Gene silencing activity of siRNA polyplexes based on thiolated N,N,N-trimethylated chitosan

N,N,N-Trimethylated chitosan (TMC) is a biodegradable polymer emerging as a promising nonviral vector for nucleic acid and protein delivery. In the present study, we investigated whether the introduction of thiol groups in TMC enhances the extracellular stability of the complexes based on this polymer and promotes the intracellular release of siRNA. The gene silencing activity and the cellular cytotoxicity of polyplexes based on thiolated TMC were compared with those based on the nonthiolated counterpart and the regularly used lipidic transfection agent Lipofectamine. Incubation of H1299 human lung cancer cells expressing firefly luciferase with siRNA/thiolated TMC polyplexes resulted in 60-80% gene silencing activity, whereas complexes based on nonthiolated TMC showed less silencing (40%). The silencing activity of the complexes based on Lipofectamine 2000 was about 60-70%. Importantly, the TMC-SH polyplexes retained their silencing activity in the presence of hyaluronic acid, while nonthiolated TMC polyplexes hardly showed any silencing activity, demonstrating their stability against competing anionic macromolecules. Under the experimental conditions tested, the cytotoxicity of the thiolated and nonthiolated siRNA complexes was lower than those based on Lipofectamine. Given the good extracellular stability and good silencing activity, it is concluded that polyplexes based on TMC-SH are attractive systems for further in vivo evaluations.     

Lipid-mediated siRNA delivery down-regulates exogenous gene expression in the mouse brain at picomolar levels

BACKGROUND: Efficient in vivo vectors are needed to exploit the enormous potential of RNA interference (RNAi). Such methods require optimisation for specific delivery routes, tissues and usages. We tested the capacity of different non-viral vectors and formulation methods for inhibition of exogenous (luciferase) gene expression when used to introduce small interfering RNA (siRNA) into the mouse brain in vivo. METHODS: Polyethylenimine (PEI)-based polyplexes and JetSI (a mixture of cationic lipids)-based lipoplexes were used to vectorise plasmid DNA encoding the firefly Photinus pyralis luciferase gene and picomolar amounts of siRNA directed against this gene. Two controls were used, DNA encoding an unrelated luciferase from Renilla reniformis and a mutated siRNA sequence. RESULTS: First, we found that linear PEI, although efficient for delivering nucleic acids to cells, did not permit development of siRNA activity within the dose range tested (<0.5 pmol). Second, various combinations of cationic lipids were tried and the best formulation was found to be a combination of JetSI with the fusogenic lipid dioleoylphosphatidylethanolamine (DOPE). Efficient inhibition of target, firefly luciferase was obtained with exceedingly low amounts of siRNA: 78 +/- 6% inhibition at 24 h post-transfection with 0.2 pmol siRNA. This inhibition was dose-dependent and specific. No effect was seen on the control gene, co-transfected Renilla luciferase, and the control mutated siRNA sequence had no effect on the targeted firefly luciferase. CONCLUSIONS: We have optimised an efficient cationic lipoplex method for delivery of siRNA into the newborn mouse brain. Specific inhibition of exogenous target gene expression is obtained with picomolar amounts of siRNA.     

Simple modifications of branched PEI lead to highly efficient siRNA carriers with low toxicity

Polymer carriers like PEI which proved their efficiency in DNA delivery were found to be far less effective for the applications with siRNA. In the current study, we generated a number of nontoxic derivates of branched PEI through modification of amines by ethyl acrylate, acetylation of primary amines, or introduction of negatively charged propionic acid or succinic acid groups to the polymer structure. The resulting products showed high efficiency in siRNA-mediated knockdown of target gene. In particular, succinylation of branched PEI resulted in up to 10-fold lower polymer toxicity in comparison to unmodified PEI. Formulations of siRNA with succinylated PEI were able to induce remarkable knockdown (80% relative to untreated cells) of target luciferase gene at the lowest tested siRNA concentration of 50 nM in Neuro2ALuc cells. The polyplex stability assay revealed that the efficiency of formulations which are stable in physiological saline is independent of the affinity of siRNA to the polymer chain. The improved properties of modified PEI as siRNA carrier are largely a consequence of the lower polymer toxicity. In order to achieve significant knockdown of target gene, the PEI-based polymer has to be applied at higher concentrations, required most probably for sufficient accumulation and proton sponge effects in endosomes. Unmodified PEI is highly toxic at such polymer concentrations. In contrast, the far less toxic modified analogues can be applied in concentrations required for the knockdown of target genes without side effects.     

