PEI Complexes Research Articles

Nona-Arginine Mediated Anti-E6 ShRNA Delivery Suppresses the Growth of Hela Cells in vitro.

The E6 oncoprotein of HPV plays a crucial role in promoting cell proliferation and inhibiting apoptosis, leading to tumor growth. Non-viral vectors such as nona-arginine (R9) peptides have shown to be potential as carriers for therapeutic molecules. This study aimed to investigate the efficacy of nona-arginine in delivering E6 shRNA and suppressing the E6 gene of HeLa cells in vitro. HeLa cells carrying E6 gene were treated with a complex of nona-arginine and E6 shRNA. The complex was evaluated using gel retardation assay and FESEM microscopy. The optimal N/P ratio for R9 peptide to transfect HeLa cells with luciferase gene was determined. Relative real-time PCR was used to evaluate the efficiency of mRNA suppression efficiency for E6 shRNA, while the effect of E6 shRNA on cell viability was measured using an MTT assay. The results indicated that R9 efficiently binds to shRNA and effectively transfects E6 shRNA complexes at N/P ratios greater than 30. Transfection with R9 and PEI complexes resulted in a significant toxicity compared to the scrambled plasmid, indicating selective toxicity for HeLa cells. Real-time PCR confirmed the reduction of E6 mRNA expression levels in the cells transfected with anti-E6 shRNA. The study suggests that R9 is a promising non-viral gene carrier for transfecting E6 shRNA in vitro, with significant transfection efficiency and minimal toxicity.

Stabilizing DNA–PEI complexes improves scalability of suspension lentiviral viral vector and AAV processes

Transient transfection is a challenging and time-sensitive upstream unit operation to scale for suspension HEK293 viral vector production. For many transfection reagents, including polyethyleneimine, careful timing of mixing and hold steps is essential to process performance but challenging to maintain at relevant manufacturing scales. We first characterized the process parameters that influence polyethyleneimine transfection reagent performance and then implemented a stabilizing strategy to preserve the activity of transfection complexes when scaling. Generating a transfection complex with an average hydrodynamic diameter between 500 and 700 nm was most predictive of transient transfection productivity, measured by viral vector titer, but is very sensitive to transfection complex concentration and hold time. The addition of human serum albumin as a stabilizing agent maintained the optimal transfection complex size for extended periods of time and improved the process control for both lentiviral vector and adeno-associated viral vector suspension processes at multiple production scales.

Oral Delivery of Nucleic Acids with Passive and Active Targeting to the Intestinal Tissue Using Polymer-Based Nanocarriers

Despite the apparent advantages for long-term treatment and local therapies against intestinal diseases, the oral delivery of nucleic acids has been challenging due to unfavorable physiological conditions for their stability. In this study, a novel nanodelivery system of PEG-PCL nanoparticles with encapsulated nucleic acids–mannosylated PEI (Man-PEI) complexes was developed for intestinal delivery. We complexed model nucleic acids with Man-PEI at the optimal N/P ratio of 20:1 for in vitro and in vivo analyses. Cells were transfected in vitro and analyzed for gene expression, receptor-mediated uptake, and PEG-PCL nanoparticles’ toxicity. We also evaluated the nucleic acid’s stability in the nanocarrier during formulation, and under simulated gastrointestinal environments or the presence of nucleases. Finally, we assessed the biodistribution for the PEG-PCL nanoparticles with encapsulated complexes and their ability to transfect intestinal cells in vivo. Nucleic acids complexed with Man-PEI were protected from degradation against nucleases. In comparison to the parent compound PEI, Man-PEI transfected the cells with an overall higher potency. Competition assay indicated receptor-mediated endocytosis promoted by mannose receptors. The PEG-PCL nanoparticles with Man-PEI/plasmid complexes indicated minimal cytotoxicity. The nanocarrier successfully protected the complexes in a simulated gastric fluid environment and released them in a simulated intestinal fluid environment, promoted by the presence of lipases. The oral administration of the PEG-PCL nanoparticles with encapsulated Man-PEI/plasmid complexes transfected intestinal cells with the plasmid in vivo, while presenting a time-dependent progression through the intestines. Conclusively, our carrier system can deliver genetic material to the GI tract and actively target mannose receptor overexpressing cells.

