Aerosolization of lipoplexes using AERx® pulmonary delivery system

The lung represents an attractive target for delivering gene therapy to achieve local and potentially systemic delivery of gene products. The objective of this study was to evaluate the feasibility of the AERx Pulmonary Delivery System for delivering nonviral gene therapy formulations to the lung. We found that “naked” DNA undergoes degradation following aerosolization through the AERx nozzle system. However, DNA formulated with a molar excess of cationic lipids (lipoplexes) showed no loss of integrity. In addition, the lipoplexes showed no significant change in particle size, zeta (ζ) potential, or degree of complexation following extrusion. The data suggest that complexation with cationic lipids had a protective effect on the formulation following extrusion. In addition, there was no significant change in the potency of the formulation as determined by a transfection study in A-549 cells in culture. We also found that DNA formulations prepared in lactose were aerosolized poorly. Significant improvements in aerosolization efficiency were seen when electrolytes such as NaCl were added to the formulation. In conclusion, the data suggest that delivery of lipoplexes using the AERx Pulmonary Delivery System may be a viable approach for pulmonary gene therapy.     

Anionic pH-sensitive pegylated lipoplexes to deliver DNA to tumors

Anionic pegylated lipoplexes have been prepared from the combined formulation of cationic lipoplexes and pegylated anionic liposomes. To this end, two original (bis- and tetra-) carboxylated cholesterol derivatives have been synthesised. Titration of the particle surface charge was realised to determine the ratio between anionic and cationic lipids that would give pH-sensitive complexes. This ratio has been optimised to form particles sensitive to pH change in the range 5.5-6.5. Compaction of DNA into these newly formed anionic complexes was checked by DNA accessibility to picogreen and DNA electrophoresis on an agarose gel. Gene expression of the formulated gene was similar for the cationic formulation taken as a control and the anionic formulations prepared. The pH-sensitive properties of these formulations was shown in vitro using bafilomycin, a vacuolar H(+)ATPase inhibitor. The efficiency of the new formulations to deliver DNA to the tumor was compared with cholesteryl hemisuccinate (CHEMS) formulations. The tetracarboxylated compound gave the most efficient formulations for tumor delivery in vivo.     

Preparation and gene delivery of alkaline amino acids-based cationic liposomes

Cationic lipids 1, 2, and 3, based on hydrophobic cholesterol linked to L-lysine, L-histidine or L-arginine, respectively, were designed and tested as gene delivery vectors. Physicochemical and biological properties of all liposomes and lipoplexes were evaluated, including lipid-DNA interactions, size, morphology, zeta potential, acid-base buffering capability, protection of DNA from DNase I digestion, and cytotoxity. The efficiency of luciferase gene transfection of lipoplexes 1-3 was compared with that of commercial dioleoyl-trimethylammonium propane (DOTAP) and polyethyleneimine (PEI) in 293T cells and HepG2 cells with or without poly(ethylene glycol) PEG stabilizer. The complexation and protection of DNA of liposome 3 was the strongest among the three liposomes. The efficiency of gene transfection of liposomes 1-3 was two-to threefold higher than that of PEI and/or DOTAP in 293T cells. Liposomes 1 and 3 in PEG as stabilizer showed sixfold higher transfection efficiency than that of PEI and/or DOTAP, whereas liposome 2 showed very low transfection efficiency. In HepG2 cells, the transfection efficiency of all the cationic liposomes was much lower than that of DOTAP. In conclusion, lipids 1-3 were efficient and non-toxic gene vectors; the headgroup of cationic lipids and the stabilizer of liposome formulation had an important influence on gene transfection.     

Gamma scintigraphic evaluation of a miniaturized AERx pulmonary delivery system for aerosol delivery to anesthetized animals using a positive pressure ventilation system.