Development of poly(amino ester glycol urethane)/siRNA polyplexes for gene silencing

Nonviral vectors, with their low immunogenicity and lack of pathogenicity, offer significant promise for siRNA therapy with fewer safety concerns. Nonviral vectors were also preferred in most transient siRNA delivery due to their ease of preparation. Previously, we incorporated tertiary amines and polyethylene glycol (PEG) into poly(ester urethane) to synthesize a soluble poly(amino ester glycol urethane), PaE(G)U, as a novel DNA transfection reagent for transgene delivery. The aim of this study was to develop PaE(G)U/siRNA polyplexes for gene silencing. We characterized the properties of PaE(G)U/siRNA polyplexes and compared them with those of PaE(G)U/DNA polyplexes. Using the Alexa Fluor 488-labeled, nonsilencing control siRNA as the reporter, we visualized cellular uptake of PaE(G)U/siRNA polyplexes and optimized the mass ratio of PaE(G)U/siRNA for delivery at 80/1. At this ratio, the average diameter of polyplexes was 540 nm, which was significantly larger than the average diameter of PaE(G)U/DNA polyplexes at 155 nm for efficient DNA delivery. Using the optimized PaE(G)U/siRNA polyplexes, transient silencing of constitutive luciferase expression (up to 92%) was achieved in our recombinant human HT-1080 fibroblast model via anti-luciferase siRNA delivery. In conclusion, PaE(G)U/siRNA polyplexes were developed and optimized for cellular uptake to allow efficient gene silencing. Engineering of soluble biodegradable polymers to incorporate amino, ester, PEG, and urethane units in the backbone constitutes a useful approach for the future design of siRNA carriers.     

Purification of polyethylenimine polyplexes highlights the role of free polycations in gene transfer

BACKGROUND: Nonviral vectors based on polyethylenimine (PEI) usually contain an excess of PEI that is not complexed to DNA. Since unbound PEI contributes to cellular and systemic toxicity, purification of polyplexes from unbound PEI is desirable. METHODS: Size exclusion chromatography (SEC) was used to purify PEI polyplexes of free PEI. Transfection properties of purified polyplexes and the effect of free PEI on gene delivery were studied in vitro and in vivo after systemic application into mice. RESULTS: SEC did not change the size and zeta-potential of polyplexes. Independent of the amount of PEI used for complex formation, purified PEI polyplexes had the same final PEI nitrogen/DNA phosphate ratio of 2.5. Notably, purified PEI polyplexes demonstrated low cellular and systemic toxicity. High transfection efficiency was achieved with purified polyplexes at high DNA concentrations (8-15 microg/ml). At low DNA concentrations (2-4 microg/ml) gene transfer with purified particles was less efficient than with polyplexes containing free PEI both in vitro and in vivo. Mechanistic studies showed that free PEI partly blocked cellular association of DNA complexes but was essential for the following intracellular gene delivery. Adding free PEI to cells treated with purified particles with a delay of up to 4 h resulted in significantly enhanced transfection efficiency compared with non-purified particles or purified particles without free PEI. CONCLUSIONS: This study presents an efficient method to remove free PEI from PEI polyplexes by SEC. Our results from transfection experiments demonstrate that free PEI substantially contributes to efficient gene expression but also mediates toxic effects in a dose-dependent manner. Purified polyplexes without free PEI have to be applied at increased concentrations to achieve high transfection levels, but exhibit a greatly improved toxicity profile.     

The impact of carboxyalkylation of branched polyethylenimine on effectiveness in small interfering RNA delivery

Background

Carboxyalkylation of branched 25 kDa polyethylenimine (PEI) was considered to reduce the positive surface charge of the polymer without reducing its ‘proton sponge’ buffering capacity, and to provide alkylene domains for hydrophobic interactions, thus generating optimized novel PEI carriers for efficient delivery of small interfering RNA (siRNA).