Enhanced anticancer activity and endocytic mechanisms by polymeric nanocarriers of n-butylidenephthalide in leukemia cells

The purpose of this study was to investigate the antitumor mechanisms of n-butylidenephthalide (BP) and to further examine the delivery efficacy of polycationic liposome containing PEI and polyethylene glycol complex (LPPC)-encapsulated BP in leukemia cells. MTS, flow cytometric and TUNEL assays were performed to assess cell viability and apoptosis. BP and BP/LPPC complex delivery efficiency was analyzed by full-wavelength fluorescent scanner and fluorescence microscope. The expressions of cell cycle- and apoptosis-related proteins were conducted by Western blotting. The results showed that BP inhibited leukemia cell growth by inducing cell cycle arrest and cell apoptosis. LPPC-encapsulated BP rapidly induced endocytic pathway activation, resulting in the internalization of BP into leukemia cells, causing cell apoptosis within 1h. LPPC encapsulation enhanced the cytotoxic activity of BP and did not influence the effects of BP induction that suggested LPPC-encapsulated BP might be developed as anti-leukemia drugs in future.

Chondroitin Sulfate Increases Transfection Efficiency by DNA–PEI Complexes

Chondroitin Sulfate Increases Transfection Efficiency by DNA–PEI Complexes

Methotrexate-Coated Complexes of Plasmid DNA and Polyethylenimine for Gene Delivery.

Folate receptors are overexpressed on the surface cancer cells. We successfully constructed a new gene delivery vector of methotrexate (MTX)-coated plasmid DNA-polyethylenimine (pDNA-PEI) complexes (PEI complexes) by electrostatic binding. The stable anionic nanoparticle was optimized at MTX charge ratios of 120 or more. pDNA-PEI-MTX complexes (MTX complexes) demonstrated gene expression efficiency as high as cationic pDNA-PEI complexes in the mouse melanoma cell line, B16-F10. The MTX complexes were taken up by the cell-specific uptake mechanisms via the folate receptor. MTX-coated complexes are useful as endocytosis ligands. The MTX120 complexes exhibited no blood aggregation. The transgene efficiency of MTX120 complexes in the liver and spleen after their intravenous administration was higher than that of PEI complexes. Therefore, MTX complexes are expected as a new gene vector in the future.

Construction of dexamethasone-conjugated PEI complexes with polycistronic SOX6, SOX9 and shANGPTL4 genes for the treatment of osteoarthritis

Purpose: In this study, we performed the gene transfection by introducing sox9/sox6 and ANGOTL4 genes into DEX-conjugated PEI for the treatment of osteoarthritis. Methods: Three genes SOX6, SOX9, and ANGPTL4 were fabricated in a single plasmid and this multicistronic plasmid was complexed with the DEX-conjugated PEI (DEX-PEI). The surface chemistries of the synthesized DEX-PEI were characterized by FTIR and 1H NMR. SEM and TEM analysis were conducted to observe the morphology of DEX-PEI complexed with the multicistronic plasmid (DEX-PEI-SOX9/SOX6/shANGPTL4). Transfection efficiency of DEX-PEI-SOX9/SOX6/shANGPTL4 complexes in hADSCs was evaluated by flow cytometry analysis and in vitro chondrogenesis of hADSCs transfected by DEX-PEI-SOX9/SOX6/shANGPTL4 complexes (hADSCSOX9/SOX6/shANG) was evaluated by RT-qPCR, western-blot and immunohistochemistry. In vivo study, the effects of intra-articular (IA) administered hADSCSOX9/SOX6/shANG were evaluated in a surgically-induced OA rat model. Results: Covalently crosslinked DEX-PEI had a diameter of 190.1±86.2 nm. The uptake of DEX-PEI-SOX9/SOX6/shANGPTL4 in hADSCs was 52.5%. Thereafter, overexpression of SOX6 and SOX9 and down-regulation of shANGPTL4 was observed. pMC_SOX9/SOX6 expressed more than control, and pMC_SOX9/SOX6/shANGPTL4 was more than pMC_SOX9/SOX6. The expression of Col2α1 and Aggrecan in hADSCSOX9SOX/6/shANG showed significantly higher than those of DEX-PEI-SOX9/SOX6 complexes-transfected hADSCSOX9/SOX6. After Safranin O staining and Immunofluorescense, it was identified that RDbSOX9 / SOX6 / shANGPTL4 expressed more than control and TGF-BMP. The OARSI and Mankin scores of hADSCSOX/9SOX/6/shANG-injected rats were significantly lower than those of vehicle and vector control. Conclusions: In conclusion, the effectiveness of DEX-PEI complexes with SOX9/SOX6/shANGPTL4 genes for the transfection of hADSCs to osteoarthritis treatment is suggested.