The purpose of this study was to characterize performance of a miniaturized AERx((R)) Pulmonary Delivery System designed for aerosol administration to large animal models. The miniaturized AERx System was developed through a systematic scaling down of the AERx System used for humans to allow for operation in certain animal models with lower inspiratory flow rates and inhaled volumes than those used for humans. We used gamma scintigraphy to characterize the in vivo particle deposition achieved with the miniaturized AERx System in two dogs. The dogs were 3-4 years old, and weighed 10.4 kg and 13.6 kg. Acepromazine was used as pre-anesthetic medication. Anesthesia was induced with 5% isoflurane. The trachea was intubated using an endotracheal tube (internal diameter 8.5 mm), and the dogs were ventilated using positive pressure during the exposure using the LRRI puff generator. An inhalation of aerosol was initiated by activation of the puff generator though the computer-controlled interface. Each dog inhaled approximately 0.8 L per puff, of which the aerosol volume comprised approximately 0.25 L, at a target flow rate of 15 L/min. The dogs were exposed to 10 AERx Strips in 10 puffs. The mass median aerodynamic diameter of the aerosolized formulation was approximately 1.25 microm with a fine particle fraction <3.5 microm of 0.976. The scintigraphic images showed uniform bilateral lung deposition following aerosol delivery with the AERx System. Total lung deposition for the two dogs was 10.7% and 18% of the loaded dose from the AERx Strip. The corresponding peripheral lung: inner lung (P/I) ratios were 0.83 and 0.75, suggestive of deposition in the deep lung. Only 0.1% to 0.2% of the loaded dose was exhaled. These results show the miniature AERx System can efficiently deliver aerosols to the deep lung of dogs. The miniaturized AERx System would be a valuable tool for conducting proof-of-concept studies as well as safety and tolerability analysis of inhaled drug candidates in large animal models.     

Anionic liposomal delivery system for DNA transfection

The present study investigates the use of novel anionic lipoplexes composed of physiological components for plasmid DNA delivery into mammalian cells in vitro. Liposomes were prepared from mixtures of endogenously occurring anionic and zwitterionic lipids, 1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (sodium salt) (DOPG) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), respectively, at a molar ratio of 17:83 (DOPG:DOPE). Anionic lipoplexes were formed by complexation between anionic liposomes and plasmid DNA molecules encoding green fluorescence protein (GFP) using Ca2+ ions. Transfection and toxicity were evaluated in CHO-K1 cells using flow cytometry and propidium iodide staining, respectively. Controls included Ca2+-DNA complexes (without lipids), anionic liposomes (no Ca2+), and a cationic liposomal formulation. Efficient delivery of plasmid DNA and subsequent GFP expression was achieved using anionic lipoplexes. Transfection efficiency increased with Ca2+ concentration up to 14 mM Ca2+, where transfection efficiency was 7-fold higher than in untreated cells, with minimum toxicity. Further increase in Ca2+ decreased transfection. Transfection efficiency of anionic lipoplexes was similar to that of cationic liposomes (lipofectAmine), whereas their toxicity was significantly lower. Ca2+-DNA complexes exhibited minimal and irregular transfection with relatively high cytotoxicity. A model was developed to explain the basis of anionic lipoplex uptake and transfection efficacy. Effective transfection is explained on the formation of nonbilayer hexagonal lipid phases. Efficient and relatively safe DNA transfection using anionic lipoplexes makes them an appealing alternative to be explored for gene delivery.     

New synthetic glycolipids for targeted gene transfer: synthesis, formulation in lipoplexes and specific interaction with lectin

Nonviral gene delivery systems are a promising approach for gene therapy applications, despite their low in vivo gene transfer efficiency. One approach to enhance this efficiency is to incorporate targeting elements into cationic lipid/DNA complexes (lipoplexes). Ligand-containing lipoplexes have to retain their efficiency while exposing accessible ligand on their surface. Physicochemical properties (particle size, surface charge, and efficacy of DNA complexation) of the lipoplexes largely determine their gene transfer efficiency. We synthesized glycolipids with various galactosylated head ligand and incorporated them into lipoplexes. We showed that incorporation of up to 33% mol of glycolipid did not change the physicochemical properties of lipoplexes. Some of our glycolipids yielded lipoplexes whose galactosyl heads were well exposed on the surface as demonstrated by a strong interaction with Ricinus communis agglutinin. Glycolipid-containing lipoplexes gave an efficient gene transfer on hepatocytes, although no ligand-targeted transfection could be observed.     

