-Chloroethyl)Ethylamino)]-Benzylamino},11-Azide (CEBA). CEBA alkylates the N7 of guanine of DNA thanks its chloroethyl group and can be coupled by a strain-promoted azide-alkyne cycloaddition to an alkynylated molecule. The optimization of the alkylation level of pDNA reveals that the expression of the encoded gene is preserved when it is randomly modified with at most 1 CEBA molecule per 150 bp. We show that the azido group of CEBA allows the linkage via click chemistry of CEBA-pDNA with a fluorophore or a peptide containing a dibenzocyclooctyne (DBCO) function. This new heterobifunctional reagent opens new ways to equip pDNA easily with signal molecules including peptides and nucleic acids without side products providing great interest for non-viral gene therapy.

Nuclear delivery of NFkappaB-assisted DNA/polymer complexes: plasmid DNA quantitation by confocal laser scanning microscopy and evidence of nuclear polyplexes by FRET imaging.

Quantification of a plasmid DNA (pDNA) and investigation of its polymer-associated state in the nucleus are crucial to evaluate the effectiveness of a gene-delivery system. This study was conducted with p3NF-luc-3NF, a pDNA-bearing optimized kappaB motif to favour NFkappaB-driven nuclear import. Here, a quantification of pDNA copies in the nucleus was performed by real-time confocal laser scanning microscopy in HeLa and C2C12 cells transfected with linear polyethylenimine or histidylated polylysine. Förster Resonance Energy Transfer (FRET) from the fluorescein-p3NF-luc-3NF donor to the co-localized rhodamine-polymer acceptor was carried out to investigate whether the pDNA was still condensed with the polymer in the nucleus. Upon 5 h of transfection, the nuclear amount of p3NF-luc3NF was approximately 1500 copies in both cell lines whereas that of pTAL-luc, a 3NF-free counterpart pDNA, was less than 250. This quantity of p3NF-luc-3NF dropped dramatically to that of pTAL-luc in the presence of the BAY 11-7085, an inhibitor of NFkappaB activation. These data strongly support a nuclear import of p3NF-luc3NF mediated by NFkappaB. Moreover, FRET experiments clearly revealed that most of nuclear pDNA were still condensed with the polymer raising the question of their passage through the nuclear pore complex and their impact on the gene-expression efficiency.

Co-delivery of NS1 and BMP2 mRNAs to murine pluripotent stem cells leads to enhanced BMP-2 expression and osteogenic differentiation.

Application of messenger RNA (mRNA) for bone regeneration is a promising alternative to DNA, recombinant proteins and peptides. However, exogenous in vitro transcribed mRNA (IVT mRNA) triggers innate immune response resulting in mRNA degradation and translation inhibition. Inspired by the ability of viral immune evasion proteins to inhibit host cell responses against viral RNA, we applied non-structural protein-1 (NS1) from Influenza A virus (A/Texas/36/1991) as an IVT mRNA enhancer. We evidenced a dose-dependent blocking of RNA sensors by NS1 expression. The co-delivery of NS1 mRNA with mRNA of reporter genes significantly increased the translation efficiency. Interestingly, unlike the use of nucleosides modification, NS1-mediated mRNA translation enhancement does not dependent to cell type. Dual delivery of NS1 mRNA and BMP-2 mRNA to murine pluripotent stem cells (C3H10T1/2), promoted osteogenic differentiation evidenced by enhanced expression of osteoblastic markers (e.g. alkaline phosphatase, type I collagen, osteopontin, and osteocalcin), and extracellular mineralization. Overall, these results support the adjuvant potentiality of NS1 for mRNA-based regenerative therapies. STATEMENT OF SIGNIFICANCE: mRNA therapy has the potential to improve the efficiency of nucleic acid based regenerative medicine. Up to now, the incorporation of expensive modified nucleotides is a common way to avoid IVT mRNA-induced detrimental immunogenicity. We here introduce co-delivery of Influenza virus immune evasion protein-NS1 coding mRNA as a strategy to suppress RNA sensors for maximizing IVT mRNA expression. An increased osteogenic commitment of pluripotent stem cells was observed after BMP2 mRNA and NS1 mRNA delivery. This study revealed how applying non-modified mRNA with NS1 could be a promising alternative as a therapeutic in bone regeneration.

Comparative binding and uptake of liposomes decorated with mannose oligosaccharides by cells expressing the mannose receptor or DC-SIGN.

Mannose Receptor (MR) and DC-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) are two mannose-specific targets for antigens carried by liposomes but DC-SIGN is more specific of DCs. Here, DC targeting is addressed by using DPPC/DOPE liposomes decorated with a series of diether lipids with a polar head of either a mannose (Man), tri-antenna of α-d-mannopyranoside (Tri-Man), [Manα1-3(Manα1-6)Man] (Man-tri), pseudo-Man4 (PMan4) or pseudo-Man5 (PMan5). Liposomes decorated with Man-Tri show the highest binding and internalization in cells expressing DC-SIGN and in human monocytes-derived DCs. Conversely, cells expressing MR bind and take up Tri-Man liposomes 3-fold higher than Man-tri liposomes. Comparatively, liposomes decorated with PMan4 and PMan5 do not show any advantages. Overall, the results indicate that liposomes decorated with Man-tri residues are more selective toward DCs than those with Tri-Man thanks to better recognition by DC-SIGN.