Intradermal delivery of STAT3 siRNA to treat melanoma via dissolving microneedles

Hyperactivity of signal transducer and activity of transcription 3 (STAT3) plays a crucial role in melanoma invasion and metastasis. Gene therapy applying siRNA targeting STAT3 is a potential therapeutic strategy for melanoma. In this article, we first fabricated safe and novel dissolving microneedles (MNs) for topical application of STAT3 siRNA to enhance the skin penetration of siRNA and used polyethylenimine (PEI, 25 kDa) as carrier to improve cellular uptake of siRNA. The results showed that MNs can effectively penetrate skin and rapidly dissolve in the skin. In vitro B16F10 cell experiments presented that STAT3 siRNA PEI complex can enhance cellular uptake and transfection of siRNA, correspondingly enhance gene silencing efficiency and inhibit tumor cells growth. In vivo experiments indicated that topical application of STAT3 siRNA PEI complex delivered by dissolving MNs into skin can effectively suppress the development of melanoma through silencing STAT3 gene, and the inhibition effect is dose-dependent. STAT3 siRNA delivery via dissolving MNs is a promising approach for skin melanoma treatment with targeting inhibition efficacy and minimal adverse effects.

Pullulan-PEI-Histidine towards gene delivery: vector unpacking with respect to molecular weight and cytoplasmic histones