Development of nanosomes using high‐pressure homogenization for gene therapy

Objectives The aim of this project was to develop a novel lipid‐based formulation suitable for gene therapy. Methods Novel nanosize liposome (nanosome) formulations containing pDNA (plasmid DNA) were developed using high‐pressure homogenization (HPH). The effect of lipid concentration was studied at two levels: 3 mm and 20 mm . The preformed nanosomes were incubated for 18–20 h with pDNA or pDNA/protamine sulfate (PS) complex. The physical properties of the pDNA nanosomes were compared by particle size distribution and zeta‐potential measurements. Their biological properties were also compared by pDNA efficiency of encapsulation/complexation, integrity, nuclease digestion, transfection efficiency and cell cytotoxicity. Key findings pDNA nanosomes prepared with 20 mm lipid (nanosomes : pDNA : PS at a ratio of 8.6 : 1 : 2) had particle sizes of 170–422 nm (90% confidence). The zeta‐potential of the formulation was 49.2 ± 1.5 mV, and the pDNA encapsulation/complexation efficiency was ～98%. pDNA nanosomes prepared with 3 mm lipid (nanosomes : pDNA : PS at a ratio of 2.09 : 1 : 2) had particle sizes of 140–263 nm (90% confidence). The zeta‐potential of this formulation was 36.4 ± 1.2 mV, and the pDNA encapsulation/complexation efficiency was ～100%. However, a comparison of the efficiency of transfection indicated that pDNA nanosomes prepared with low‐concentration lipids (3 mm ) showed significantly higher transfection efficiency compared with the pDNA nanosomes prepared with high‐concentration lipids (20 mm ), as well as those prepared with Fugene‐6 (a commercially available transfection reagent). This particular formulation (pDNA nanosomes, 3 mm lipids) also showed significantly less cytotoxicity compared with the other pDNA nanosome formulations. Conclusions To conclude, these results indicate that condensing pDNA with PS followed by subsequent complexation with low‐concentration nanosomes generated from HPH can produce a pDNA nanosome formulation that will boost transfection efficiency, while minimizing cytotoxicity. This new technology appears to be an efficient tool for future commercial or large‐scale manufacture of DNA delivery systems for gene therapy.     

Peptide-mediated lipofection is governed by lipoplex physical properties and the density of surface-displayed amines

Peptides can potentiate lipid-mediated gene delivery by modifying lipoplex physiochemical properties to overcome rate-limiting steps to gene transfer. The objectives of this study were to determine the regimes over which cationic peptides enhance lipofection and to investigate the mechanism of action, such as increased cellular association resulting from changes in lipoplex physical properties. Short, cationic peptides were incorporated into lipoplexes by mixing peptide, lipid and DNA. Lipoplexes were characterized using gel retardation, dynamic light scattering, and fluorescent microscopy, and the amount of surface-displayed amines was quantified by fluorescamine. Size, zeta potential, and surface amines for lipoplexes were dependent on peptide/DNA ratio. Inclusion of peptides in lipoplexes resulted in up to a 13-fold increase in percentage of cells transfected, and up to a 76-fold increase in protein expression. This transfection enhancement corresponded to a small particle diameter and positive zeta potential of lipoplexes, as well as increased amount of surface-displayed amines. Relative to lipid alone, these properties of the peptide-modified lipoplexes enhanced cellular association, which has been reported as a rate-limiting step for transfection with lipoplexes. The addition of peptides is a simple method of lipofection enhancement, as direct chemical modification of lipids is not necessary for increased transfection.     