An optimized extended DNA kappa B site that enhances plasmid DNA nuclear import and gene expression.

BACKGROUND: The nuclear factor kappa B (NF kappaB) transcription factor, which shuttles between the cytoplasm and the nucleus under specific conditions, is a suitable intracellular target to increase the nuclear import of plasmid DNA. We report the design of an optimized and extended NF kappaB DNA binding sequence that promotes an efficient plasmid nuclear import. METHODS: On the basis of structural studies, the 5'-CTGGGGACTTTCCAGCTGGGGACTTTCCAGCTGGGGACTTTCCAGG-3' segment (termed 3NF) comprising three 10-bp kappaB sites (GGGACTTTCC) separated by a 5-bp optimized spacer (AGCTG) was selected for its capacity to ensure the best structural fit with NF kappaB and to fix simultaneously three proteins. Plasmids encoding luciferase and bearing this sequence (3NF-plasmids) were constructed and their nuclear import and gene expression efficiencies compared with that of plasmids containing classical kappaB motifs. RESULTS: A high luciferase expression was associated with plasmids containing one (p3NF-luc) or two (p3NF-luc-3NF) 3NF sequences. In situ hybridization experiments and quantitative measurement of the number of plasmid copies demonstrated that the nuclear delivery of 3NF-plasmids was more efficient than that of 3NF-free plasmids. Cross-linked immunoprecipitation showed that 3NF-plasmids were recognized by NF kappaB inside cells upon transfection. The nuclear delivery was inhibited with BAY 11-7085, an inhibitor of NF kappaB activation. Finally, p3NF-luc-3NF, the most efficient construct for in vitro transfection, had a long-lived luciferase expression in vivo. CONCLUSIONS: The results obtained in the present study demonstrate the NF kappaB-mediated nuclear delivery of 3NF-plasmids. Due to its high affinity for fixing several NF kappaB, the 3NF sequence is a very promising helper for a nonviral gene delivery system.

Novel neutral imidazole-lipophosphoramides for transfection assays.

New helper lipids, possessing an imidazole polar head, have been synthesized and included in formulations for transfection assays; these new helper lipids can improve the transfection by a factor of up to 100 compared to the use of DOPE as co-lipid.

Poly(N-methylvinylamine)-Based Copolymers for Improved Gene Transfection.

Poly(N-methylvinylamines) with secondary amines can form complexes with plasmid DNA (pDNA) and provide transfection efficiency in HeLa cells in the same order as linear polyethyleneimine but with higher cell viability. Chemical modifications of poly(N-methylvinylamine) backbones are performed to further improve transfection efficiency while maintaining low degree of cytotoxicity. In a first type of polymer, primary amino groups are incorporated via a copolymerization strategy. In a second one, primary amino and imidazole groups are incorporated also via a copolymerization strategy. In a third one, secondary amino groups are substituted with methylguanidine functions through a postpolymerization reaction. Thus, novel polymers of various molecular masses are synthesized, characterized, and their interaction with pDNA studied. Then, their transfection efficiency and cytotoxicity are tested in HeLa cells. Two polymethylvinylamine-based copolymers, one containing 20% of imidazole moieties and another one composed of 12% of guanidinyl units allow remarkable transfection efficiency of HeLa, pulmonary (16HBE), skeletal muscle (C2C12), and dendritic (DC2.4) cells. Overall, this work thus identifies new promising DNA carriers and chemical modifications that improve the transfection efficiency while maintaining low degree of cytotoxicity.

Peptides mediating DNA transport on microtubules and their impact on non-viral gene transfer efficiency.

Synthetic vectors such as cationic polymers and cationic lipids remain attractive tools for non-viral gene transfer which is a complex process whose effectiveness relies on the ability to deliver a plasmid DNA (pDNA) into the nucleus of non-dividing cells. Once in the cytosol, the transport of pDNAs towards the nuclear envelope is strongly impaired by their very low cytosolic mobility due to their large size. To promote their movement towards the cell nucleus, few strategies have been implemented to exploit dynein, the microtubule's (MT's) motor protein, for propagation of cytosolic pDNA along the MTs towards the cell nucleus. In the first part of this review, an overview on MTs, dynein, dynein/virus interaction feature is presented followed by a summary of the results obtained by exploitation of LC8 and TCTEL1 dynein light chain association sequence (DLC-AS) for non-viral transfection. The second part dedicated to the adenoviral protein E3-14.7K, reports the transfection efficiency of polyplexes and lipoplexes containing the E3-14.7K-derived P79-98 peptide linked to pDNA. Here, several lines of evidence are given showing that dynein can be targeted to improve cytosolic pDNA mobility and accumulate pDNA near nuclear envelope in order to facilitate its transport through the nuclear pores. The linkage of various DLC-AS to pDNA carriers led to modest transfection improvements and their direct interaction with MTs was not demonstrated. In contrast, pDNA linked to the P79-98 peptide interacting with TCTEL1 via a cytosolic protein (fourteen seven K-interacting protein-1 (FIP-1)), interaction with MTs is evidenced in cellulo and transfection efficiency is improved.