Event Abstract Back to Event Pullulan-PEI-Histidine towards gene delivery: vector unpacking with respect to molecular weight and cytoplasmic histones Rekha M. Ramesan1 1 Sree Chitra Tirunal Institute for Medical Sciences & Technology, Biosurface Technology, BiomedicalTechnology Wing, India Introduction: Cationic polymers, one of the most important nonviral vectors have a number of advantages over viral vectors. However cationic surface charge, the key factor which determines the transfection efficiency, is also one of the limitations associated with it. Major limiting factors are cytotoxicity, biocompatibility and the release of the cargo from the vector intracellularly. The tightly formed nanoplexes gains entry into the cells, however it doesn’t lead to good transfection if the DNA remains tightly bound to carrier. Schaffer et.al proved that unpacking of the DNA from the vectors is a major limiting step in the process of non-viral gene delivery[1]. Earlier we reported the possible role of histones in unpacking in which a combination of all histones were used[2]. In this work we attempted to evaluate the possible role played by the molecular weight of the polymers and which of the cytoplasmic histones aids more in vector unpacking. Histone 1 (H1) and histone 3 (H3) reported to be found in cytoplasm were used for the study. Materials and Methods: Pullulan a hydrophilic, nonionic polysaccharide was conjugated with PEI (10 and 25KDa) to which histidine was introduced to improve its transfection efficiency (PPE10H and PPE25H). The conjugation was extablished by variuos techniques including NMR. The particle size and zetapotential of nanoplexes was evaluated with DLS. The cellular uptake and transfection efficiency was done in C6 glioma cells. For vector unpacking studies, cellulose-DNA beads were used by which the displacement of the polymer was assessed. Labelled PPEH and histones (H 1 and H 3) were used to study the unpacking in cell lines. Results and Discussion: The derivatisation was established using techniques including NMR and Raman spectroscopy. The optimum size of PPE10H and PPE25H nanoplexes were 78.4 ± 0.72 and 101.6 ± 2.3 respectively at 1:4 ratio with a zeta potential in the range 13-15 mV. The nanoplexes of both polymers demonstrated very high intracellular uptake and PPE10H had higher transfection efficiency in comparison with PEI10K and PPE10KH. Among the histones studied H3 was found to be more efficient in vector unpacking (Fig.1). In the absence of histone intact nanoplex is internalised as TRITC labeled PPE10H is found in the cytoplasm and tagged DNA in the nucleus. The nanoplex treated with H1 and H3 resulted in unpacking outside the cell and the labeled polymers are excluded from the entry into the cells (Fig. 1 B to E). However H1 is not as efficient as H3 in unpacking the DNA from the polyplex. The vector unpacking was higher with PPEH10K which might be the reason for better transfection efficacy which could be attributed to its low cationicity (Fig. 2). Figure 1. Internalization of TRITC labeled PPE10KH nanoplex in C6 cells. Nanoplex alone (A), nanoplex treated with 500 ng histone H1 (B), 1 micro g histone H1 (C), 500 ng histone H3 (D) and 1 micro g histone H3 (E) . Absence of TRITC labeled polymer in D & E indicate the better displacement of polymer with H3. Figure 2. Luciferase expression in C6 cells transfected by PPE10H and PEI complexes at ratios 1:3 and 1:4. ** p<0.05 Conclusion: PPE10H was found to be a better transfecting agent. It was observed that H3 is more effiecient in displacing the plasmid DNA from the vector. The extent of cationicity also found to play a role in release of DNA from the vector in presence of histones.

ATP triggered drug release and DNA co-delivery systems based on ATP responsive aptamers and polyethylenimine complexes.

Stimuli-responsive nanocarriers for anticancer drug and gene co-delivery are a promising strategy in cancer therapy due to their combination of chemotherapy and gene therapy. In this work, we developed a facile and effective method to fabricate stimuli-responsive nanocarriers for anticancer drug and gene co-delivery based on complexes of polyethylenimine (PEI) with an adenosine triphosphate (ATP) responsive aptamer duplex (ARAD). No chemical reactions or complex modifications were used in the construction processes. In this system, Doxorubicin-loaded aptamer duplex and plasmid DNA (p53) can be bound by PEI by electronic interactions to form stable complexes which effectively protect the aptamer and p53 from DNase degradation. The intercalated Dox can be released on-demand by a structural change in the aptamer duplex in an ATP-rich environment. The morphology and average size of the nanocarriers were characterized by zeta potential and transmission electron microscopy (TEM). The nanocarriers exhibit lower cell toxicity in HeLa cell lines relative to PEI. RT-PCR and Western blot analysis confirmed that p53 could be effectively delivered and expressed in HeLa cells by PEI/ARAD/p53 complexes. Moreover, the apoptosis percentage of HeLa cells treated with PEI/ARAD/Dox/p53 complex increased to 40.8%, compared to 24.7% for PEI/ARAD/Dox complex and 11.5% for PEI/ARAD/p53, respectively. The result demonstrated that the combinatorial delivery of Dox and p53 by nanocarriers could induce synergistic actions and lead to effective cancer cell apoptosis.