Comparison of in vitro and in vivo efficiencies of a novel unit-dose liquid aerosol generator and a pressurized metered dose inhaler

Gamma scintigraphic imaging was employed in 10 healthy volunteers to compare the total and regional lung deposition of aerosols generated by two delivery platforms that permitted microprocessor-controlled actuation at an optimal point during inhalation. An aqueous solution containing 99mTc-DTPA was used to assess the deposition of aerosols delivered by inhalation from two successive unit-dosage forms (44 microl volume) using a prototype of a novel liquid aerosol system (AERx Pulmonary Delivery System). This was compared with aerosol deposition after inhalation of two 50 microl puffs of a 99mTc-HMPAO-labeled solution formulation from a pressurized metered dose inhaler (MDI). The in vitro size characteristics of the radiolabeled aerosols were determined by cascade impaction. For the AERx system, the predicted lung delivery efficiency based on the product of emitted dose (60.8%, coefficient of variation (CV)=12%) and fine particle fraction (% by mass of aerosol particles <5.7 microm in diameter) was 53.3% (CV=13%). For the solution MDI, the emitted dose was 62.9% (CV=13%) and the predicted lung dose was 44. 9% (CV=15%). The AERx system demonstrated efficient and reproducible dosing characteristics in vivo. Of the dose loaded into the device, the mean percent reaching the lungs was 53.3% (CV=10%), with only 6. 9% located in the oropharynx/stomach. In contrast, the lung deposition from the solution MDI was significantly less (21.7%) and more variable (CV=31%), with 42.0% of the radiolabel detected in the oropharynx/stomach. Analysis of the regional deposition of the radioaerosol indicated a homogeneous pattern of deposition after delivery from the AERx system. A predominantly central pattern of distribution occurred after MDI delivery, where the pattern of deposition was biased towards a central zone depicting the conducting airways. The AERx system, in contrast to MDIs, seems highly suited to the delivery of systemically active agents via pulmonary administration.     

New Synthetic Glycolipids for Targeted Gene Transfer: Synthesis,Formulation in Lipoplexes and Specific Interaction with Lectin

Nonviral gene delivery systems are a promising approach for gene therapy applications, despite their low in vivo gene transfer efficiency. One approach to enhance this efficiency is to incorporate targeting elements into cationic lipid/DNA complexes (lipoplexes). Ligand-containing lipoplexes have to retain their efficiency while exposing accessible ligand on their surface. Physicochemical properties (particle size, surface charge, and efficacy of DNA complexation) of the lipoplexes largely determine their gene transfer efficiency. We synthesized glycolipids with various galactosylated head ligand and incorporated them into lipoplexes. We showed that incorporation of up to 33% mol of glycolipid did not change the physicochemical properties of lipoplexes. Some of our glycolipids yielded lipoplexes whose galactosyl heads were well exposed on the surface as demonstrated by a strong interaction with Ricinus communis agglutinin. Glycolipid-containing lipoplexes gave an efficient gene transfer on hepatocytes, although no ligand-targeted transfection could be observed.     

Biotinylated Cyclen‐Contained Cationic Lipids as Non‐Viral Gene Delivery Vectors

A series of 1, 4, 7, 10‐tetraazacyclododecane (cyclen)‐based cationic lipids, namely 5a–c bearing a biotin moiety and a variety of end groups (cholesterol, diosgenin, and α‐tocopherol) via biodegradable carbamate bond linkage were prepared and applied as non‐viral gene delivery vectors. The liposomes formed from 5 and dioleoylphosphatidylethanolamine could bind and condense plasmid DNA into nanoparticles with appropriate size and zeta potentials. All biotinylated cyclen cationic lipids showed higher cell viability than commercially available lipofectamine 2000 even at high N/P ratios, while their transfection efficiency was relatively lower. Further, results indicate that among the three lipids, α‐tocopherol‐containing compound 5c has higher DNA‐binding ability, lower cytotoxicity, and higher transfection efficiency. Transfection in two different cell lines revealed that these lipoplexes have higher gene delivery efficiency toward tumor cells.     