Methods

Substitution of PEI was evaluated in the range of 6 to > 50 mole percentage of primary amines. Additionally, variation of the carboxyalkyl chain (one to 15 methylene groups) was explored to modulate the carrier hydrophobicity. Carriers were characterized in their buffering capacity, capability of siRNA polyplex formation, and cytotoxicity. Marker gene‐silencing efficacy was evaluated using Neuro2A‐eGFPLuc neuroblastoma cells.

Results

Carboxyalkylation strongly reduced cytotoxicity of PEI and improved siRNA mediated luciferase gene knockdown. An optimum silencing activity was observed at an alkylcarboxylation degree of 6–9 mole percentage of primary amines and with a broad range of carboxyalkylene chains (containing one to 15 methylene groups). Strongly enhanced gene‐silencing efficacy also was observed when the biocompatible polymers were separately added at 1 h after transfection with tolerated doses of standard PEI25/siRNA polyplexes.

Conclusions

Carboxyalkylation of branched 25 kDa PEI resulted in polymers with strongly reduced cytotoxicity and improved silencing efficacy. Mechanistic studies demonstrated that the presence of a surplus of free carboxyalkylated polymer is responsible for the improved siRNA delivery. Copyright © 2010 John Wiley & Sons, Ltd.     

Tumor necrosis factor receptor-1-induced neuronal death by TRADD contributes to the pathogenesis of Japanese encephalitis

While a number of studies have documented the neurotropism of Japanese encephalitis virus (JEV), little is known regarding the molecular mechanism of neuronal death following viral infection. The tumor necrosis factor receptor (TNFR)-associated death domain (TRADD) has been suggested to be the crucial signal adaptor that mediates all intracellular responses from TNFR-1. Using mouse (Neuro2a) and human (SK-N-SH) neuroblastoma cell lines, we have shown that the altered expression of TNFR-1 and TRADD following JEV infection regulates the downstream apoptotic cascades. Activation of TRADD led to mitochondria-mediated neuronal apoptosis. As TRADD-knockout animals or deficient cell lines are unavailable, it has been difficult to definitively address the physiological role of TRADD in diseases pathology following JEV infection. We circumvented this problem by silencing TRADD expression with small-interfering RNA (siRNA) and have found that TRADD is required for TNFR-1-initiated neuronal apoptosis following in vitro infection with JEV. Interestingly, siRNA against TRADD also decreased the viral load in Neuro2a cells. Furthermore, siRNA against TRADD increased the survival of JEV-infected mice by altering the expression of pro apoptotic versus antiapoptotic molecules. These studies show that the engagement of TNFR-1 and TRADD following JEV infection plays a crucial role in neuronal apoptosis.     

PEGylation significantly affects cellular uptake and intracellular trafficking of non-viral gene delivery particles

In vitro studies of non-viral gene delivery vectors are typically not performed at physiological conditions, and thus may not provide meaningful results for in vivo investigations. We determine if polycation-plasmid DNA complexes (polyplexes) exploited for in vitro studies behave similarly to variants more applicable to in vivo use by examining their cellular uptake and trafficking. Branched polyethylenimine (25 kDa) or a linear beta-cyclodextrin-containing polymer are each used to formulate polyplexes, which can be PEGylated (PEG: poly(ethylene glycol)) to create particles stable in physiological salt concentrations. Particle size, cellular uptake, intracellular trafficking, and reporter gene expression are reported for polyplexes and for their PEGylated variants. PEGylation confers salt stability to particles but produced a reduction in luciferase expression. Examination of in vitro particle internalization by transmission electron microscopy shows unmodified polyplexes entering cells as large aggregates while PEGylated particles remain small and discrete, both outside and within cells. Unmodified and PEGylated particles enter cells through the endocytic pathway and accumulate in a perinuclear region. Immunolabeling reveals unpackaged exogenous DNA in the cytoplasm and nuclei. It appears all particle types traffic towards the nucleus within vesicles and undergo degradation in vesicles and/or cytoplasm, and eventually some exogenous DNA enters the nucleus, where it is transcribed. In comparing polyplexes and their PEGylated variants, significant differences in particle morphology, cellular uptake, and resultant expression suggest that in vitro studies should be conducted with particles prepared for physiological conditions if the results are to be relevant to in vivo performance.     