Synthesis of Mannosylated Polyethylenimine and Its Potential Application as Cell-Targeting Non-Viral Vector for Gene Therapy

Mannose polyethylenimine with a molecular weight of 25 k (Man-PEI25k) was synthesized via a phenylisothiocyanate bridge using mannopyranosylphenyl isothiocyanate as a coupling reagent, and characterized by 1H NMR (nuclear magnetic resonance) and FT-IR (Fourier transform infrared spectroscopy) analysis. Spherical nanoparticles were formed with diameters of 80–250 nm when the copolymer was mixed with DNA at various charge ratios of copolymer/DNA (N/P). Gel electrophoresis demonstrated that the DNA had been condensed and retained by the PEI derivates at low N/P ratios. The Man-PEI25k/DNA complexes were less cytotoxic than the PEI complexes with a molecular weight of 25 k (PEI25k) at the same N/P ratio. Laser scan confocal microscopy and flow cytometry confirmed that the Man-PEI25k/DNA complexes gave higher cell uptake efficiency in (Dendritic cells) DC2.4 cells than HeLa cells. The transfection efficiency of Man-PEI25k was higher than that of PEI25k towards DC2.4 cells. These results indicated that Man-PEI25k could be used as a potential DC-targeting non-viral vector for gene therapy.

Gene transfection in complex media using PCBMAEE-PCBMA copolymer with both hydrolytic and zwitterionic blocks

Safety and high efficacy of vectors are essential requirements for gene therapy. To address these challenges, poly(carboxy betaine methacrylate ethyl ester)-poly(carboxy betaine methacrylate) (PCBMAEE-PCBMA) diblock copolymers were synthesized to form core–shell vector for gene delivery. The hydrophobic PCBMAEE segment, a polyzwitterionic precursor, can condense plasmid DNA (pDNA) into a hydrophobic core, which improves pDNA protection from nuclease attack and maintains the condensed structure against dilution. Moreover, the hydrolysis of PCBMAEE in uptaken gene vectors can enhance the pDNA release and reduce the cytotoxicity caused by the cationic polymer accumulation in the host cells. The PCBMA segment, zwitterionic fouling resistant material, is utilized to stabilize the gene vector in the complex medium and reduce the interference from serum proteins without impeding the endocytosis of DNA vector like PEG protection layer. Results showed that the complex formed by PCBMAEE50-PCBMA14 with luciferase or pEGFP gene exhibit higher transfection efficacy of pDNA than that formed by PEI 25 kDa or Lipofectamine® 2000 in tested cell lines (COS-7, HepG-2, HeLa, and HUVEC), especially, in difficult-to-transfect ones, such as HeLa and HUVEC. The luciferase expression level infected by the vectors of PCBMAEE50-PCBMA14/pGL-4 at N/P = 20/1 is 27 times of the branched PEI 25 kDa in COS-7 cells and 16 times of Lipofectamine® 2000 in HUVEC. Furthermore, the complex formed by PCBMAEE50-PCBMA14 also show advantages in transfection rate, dosage effectiveness and preservation of transfecting activity in serum contained growth medium. The luciferase expression of the vectors of PCBMAEE50-PCBMA14/pGL-4 at N/P = 20/1 is 230 times higher than that of PEI complex at low vector dosage (the 5% standard dosage). And the transfection rate is 25 times higher than that of PEI complex in 10% serum contained growth medium. In short, all these results indicated that the polymeric gene vector, consisted of convertible hydrophobic polyzwitterionic precursor and fixed polyzwitterionic fouling resistant segment, is a promising candidate for high and stable gene transfection in complex growth medium.