Cationic lipids for gene delivery in vitro and in vivo

Certain disease states can be corrected by using nucleic acids as therapeutic agents. To achieve this, nucleic acids must be delivered into the affected cells efficiently. At the core of a successful gene therapy protocol is the design of the nucleic acid carrier. Cationic lipids, as one of the gene delivery systems, have a wide potential in delivering nucleic acids both in vivo and in vitro. They are synthetic in origin and, hence, can be produced in required quantities and are biologically safe. Significant inputs from synthetic chemists in the recent past have resulted in the exploration of cationic lipids with very interesting functionalities. Transfection efficiencies of cationic lipids are comparable to viral-mediated transfection in vitro. However, viral-based methods for gene delivery in vivo are comparatively more efficient. Current understanding of lipid-mediated transfection is partially due to incomplete characterisation of the lipoplex, poor understanding of cell biology of transfection and cell type variations in transfection efficiencies. The published patents and research demonstrates the need for incorporation of the biological information in the design of the gene delivery formulations. In this review, the cell biological aspects critical for lipid-mediated transfection are emphasised. The parameters that influence the colloidal stability of the lipoplexes, cell biological processes relevant to gene delivery, such as cell association/uptake, cytoplasmic stability of the DNA and nuclear import, are discussed. The main focus of this review is patents published in the last 5 years.     

Spacer-arm modulated gene delivery efficacy of novel cationic glycolipids: design, synthesis, and in vitro transfection biology

Design, syntheses and relative in vitro gene delivery efficacies of six novel cationic glycolipids 1-6 containing open-form galactosyl units in CHO, COS-1, MCF-7 and A549 cells are described. The results of the present structure-activity investigation convincingly demonstrate that the in vitro gene delivery efficacies of galactosylated cationic glycolipids are strikingly dependent on the absence of a spacer-arm between the open-form galactose and the positively charged nitrogen atom in their headgroup region. While the cationic glycolipids 1-3 with no headgroup spacer unit between the positively charged nitrogen and galactose showed high in vitro gene transfer efficacies in all four cells (lipids 1 and 2 with myristyl and palmityl tails, respectively, being the most efficacious), lipids 4-6 with five-carbon spacer units between the quaternized nitrogen and galactose heads were essentially transfection incompetent. The transfection inhibiting role of the five-carbon spacer unit in the headgroup region of the present novel class of cationic lipids was demonstrated by both beta-galactosidase reporter gene expression and histochemical X-gal staining assays. Results of MTT assay-based cell viability measurements in representative MCF7 cells show that cell viabilities of lipoplexes (lipid:DNA complexes) prepared from all the lipids 1-6 are remarkably high. Thus, possibilities of differential cellular cytotoxicities playing any key role behind the strikingly contrasting transfection properties of lipids 1-3 with no spacer and lipids 4-6 with a spacer unit in the headgroup regions was ruled out. Electrophoresis gel patterns in DNase I sensitivity assays are consistent with more free DNA (accessible to DNase I) being present in lipoplexes of lipids 4-6 than in lipoplexes of lipids 1-3. Thus, the results of our DNase I protection experiments support the notion that enhanced degradation of DNA associated with lipoplexes of lipids 4-6 may play an important role in abolishing their in vitro gene transfer efficacies.     

Folate-associated lipoplexes mediate efficient gene delivery and potent antitumoral activity in vitro and in vivo

The lack of suitable vectors for efficient nucleic acid delivery into target cells represents a major hurdle for the successful application of gene therapy. Cationic liposomes exhibit attractive features for gene delivery, but their efficacy is still unsatisfactory, particularly for in vivo applications, which justifies the drive to further improve their performance by developing novel and efficient formulations. In the present study, we generated a new formulation of lipoplexes through electrostatic association of folate (FA) to 1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine (EPOPC):cholesterol (Chol) liposomes, prepared at different lipid/DNA charge ratios, and explored their potential to mediate gene delivery. The optimal FA-lipoplex formulation was evaluated for its efficacy to mediate antitumoral activity upon application of HSV-tk suicide gene therapy, both in vitro and in an animal model of oral cancer. Our results demonstrate that FA-EPOPC:Chol/DNA lipoplexes were able to promote a great enhancement of transfection and high in vitro antitumoral activity compared to plain lipoplexes in two different cancer cell lines. Most importantly, a considerable reduction of tumor growth was achieved with the developed FA-lipoplexes as compared to that observed for control FA-lipoplexes or plain lipoplexes. Overall, our study shows that FA-EPOPC:Chol/DNA lipoplexes constitute a promising system for the successful application of suicide gene therapy aiming at treating solid tumors.     