SPARC overexpression inhibits cell proliferation in neuroblastoma and is partly mediated by tumor suppressor protein PTEN and AKT

Secreted protein acidic and rich in cysteine (SPARC) is also known as BM-40 or Osteonectin, a multi-functional protein modulating cell-cell and cell-matrix interactions. In cancer, SPARC is not only linked with a highly aggressive phenotype, but it also acts as a tumor suppressor. In the present study, we sought to characterize the function of SPARC and its role in sensitizing neuroblastoma cells to radio-therapy. SPARC overexpression in neuroblastoma cells inhibited cell proliferation in vitro. Additionally, SPARC overexpression significantly suppressed the activity of AKT and this suppression was accompanied by an increase in the tumor suppressor protein PTEN both in vitro and in vivo. Restoration of neuroblastoma cell radio-sensitivity was achieved by overexpression of SPARC in neuroblastoma cells in vitro and in vivo. To confirm the role of the AKT in proliferation inhibited by SPARC overexpression, we transfected neuroblastoma cells with a plasmid vector carrying myr-AKT. Myr-AKT overexpression reversed SPARC-mediated PTEN and increased proliferation of neuroblastoma cells in vitro. PTEN overexpression in parallel with SPARC siRNA resulted in decreased AKT phosphorylation and proliferation in vitro. Taken together, these results establish SPARC as an effector of AKT-PTEN-mediated inhibition of proliferation in neuroblastoma in vitro and in vivo.     

Expression of macrophage migration inhibitory factor by neuroblastoma leads to the inhibition of antitumor T cell reactivity in vivo

Neuroblastomas and many other solid tumors produce high amounts of macrophage migration inhibitory factor (MIF), which appears to play a role in tumor progression. We found that MIF expression in neuroblastoma inhibits T cell proliferation in vitro, raising the possibility that MIF promotes tumorigenesis, in part, by suppressing antitumor immunity. To examine whether tumor-derived MIF leads to suppression of T cell immunity in vivo, we generated MIF-deficient neuroblastoma cell lines using short hairpin small interfering RNAs (siRNA). The MIF knockdown (MIFKD) AGN2a neuroblastoma cells were more effectively rejected in immune-competent mice than control siRNA-transduced or wild-type AGN2a. However, the increased rejection of MIFKD AGN2a was not observed in T cell-depleted mice. MIFKD tumors had increased infiltration of CD8(+) and CD4(+) T cells, as well as increased numbers of macrophages, dendritic cells, and B cells. Immunization with MIFKD AGN2a cells significantly increased protection against tumor challenge as compared with immunization with wild-type AGN2a, and the increased protection correlated with elevated frequencies of tumor-reactive CD8(+) T cells in the lymphoid tissue of treated animals. Increased numbers of infiltrating tumor-reactive CD8(+) T cells were also observed at the site of tumor vaccination. In vitro, treatment of AGN2a-derived culture supernatants with neutralizing MIF-specific Ab failed to reverse T cell suppressive activity, suggesting that MIF is not directly responsible for the immune suppression in vivo. This supports a model whereby MIF expression in neuroblastoma initiates a pathway that leads to the suppression of T cell immunity in vivo.     

Phosphonium-containing diblock copolymers for enhanced colloidal stability and efficient nucleic Acid delivery

RAFT polymerization successfully controlled the synthesis of phosphonium-based AB diblock copolymers for nonviral gene delivery. A stabilizing block of either oligo(ethylene glycol(9)) methyl ether methacrylate or 2-(methacryloxy)ethyl phosphorylcholine provided colloidal stability, and the phosphonium-containing cationic block of 4-vinylbenzyltributylphosphonium chloride induced electrostatic nucleic acid complexation. RAFT polymerization generated well-defined stabilizing blocks (M(n) = 25000 g/mol) and subsequent chain extension synthesized diblock copolymers with DPs of 25, 50, and 75 for the phosphonium-containing block. All diblock copolymers bound DNA efficiently at ± ratios of 1.0 in H(2)O, and polyplexes generated at ± ratios of 2.0 displayed hydrodynamic diameters between 100 and 200 nm. The resulting polyplexes exhibited excellent colloidal stability under physiological salt or serum conditions, and they maintained constant hydrodynamic diameters over 24 h. Cellular uptake studies using Cy5-labeled DNA confirmed reduced cellular uptake in COS-7 and HeLa cells and, consequently, resulted in low transfection in these cell lines. Serum transfection in HepaRG cells, which are a predictive cell line for in vivo transfection studies, showed successful transfection using all diblock copolymers with luciferase expression on the same order of magnitude as Jet-PEI. All diblock copolymers exhibited low cytotoxicity (>80% cell viability). Promising in vitro transfection and cytotoxicity results suggest future studies involving the in vivo applicability of these phosphonium-based diblock copolymer delivery vehicles.     