Development of a semi-automated high throughput transient transfection system

Transient transfection of mammalian cells provides a rapid method of producing protein for research purposes. Combining the transient transfection protein expression system with new automation technologies developed for the biotechnology industry would enable a high throughput protein production platform that could be utilized to generate a variety of different proteins in a short amount of time. These proteins could be used for an assortment of studies including proof of concept, antibody development, and biological structure and function. Here we describe such a platform: a semi-automated process for PEI-mediated transient protein production in tubespins at a throughput of 96 transfections at a time using a Biomek FXP liquid handling system. In one batch, 96 different proteins can be produced in milligram amounts by PEI transfection of HEK293 cells cultured in 50mL tubespins. Methods were developed for the liquid handling system to automate the different processes associated with transient transfections such as initial cell seeding, DNA:PEI complex activation and DNA:PEI complex addition to the cells. Increasing DNA:PEI complex incubation time resulted in lower protein expression. To minimize protein production variability, the methods were further optimized to achieve consistent cell seeding, control the DNA:PEI incubation time and prevent cross-contamination among different tubespins. This semi-automated transfection process was applied to express 520 variants of a human IgG1 (hu IgG1) antibody.

Retro-Inverso CendR Peptide-Mediated Polyethyleneimine for Intracranial Glioblastoma-Targeting Gene Therapy

The development of nonviral gene delivery vectors offers the potential to provide effective treatment for glioblastoma in the form of gene therapy. Here, we report the use of retro-inverso C-end rule (CendR) peptide D(RPPREGR) as a targeting ligand to prepare a D(RPPREGR)-PEG-PEI gene vector. D(RPPREGR) peptide specifically recognized the neuropilin-1 receptor that was overexpressed on U87 glioma cells, and showed enhanced tumor spheroid penetration ability. Compared with parental RGERPPR, D(RPPREGR) possessed improved biological stability and had a higher affinity for U87 glioma cells; it also showed enhanced penetration of the tumor spheroid. mPEG-PEI/pDNA and D(RPPREGR)-PEG-PEI/pDNA complexes were prepared and MTT assay results revealed that the cytotoxicity of D(RPPREGR)-PEG-PEI complexes was significantly lower than that of PEI complexes, with cell survival rates above 80%. Qualitative and quantitative in vitro transfection results revealed that D(RPPREGR)-PEG-PEI complex transfection efficiencies were 1.9 times higher than those of mPEG-PEI. Fluorescent imaging and frozen sections of brain tissue demonstrated that the D(RPPREGR) modification improved the in vivo transfection efficiency of mPEG-PEI in nude mice bearing U87 gliomas. An antiglioblastoma assay revealed that D(RPPREGR)-PEG-PEI carrying the therapeutic gene pORF-hTRAIL significantly prolonged the survival time of intracranial U87 glioma-bearing mice from 25 to 30 days. Therefore, D(RPPREGR)-PEG-PEI appears to be suitable for use as a safe and efficient gene delivery vehicle with potential applications in glioblastoma gene therapy.

DNA complexed with TAT peptide and condensed using calcium possesses unique structural features compared to PEI polyplexes

Complexes of the TAT peptide with plasmid DNA (pDNA) show unusually high transfection efficiency when condensed via "soft" calcium cross links. In this study, we characterize the structure of pDNA within TAT complexes and compare it with that of branched polyethylenimine (PEI, 25 kDa) complexes. Dynamic light scattering (DLS), second derivative ultraviolet absorption spectroscopy, extrinsic dye fluorescence, Fourier transform infrared (FTIR) spectroscopy, circular dichroism (CD) spectroscopy and differential scanning calorimetry (DSC) were employed to access various aspects of the structure of the pDNA. TAT complexes showed the highest transfection efficiency at an N/P ratio of 25 in A549 cells. FTIR and CD spectra of complexes demonstrated that the pDNA remained in the B conformation when associated with TAT or PEI. DSC showed that both TAT and PEI stabilized all forms of pDNA, with TAT increasing the melting temperature of pDNA compared to PEI complexes. Second derivative ultraviolet spectroscopy of TAT complexes showed a substantial reduction in the absorbance and an increase in the wavelengths of the peaks of pDNA at N/P>13; however, a clear correlation between pDNA structure and transfection efficiency was not readily apparent.