阳性脂质体介导基因转染及其研究进展*

基因治疗是一种很有前景的治疗模式,而阳性脂质体介导的基因转染是目前基因治疗的研究热点之一。现综述近5年来有关阳性脂质体的文献,介绍了阳性脂质体的基本组成,并从生物学、理化性质及制剂学等几个方面介绍了影响阳性脂质体/DNA复合物转染效率的主要因素,最后从新的阳性脂质成分及阳性脂质体或阳性脂质体/DNA复合物的表面修饰等方面介绍了近年来有关改善阳性脂质体介导基因转染的研究进展。     

Peptide-based cationic molecules for the production of positive charged liposomes and micelles

This paper describes the synthesis and the physico-chemical characterization of cationic peptides (CPs) for possible application as non-viral gene delivery systems. Particularly, the production of cationic liposomes and micelle solutions was considered. Liposomes were prepared by REV-phase and extrusion presenting an average diameter reflecting the pore size of the membrane used for the extrusion. After DNA complexation the mean diameter of complexes decreased by increasing the number of positive charges. The non-complexed liposome preparations showed a net positive zeta potential comprised between 17.8-30 mV. After adding Defibrotide (DFT) to liposomes (at a 1:4 +/- molar ratio) the zeta potential fell down to a net negative value indicating the formation of the ionic complex. Concerning micelles, before complexation it was not possible to measure their size by PCS. However, after DFT complexation the size of complexes highly increased. In addition, as previously seen for liposomes, before complexation, the five CPs solutions showed a positive zeta potential ranging from 10-17.8 mV, while after addition of DFT the zeta potential fell to negative values. Concerning toxicity studies, in general CP-liposomes displayed a lower toxicity towards K562 cells as compared to the corresponding CP-solution. Taking into account these results, the studied CPs could be efficiently used to obtain both cationic liposomes and micelles. Moreover they are able to complex DNA with different interaction strength, depending on the type of peptide-based cationic molecule used.     

Peptide-based cationic molecules for the production of positive charged liposomes and micelles

This paper describes the synthesis and the physico-chemical characterization of cationic peptides (CPs) for possible application as non-viral gene delivery systems. Particularly, the production of cationic liposomes and micelle solutions was considered. Liposomes were prepared by REV-phase and extrusion presenting an average diameter reflecting the pore size of the membrane used for the extrusion. After DNA complexation the mean diameter of complexes decreased by increasing the number of positive charges. The non-complexed liposome preparations showed a net positive zeta potential comprised between 17.8–30 mV. After adding Defibrotide (DFT) to liposomes (at a 1:4?±?molar ratio) the zeta potential fell down to a net negative value indicating the formation of the ionic complex. Concerning micelles, before complexation it was not possible to measure their size by PCS. However, after DFT complexation the size of complexes highly increased. In addition, as previously seen for liposomes, before complexation, the five CPs solutions showed a positive zeta potential ranging from 10–17.8 mV, while after addition of DFT the zeta potential fell to negative values. Concerning toxicity studies, in general CP-liposomes displayed a lower toxicity towards K562 cells as compared to the corresponding CP-solution. Taking into account these results, the studied CPs could be efficiently used to obtain both cationic liposomes and micelles. Moreover they are able to complex DNA with different interaction strength, depending on the type of peptide-based cationic molecule used.     