Downregulation of MDM2 expression by RNAi inhibits LoVo human colorectal adenocarcinoma cells growth and the treatment of LoVo cells with mdm2siRNA3 enhances the sensitivity to cisplatin

To investigate the biological effect of mdm2 in human colorectal adenocarcinoma LoVo cells, three mdm2siRNA constructions were recombined and transient transfected into human colorectal adenocarcinoma LoVo cells with low differentiation character in vitro. The results showed that mdm2siRNA3 reduced mRNA level of mdm2 and protein level of mdm2, leading to proliferation inhibition on LoVo cells, and reduced tumor growth in nude mice. It was found that depletion of MDM2 in this pattern promoted apoptosis of LoVo cells and Cisplatin (DDP) treated in the mdm2siRNA3 transfected cell population would result in a substantial decrease by MTT colorimetry. Decreasing the MDM2 protein level in LoVo cells by RNAi could significantly inhibit tumor growth both in vitro and in vivo, which indicated that mdm2 gene played a definite role in the development and aggressiveness of human colon carcinoma. It also could be a therapeutic target in colorectal carcinoma. The synergistic activation of RNAi and cell toxicity agents indicated that the combination of chemotherapy and gene therapy will be a promising approach in the future.     

Cationic lipid polymerization as a novel approach for constructing new DNA delivery agents

In vivo gene delivery mediated by cationic lipids is often compromised by aggregation due to complexation with proteins in the blood. To improve the stability of cationic lipid-DNA complexes, the present study aimed to develop a novel approach in which a poly(cationic lipid) (PCL) is utilized to form stable cationic polyplexes for gene transfection. Hydrogenation of the acrylamide analogue of betaAE-DMRI, the polymerizable precursor of PCL, provided a monomeric lipid derivative (MHL) which was used for direct comparison of corresponding lipoplex stability, toxicity, and transfection activity. Various formulations of cationic liposomes, such as MHL, MHL-cholesterol (Chol), PCL, PCL-Chol, DOTAP-Chol, and commercially available lipofectamine were generated and examined in this study. The new poly(cationic lipid) did not display any significant toxicity to rat hepatocytes or Hep G2 cells as indicated by an LDH leakage assay. Furthermore, PCL was significantly less toxic than MHL, DOTAP-Chol or lipofectamine. Suspensions of PCL were resistant to aggregation even after 24 h of exposure to solutions containing 50 and 100% fetal bovine serum (FBS). In contrast, suspensions of lipofectamine extensively aggregated after 24 h of exposure to 50% FBS. To examine the influence of lipid polymerization on gene transfer activity, liposome-mediated transfections of a luciferase vector (pGL3) were performed in Hep G2 and Alexander cell lines. The luciferase activity of the PCL formulations in Hep G2 cells were similar to those of the MHL, DOTAP-Chol and lipofectamine formulations, demonstrating that lipid polymerization does not compromise transfection activity. In comparison to the monomeric precursor MHL and to the industry transfection standards DOTAP and lipofectamine, the novel poly(cationic lipid) exhibited the lowest cytotoxicity, was the most resistant to serum-induced aggregation and had comparable transfection activity when coformulated with cholesterol. This novel polymerization approach for the development of stable and active polyplexes may prove a valuable alternative for in vivo gene delivery.     