Examination of the dynamic cellular internalization processes of siRNA mediated by nonviral vectors

The study investigated the dynamic process of internalization and intracellular processing of DOTAP/siRNA Lipoplexes and PEI/ siRNA, HPAMAM-PHE45/siRNA Polyplexes by H1299 cells. Fluorescence colocalization studies with a lysosomal marker-LysoTracker, revealed that DOTAP complexes could be quickly take up by cells into the cytoplasm, and most were released from endosomal pathway by 6 h. HPAMAM-PHE45 or PEI complexes were found to attach to the surface of the cell membrane initially. Some of the complexes followed the endosomal pathway, but also a part of complexes enter cells through caveolae-mediated endocytosis. We conclude that lipoplex uptake proceeds only by clathrin-mediated endocytosis, while Polyplexes are taken up by two mechanisms, involving caveolae and clathrin-mediated endocytosis. In this study, we compared the different siRNA uptake and intracellular processing of Polyplexes and Lipoplexes delivery systems basis optimization study in the future.

Biocompatibility and Efficacy of Oligomaltose-Grafted Poly(ethylene imine)s (OM-PEIs) forin VivoGene Delivery

Polycationic polymers like poly(ethylene imine)s (PEIs) are extensively explored for the nonviral transfer of DNA or small RNAs (siRNAs). To enhance biocompatibility and alter pharmacokinetic properties, hyperbranched PEI was recently grafted with the nonligand oligosaccharides maltose or maltotriose at various degrees in a systematic study to yield (oligo-)maltose PEIs (OM-PEIs). In this paper, we investigate the in vivo biocompatibility and efficacy of a whole set of (OM-)PEIs and the corresponding (OM-)PEI-based DNA or siRNA complexes upon systemic (intravenous, i.v.) administration in mice. We determine the overall survival and animal welfare, hepatotoxicity, immune stimulation, erythrocyte aggregation, and the efficacy of DNA delivery in vivo. Higher-degree oligomaltose-grafting of PEI substantially decreases weight loss, abolishes lethality upon repeated treatment with the free polymers or with complexes, and abrogates hepatotoxicity, as determined by serum levels of liver enzymes. Immunostimulatory effects (TNF-α, IFN-γ) and erythrocyte aggregation are mainly observed upon treatment with partially maltotriose-grafted PEI or PEI-based complexes and are largely abolished upon higher-degree grafting. In vivo transfection experiments in mice bearing subcutaneous (s.c.) tumor xenografts reveal a strong dependence of reporter gene expression in a given organ on the mode of complex administration (i.v. vs intraperitoneal injection) and the OM-PEI architecture, with high-level maltose-grafted PEI (PEI-(2-Mal)) being most efficient for DNA delivery. We conclude that distinct differences between different patterns of maltose- or maltotriose-grafting are observed with regard to both biocompatibility and in vivo efficacy and identify optimal oligomaltose-PEIs for therapeutic applications.

Targeted gene delivery to glioblastoma using a C-end rule RGERPPR peptide-functionalised polyethylenimine complex

Safe and efficient systems capable of specifically targeting brain tumour cells represent a promising approach for the treatment glioblastoma multiforme. Neuropilin-1 (NRP-1) is over-expressed in U87 glioma cells. In the current study, the tumour specific peptide RGERPPR, which binds specifically to NRP-1, was used as a targeting ligand in a gene delivery strategy for glioblastoma. The RGERPPR peptide was coupled to branched polyethylenimine (PEI, 25kDa) using heterobifunctional Mal-PEG-NHS, resulting in a novel gene delivery polymer. Polymer/plasmid DNA (pDNA) complexes were formed and their sizes and zeta potentials were measured. Compared with the unmodified mPEG-PEI/pDNA complexes, the RGERPPR-PEG-PEI/pDNA complex led to a significant enhancement in intracellular gene uptake and tumour spheroid penetration. Furthermore, the RGERPPR-PEG-PEI/pDNA complex facilitated enhanced transfection efficiency levels, as well as a reduction in cytotoxicity when tested in U87 glioma cells in vitro. Most significantly of all, when complexes formed with pDsRED-N1 were injected into the tail vein of intracranial U87 tumour-bearing nude mice, the RGERPPR-PEG-PEI complexes led to improved levels of red fluorescence protein expression in the brain tissue. Taken together, the results show that RGERPPR-PEG-PEI could be used as a safe and efficient gene delivery vehicle with potential applications in glioblastoma gene delivery.