Efficient and safe delivery of siRNA using anionic lipids: Formulation optimization studies

Novel formulations based on physiologically occurring anionic lipids have been designed to achieve safe and efficient siRNA delivery. Anionic liposomes (DOPG/DOPE) were complexed with siRNA using calcium ion bridges to prepare anionic lipoplexes. Various formulation parameters (liposome composition, lipid and calcium concentration) were evaluated and optimized to achieve efficient silencing and high cell viability in breast cancer cells. The optimal anionic lipoplexes composed of 1μg/mL lipid (40:60 (DOPG/DOPE m/m)), 2.4mM calcium and 10nM siRNA, showed maximum silencing (～70% knockdown) without being cytotoxic. These lipoplexes also showed stability and high efficiency in the presence of serum. Additionally, optimal anionic lipoplexes showed efficient intracellular uptake and endosomal escape. Characterization studies indicated the optimal anionic formulations were 324.2±19.6nm with a surface charge of (-22.9±0.1)mV and 98.5±1.4% encapsulation efficiency. Control cationic lipoplexes (Lipofectamine 2000) showed silencing comparable to the anionic lipoplexes but were highly cytotoxic as indicated by IC50 values (cationic - 22.9μg/mL, compared to anionic - greater than 10(7)μg/mL). Calcium-siRNA complexes (without liposomes) showed low efficiency (～50% silencing), and highly variable results. The optimized anionic formulations may offer a safer alternative to conventional cationic based systems for efficient in vitro as well as in vivo delivery of therapeutic siRNAs.     

A Novel Formulation Based on 2,3-Di(tetradecyloxy)propan-1-amine Cationic Lipid Combined with Polysorbate 80 for Efficient Gene Delivery to the Retina

Purpose

The aim of the present study was to evaluate the potential application of a novel formulation based on a synthesized cationic lipid 2,3-di(tetradecyloxy)propan-1-amine, combined with polysorbate 80 to deliver the pCMS-EGFP plasmid into the rat retina.

Methods

We elaborated lipoplexes by mixing the formulation containing the cationic lipid and the polysorbate 80 with the plasmid at different cationic lipid/DNA ratios (w/w). Resulted lipoplexes were characterized in terms of size, charge, and capacity to condense, protect and release the DNA. In vitro transfection studies were performed in HEK-293 and ARPE-19 cells. Formulations were also tested in vivo by monitoring the expression of the EGFP after intravitreal and subretinal injections in rat eyes.

Results

At 2/1 cationic lipid/DNA mass ratio, the resulted lipoplexes had 200 nm of hydrodynamic diameter; were positive charged, spherical, protected DNA against enzymatic digestion and transfected efficiently HEK-293 and ARPE-19 cultured cells exhibiting lower cytotoxicity than LipofectamineTM 2000. Subretinal administrations transfected mainly photoreceptors and retinal pigment epithelial cells; whereas intravitreal injections produced a more uniform distribution of transfection through the inner part of the retina.

Conclusions

These results hold great expectations for other gene delivery formulations based on this cationic lipid for retinal gene therapy purposes.     

Advances in noninvasive pulmonary gene therapy

One of the most noninvasive approaches to drug delivery is via inhalation. The delivery of genes via aerosol holds promise for the treatment of a broad spectrum of pulmonary disorders and offers numerous advantages over more invasive modes of delivery. Delivery of genes expressing secretory therapeutic proteins or peptides may even have application to a number of nonpulmonary diseases. After the cloning of the cystic fibrosis gene, there was great interest in the delivery of genes directly to the lung surfaces via inhalation and most early efforts focused on the use of nonviral vectors, particularly cationic lipids. Early on, nebulization shear forces, inefficient penetration of mucous barriers and inhibitory effects of surfactant and other lung specific features generally resulted in a lack of therapeutic effect. But in recent years, a number of other nonviral and even viral vectors have been delivered successfully in this manner. Polyethyleneimine (PEI)-based formulations have proven stable during nebulization and result in transfection of a very large proportion of epithelial cells throughout the airways (though the level of transgene expression per cell may be relatively low), as well as significant, though lower levels of transfection throughout the lung parenchyma. Most importantly, therapeutic responses have been obtained in several animal lung tumor models when PEI-based complexes of p53 and IL-12 genes were delivered by aerosol. This approach may also prove useful as a means of localized genetic immunization. In addition, inhalation delivery of some formulations seems to be associated with surprisingly low toxicity and has resulted in little or no immunostimulatory response to the unmethylated CpG sequences in bacterially-produced plasmid DNA, which has presented a challenge to repeated gene therapy via many other modes of delivery.