Role of biophysical parameters on ex vivo and in vivo gene transfer to the airway epithelium by polyethylenimine/albumin complexes

Efficient gene transfer to the airways by nonviral vectors is a function of different parameters, among which the size and the charge of the transfecting particles. The aim of this study was to determine the transfection efficiency of polyethylenimine (PEI)/albumin polyplexes in ex vivo and in vivo models of respiratory epithelium and to correlate it with biophysical characteristics of the particles. Complexes were obtained by adding different amounts of human serum albumin (HSA) to PEI polyplexes preformed in saline. The presence of HSA caused the formation of bigger and more negative polyplexes and increased PEI transfection efficiency in primary respiratory epithelial cells by 4-6-fold. For in vivo administration to the lung, PEI polyplexes were formed in water and optimized with respect to the N/ P ratio. PEI/pC-Luc complexes gave the highest luciferase expression at N/ P 15 when administered through the trachea. At this N/ P ratio, the size and the surface charge of albumin-containing polyplexes were not different as compared with plain PEI polyplexes. Formulation of PEI polyplexes in the presence of HSA or murine serum albumin (MSA) resulted in a 2-fold increase in luciferase expression. In mice treated with PEI or PEI/MSA polyplexes containing the nuclear beta-gal gene, X-gal staining revealed that transfected cells localized at the bronchiolar epithelium and that PEI/MSA transfected four times as many cells as PEI ( p < 0.05). Finally, double administration of PEI/MSA polyplexes resulted in a further enhancement of transfection of the lung. Our data show that serum albumin enhances PEI-mediated gene transfer to airway epithelial cells in vivo, likely facilitating the uptake of polyplexes, and indicate that this formulation would fulfill the requirement of repeated administration, as necessary in chronic lung diseases like cystic fibrosis.     

PEGylation of poly(ethylene imine) affects stability of complexes with plasmid DNA under in vivo conditions in a dose-dependent manner after intravenous injection into mice

The influence of PEGylation on polyplex stability from poly(ethylene imine), PEI, and plasmid DNA was investigated both in vitro and after intravenous administration in mice. Polyplexes were characterized with respect to particle size (dynamic light scattering), zeta-potential (laser Doppler anemometry), and morphology (atomic force microscopy). Pharmacokinetics and organ accumulation of both polymers and pDNA were investigated using 125I and 32P radioactive labels, respectively. Furthermore gene expression patterns after 48 h were measured in mice. To elucidate the effect of different doses, all experiments were performed using ca. 1.5 microg and 25 microg of pDNA per mouse. Our studies demonstrated that both PEI and PEG-PEI form stable polyplexes with DNA with similar sizes of 100-130 nm. The zeta potential of PEI/pDNA polyplexes was highly positive, whereas PEG-PEI/pDNA showed a neutral surface charge as expected. The pharmacokinetic and organ distribution profiles after 2 h show similarities for both PEI and pDNA blood-level time curves from polyplexes at both doses indicative for significant stability in the bloodstream. A very rapid clearance from the bloodstream was observed and as major organs of accumulation liver and spleen were identified. PEG-PEI/pDNA complexes at a dose of approximately 25 microg exhibit similar profiles except a significantly lower deposition in the lung. At the lower dose of approximately 1.5 microg pDNA, however, for polyplexes from PEG-PEI, significant differences in blood level curves and organ accumulation of polymer and pDNA were found. In this case PEG-PEI shows a greatly enhanced circulation time in the bloodstream. By contrast, pDNA was rapidly cleared from circulation and significant amounts of radioactivity were found in the urine, suggesting a rapid degradation possibly by serum nucleases after complex separation. Regarding in vivo gene expression, no luciferase expression could be detected at approximately 1.5 microg dose in any organ using both types of complexes. At 25 microg only in the case of PEI/pDNA complexes were significant levels of the reporter gene detected in lung, liver, and spleen. This coincided with high initial accumulation of pDNA complexed with PEI and a high acute in vivo toxicity. For PEG-PEI, initial accumulation was much lower and no gene expression as well as a low acute toxicity was found. In summary, our data demonstrate that PEG-PEI used in this study is not suitable for low dose gene delivery. At a higher dose of approximately 25 microg, however, polyplex stability is similar to PEI/pDNA combined with a more favorable organ deposition and significantly lower acute in vivo toxicity. These findings have consequences for the design of PEG-PEI-based gene delivery systems for in vivo application.     