Synthesis of α-amino-lipophosphonates as cationic lipids or co-lipids for DNA transfection in dendritic cells.

Cationic lipid/co-lipid combinations have been extensively explored in gene delivery as alternatives to viral vectors. To be established as a gold standard of chemical vectors, considerable improvement in their transfection efficiency is however required. Herein, we report a simple procedure to synthesize new cationic lipids and co-lipids for the DNA transfection of dendritic cells (DCs). Seven α-amino-lipophosphonates featuring two aza-heterocycles with protonable sites (imidazole or pyridine) were synthesized and used as co-lipids in liposomes with cationic lipids. For each liposome, the cationic lipid is either an imidazolium lipophosphoramidate (lipid 2) or an α-amino-lipophosphonate containing a basic tertiary aliphatic amine in the polar head group (lipid 3b). The cationic lipids either with new co-lipids or DOPE formed positively charged nano-sized stable liposomes that effectively interact with plasmid DNA (pDNA) to produce lipoplexes. Membrane fusion studies showed that α-amino-phosphonates featuring an imidazole moiety in the polar head group exhibited higher fusion at pH 5.5 than pH 7.4. This study suggests that the best formulations for the transfection of DCs (based on the % transfected cells and the intensity of EGFP-based fluorescence) are lipid 2 associated with either 3a, 3d or DOPE and cationic lipid 3b formulated with 3a or DOPE as a helper lipid. Furthermore, lipid 3a could be used as an alternative to DOPE as a helper lipid. Overall, these results indicate that novel imidazole containing α-amino-phosphonates can serve as effective transfection agents for DC-based vaccines.

Curcumin/poly(2-methyl-2-oxazoline-b-tetrahydrofuran-b-2-methyl-2-oxazoline) formulation: An improved penetration and biological effect of curcumin in F508del-CFTR cell lines.

Neutral amphiphilic triblock ABA copolymers are of great interest to solubilize hydrophobic drugs. We reported that a triblock ABA copolymer consisting of methyl-2-oxazoline (MeOx) and tetrahydrofuran (THF) (MeOx6-THF19-MeOx6) (TBCP2) can solubilize curcumin (Cur) a very hydrophobic molecule exhibiting multiple therapeutic effects but whose insolubility and low stability in water is a major drawback for clinical applications. Here, we provide evidences by flow cytometry and confocal microscopy that Cur penetration in normal and ΔF508-CFTR human airway epithelial cell lines is facilitated by TBCP2. When used on ΔF508-CFTR cell lines, the Cur/TBCP2 formulation promotes the restoration of the expression of the CFTR protein in the plasma membrane. Furthermore, patch-clamp and MQAE fluorescence experiments show that this effect is associated with a correction of a Cl- selective current at the membrane surface of F508del-CFTR cells. The results show the great potential of the neutral amphiphilic triblock copolymer MeOx6-THF19-MeOx6 as carrier for curcumin in a Cystic Fibrosis context. We anticipate that other MeOxn-THFm-MeOxn copolymers could have similar behaviours for other highly insoluble therapeutic drugs or cosmetic active ingredients.

Synthesis of a trimannosylated-equipped archaeal diether lipid for the development of novel glycoliposomes.

An archaeal diether lipid possessing a tri-antenna of α-D-mannopyranoside linked via an oligoethylene spacer to a (2S)-2-(phytanyloxy)-3-(hexadecyloxy)propanoic acid backbone (TriMan-Diether) was designed and synthesized. This new mannosylated lipid inserted in liposomes would show both DC-targeting and adjuvant properties thanks to the TriMan structure and the diether tail part, respectively.

Gene delivery efficiency and intracellular trafficking of novel poly(allylamine) derivatives.

Non-viral gene carriers for safe and efficient gene transfection have become of particular interest among researchers of different disciplines ranging from physical chemistry to biotechnology. Recently polymeric vectors have been extensively studied as potentially new gene transfer agents. Until now most of the research efforts were made to optimize the gene-to-polymer weight ratio of polyplexes for safe and efficient gene transfection. In this work, we report on the development of novel poly(allylamine) derivatives with different balance of the primary, secondary, tertiary, and quaternary amino groups. All derivatives were able to complex pDNA into polyplexes at low gene-to-polymer weight ratios i.e., 1:1 or 1:2. Moreover, the examined polyplexes were less cytotoxic and showed better transfection efficiency when compared to linear poly(ethyleneimine). These results indicate that the presence of quaternary ammonium groups is important in the formation of stable polyplexes. Polymers with all types of amino groups showed large potential for gene delivery. Furthermore, polyplexes with such derivatives were well internalized by cells and ended up into acidic late endosomes.