Phospholipid-modified polyethylenimine-based nanopreparations for siRNA–mediated gene silencing: Implications for transfection and the role of lipid components

The clinical application of gene silencing mediated by small interfering RNA (siRNA) has been limited by the lack of efficient and safe carriers. Phospholipid modification of low molecular weight polyethylenimine (PEI 1.8kDa) dramatically increased its gene down-regulation capacity while keeping cytotoxicity levels low. The silencing efficacy was highly dependent on the nature of the lipid grafted to PEI and the polymer/siRNA ratio employed. Phosphoethanolamine (DOPE and DPPE) and phosphocholine (PC) conjugation did not change the physicochemical properties and siRNA binding capacity of PEI complexes but had a large impact on their transfection and ability to down-regulate Green Fluorescent Protein (GFP) expression (60%, 30% and 5% decrease of GFP expression respectively). We found that the micelle-forming structure of DOPE and DPPE-PEI dramatically changed PEI's interaction with cell membranes and played a key role in promoting PEI 1.8kDa transfection, completely ineffective in the absence of the lipid modification. From the Clinical EditorWhile siRNA-based gene silencing methods could have numerous clinical applications, efficient delivery remains a major challenge. This team reports that DOPE-PEI and DPPE-PEI based micelle-forming nanostructures may be able to provide an efficient vector for siRNA transfection.

Ternary complexes DNA–polyethylenimine–Fe(iii) with linear and branched polycations: implications on condensation, size, charge and in vitro biocompatibility

The successful use of nucleic acids in medicine requires efficient DNA condensation and protection, as well as a low cytotoxicity of the vector. In previous work, we have used a ternary DNA–PEI–Fe(III) complex and have shown that the addition of Fe(III) to the DNA–PEI complexes enhances DNA condensation, whereas allows an easier DNA release from the complexes, in the presence of a decompacting agent. The focus of the present work was to investigate the influence of the polycation architecture on the DNA–PEI–Fe(III) ternary system, and also to develop a potential gene delivery system with reduced toxicity. Herein, lPEI (2.5 and 25 kDa) and bPEI (1.2 and 10 kDa) were used in conjunction with Fe(III), and the complexes formed were physico-chemically characterized using precipitation and gel electrophoresis assays, and size and zeta potential determinations. It was observed that the addition of Fe(III) ions equalizes the charge ratios for the onset of DNA condensation irrespective of the PEI used, although a more pronounced condensation effect is still visible with the polycations with a lower Mw. At low N/P ratios, the presence of Fe(III) caused the zeta potential to change from negative to positive values. Also, a reduction in polyplex size is visible before aggregation takes place. The hemolytic activity of the condensing agents was found to be dependent on the pH of the medium, but no perturbation of erythrocytes integrity was detected. Furthermore, the cytotoxicity of each condensing agent individually and of the complexes towards 3T3 fibroblast cell line and HeLa cell line was monitored using the MTT and LDH assays. The magnitude of the cytotoxic effects of the polycations and Fe(III) was found to be dependent on the concentration of the condensing agents, the molecular weight of the polycations, and the pH of the culture medium. The combination of Fe(III) with PEI determines higher IC50 values than in the absence of the metal ion. Additionally, a lower cytotoxicity of DNA–PEI–Fe(III) complexes was observed, when compared to native PEI–DNA complexes, at the same degree of condensation and values of zeta potential. The results show that Fe(III) reinforces the DNA–PEI condensation in both PEI architectures. It allows the cytotoxicity of PEIs to be mitigated and the concentration of the latter needed to obtain stable polyplexes to be decreased.