Acetal linked oligoethylenimines for use as pH-sensitive gene carriers

Two types of acid-degradable nonviral gene carriers, OEI-MK and OEI-BAA, were synthesized by polymerizing oligoethylenimine of 800 Da (OEI800) with the pH-sensitive acetone ketal cross-linker 2,2-bis(N-maleimidoethyloxy) propane (MK) or the 4-methoxybenzaldehyde bisacrylate acetal cross-linker 1,1-bis-(2-acryloyloxy ethoxy)-[4-methoxy-phenyl]methane) (BAA). Corresponding acid-insensitive counterparts (OEI-BM and LT-OEI-HD) were synthesized as well, representing control polymers. Kinetics of hydrolysis were measured and confirmed the pH-dependent degradation profile of the acetal functions, with short half-lives of 3 min at pH 5.0, and 5 h (OEI-MK) or 3.5 h (OEI-BAA) at physiological pH 7.4 and 37 degrees C. DNA polyplexes of a luciferase expression plasmid were tested for gene transfer efficiency and biocompatibility in two cell lines (B16F10 and Neuro2A). Polyplexes with acid-labile polymers showed an improved toxicity profile compared to those made with acid-stable polymer analogues. At low cation/plasmid (c/p) w/w ratios the transfection efficiency of pH-sensitive polymers was slightly reduced, but it became similar or superior to the efficiency of acid-stable polymers at higher c/p ratios. An improved in vivo biocompatibility of the acid-degradable polymers over the stable control polymers was confirmed by liver histology after systemic administration of polymers in Balb/c mice.     

Extracellular barriers to in Vivo PEI and PEGylated PEI polyplex-mediated gene delivery to the liver

Polyplex-mediated gene therapy is a promising alternative to viral gene therapy. One challenge to these synthetic carriers is reduced transfection efficiencies in vivo compared to those achieved in vitro. Many of the intracellular barriers to gene delivery have been elucidated, but similar quantification of extracellular barriers to gene delivery remains a need. In this study, the unpackaging of polyplexes by serum proteins, soluble glycosaminoglycans, and an extracellular matrix extract was demonstrated by a YOYO-1 fluorescence quenching assay. Additionally, exposing polyplexes to serum or proteoglycans before in vitro transfection caused decreased cellular uptake of DNA. Lastly, PEI polyplexes and PEGylated PEI polyplexes were injected into the portal vein of mice, and the intrahepatic distributions of labeled DNA and polymer were assessed by confocal microscopy. PEI polyplexes delivered DNA to the liver, but extensive vector unpackaging was observed, with PEI primarily colocalized with the extracellular matrix. PEGylated polyplexes mediated less DNA delivery to the liver, possibly due to premature vector unpackaging in the blood. Through this work, both the blood and the extracellular matrix have been determined to be significant extracellular barriers to polyplex-mediated in vivo gene delivery to the liver.     

小干扰RNA靶向VEGF基因体内外抑制乳腺癌细胞MCF-7的增殖

 血管生成与肿瘤生长、侵袭、转移密切相关.血管内皮生长因子能特异地促进内皮细胞分裂、增殖及迁移,在肿瘤新生血管生成过程中起着至关重要的作用.通过RNAi抑制VEGF表达的抗血管生成疗法可有效应用于肿瘤治疗.本研究采用化学修饰的siRNA在体内外抑制VEGF基因表达,探讨化学修饰的siRNA介导的RNA干扰技术在乳腺癌基因治疗的可行性和特异性.选用阳离子脂质体Lipofectamine^TM2000作为转染试剂,将针对人VEGF基因的小干扰RNA(small interfering RNA,siRNA)转染人类乳腺细胞株MCF-7和裸鼠移植瘤,在体内外诱导RNAi.采用四甲基偶氮唑蓝（MTT）法,逆转录聚合酶链反应(RT-PCR),蛋白印迹实验等检测siRNA治疗组和对照组VEGF基因表达及细胞增殖变化.体外实验结果显示：靶向VEGF基因siRNA转染乳腺癌MCF-7细胞后,细胞生长抑制率达52.5%;VEGF的mRNA和蛋白表达水平显著降低（P<0.01）;裸鼠体内实验结果显示：siRNA治疗组瘤组织的增长受到明显抑制;RT-PCR结果同时表明治疗组VEGF表达下调.体内外对照组各指标无显著变化.化学修饰的siRNA介导的RNAi在体内外均能成功下调靶基因VEGF的表达,抑制MCF-7细胞增殖,是潜在的肿瘤治疗新方法.