Eleonora Vighi

pDNA condensation capacity and in vitro gene delivery properties of cationic solid lipid nanoparticles

Cationic solid lipid nanoparticles (SLN) are promising nonviral gene delivery carriers suitable for systemic administration. The objective of this study was to investigate the relationship between the composition of cationic SLN and their ability to condense plasmid DNA (pDNA) and to transfer it in neuroblastoma cells. The SLN were prepared by using stearic acid and stearylamine as lipid core along with Esterquart 1 (EQ1) or Protamine obtaining two samples (SLN-EQ1 and SLN-Protamine, respectively). The cationic SLN were freeze-dried after preparation and their physical-chemical properties, including the surface composition and the transfection efficiency were investigated. The results showed that the two samples had similar size, zeta potential and pDNA binding properties but SLN-Protamine were able to condense pDNA more efficaciously than SLN-EQ1 forming smaller and less positive complexes. SLN-Protamine:pDNA complexes demonstrated to be less cytotoxic and more efficient in the transfection of Na1300 cell line than SLN-EQ1:pDNA. These findings were attributed to the different surface composition of the two samples and in particular to the localization of the Protamine on the surface of the particle while EQ1 in the lipid core. In conclusion the results here suggest that not only the z-potential but also the surface composition may affect the pDNA condensation proprieties and thus the transfection efficiency of nonviral gene nanocarriers.

Nuclear localization of cationic solid lipid nanoparticles containing Protamine as transfection promoter.

Protamine has attracted much attention as DNA condenser and nuclear transfer enhancer although the excess of hydrophilicity and the strong DNA pack restrain its potentialities. In order to overcome this limitation, we added Protamine in the composition of solid lipid nanoparticles (SLN-Protamine) and we compared this carrier with the same kind of SLN containing Esterquat 1 instead of Protamine (SLN-EQ1). Carriers cytotoxicity was assessed on COS-I cells evaluating the cell cycle by propidium iodide test, while the transfection efficiency was studied using pEGFP as plasmid model. The cell penetrating activity of Protamine inside the lipid vectors was evaluated studying cell internalization by confocal microscopy using Red Nile-labeled carriers. SLN-Protamine:pDNA showed a mean diameter five-times smaller than the size of SLN-EQ1:pDNA and a remarkably lesser cytotoxicity. Transfection by SLN-Protamine:pDNA was seven-times more effective compared with the Protamine:pDNA polyplexes while no transfection capacity was observed for SLN-EQ1:pDNA complexes due to their inability to be internalized owing to their larger dimension. Red Nile-SLN-Protamine were localized in endocytic-like vesicles into the nuclear membrane suggesting the inclusion of Protamine in nano-lipophilic systems may enhance the reduction in the complex dimensions, the nuclear pDNA translocation and the pDNA release in the cells.

The role of protamine amount in the transfection performance of cationic SLN designed as a gene nanocarrier

Cationic solid lipid nanoparticles (SLN) have been recently proposed as non-viral vectors in systemic gene therapy. The aim of this study was to evaluate the effect of the protamine amount used as the transfection promoter in SLN-mediated gene delivery. Three protamine-SLN samples (Pro25, Pro100, and Pro200) prepared by adding increasing amounts of protamine were characterized for their size, zeta potential, and protamine loading level. The samples were evaluated for pDNA complexation ability by gel-electrophoresis analysis and for cytotoxicity and transfection efficiency by using different cell lines (COS-I, HepG2, and Na1300). The size of SLN was ~230 nm and only Pro200 showed few particle aggregates. Unlike the Pro25 sample with the lowest protamine loading level, the others SLN samples (Pro100 and Pro200) exhibited a good ability in complexing pDNA. A cell-line dependent cytotoxicity lower than that of the positive control PEI (polyethilenimmine) was observed for all the SLN. Among these, only Pro100, having an intermediate amount of protamine, appeared able to promote pDNA cell transfer, especially in a neuronal cell line (Na1300). In conclusion, the amount of protamine as the transfection promoter in SLN affects not only the gene delivery ability of SLN but also their capacity to transfer genes efficiently to specific cell types.

Fabrication via a nonaqueous nanoprecipitation method, characterization and in vitro biological behavior of N6-cyclopentyladenosine-loaded nanoparticles

A novel nonaqueous nanoprecipitation method was proposed to achieve the encapsulation of a small weight hydrophilic drug (N(6)-cyclopentyladenosine, CPA) in PLGA nanoparticles using a mixture of cottonseed oil and Tween-80 as nonsolvent phase. The nanoparticles were characterized in vitro as concerns size, morphology, drug loading, drug release, and drug stability in human blood. Human retinal pigment epithelium (HRPE) cells were employed to study intracellular accumulation of encapsulated or free CPA with and without unloaded particles, in the presence or absence of an equilibrative nucleoside transporter inhibitor. The particles displayed a mean size lower than 300 nm and a drug loading considerably higher than that found by conventional encapsulation methods. The suitable in vitro release properties permitted to obtain good drug stabilization in the blood. Studies on HRPE cells suggested that CPA can permeate their membrane by both diffusive- and transport-mediated mechanisms. The loaded and unloaded nanoparticles appeared able to increase the permeation rate of the diffusive mechanism, without interfering with the transporter.

Design flexibility influencing the in vitro behavior of cationic SLN as a nonviral gene vector.

Several advanced in vitro and in vivo studies have proved the broad potential of cationic solid lipid nanoparticles (SLN) as nonviral vectors. However, a few data are available about the correlation between lipid component of the SLN structure and in vitro performance in terms of cell tolerance and transfection efficiency on different cell lines. In this paper SLN were prepared using stearic acid as main lipid component, stearylamine as cationic agent and protamine as transfection promoter and adding phosphatidylcholine (PC), cholesterol (Chol) or both to obtain three different multicomponent SLN (SLN-PC, SLN-Chol and SLN-PC-Chol, respectively). Cytotoxicity and transfection efficiency of the obtained SLN:pDNA complexes were evaluated on three different immortalized cell lines: COS-I (African green monkey kidney cell line), HepG2 (human hepatocellular liver carcinoma cell line) and Na1300 (murine neuroblastoma cell line). Samples were characterized for the exact quantitative composition, particle size, morphology, zeta potential and pDNA binding ability. All the three SLN samples were about 250-300 nm in size with a positive zeta potential, whereas SLN:pDNA complexes were about 300-400 nm in size with a less positive zeta potential, depending on the SLN composition. Concerning the cell tolerance, the three samples showed a level of cytotoxicity lower than that of the positive control polyethylenimine (PEI), regardless of the cell lines. The best transfection performance was observed for SLN-PC-Chol on COS-I cells while a transfection level lower than PEI was observed on HepG2 cells, regardless the SLN composition. On Na1300 cells, SLN-Chol showed a double efficiency with respect to PEI. Comparing these results to those obtained with the same kind of SLN without PC and/or Chol, it is possible to conclude that the addition of Chol and/or PC to the composition of cationic SLN modify the cell tolerance and the transfection efficiency of the gene vector in a manner strictly dependent on the cell type and the internalization pathways.

Particulate adducts based on sodium risedronate and titanium dioxide for the bioavailability enhancement of oral administered bisphosphonates

Adducts based on a bisphosphonate drug (sodium risedronate) and titanium dioxide (TiO(2)) particles have been developed and characterized in order to improve the bioavailability of orally administrated bisphosphonates. Nanocrystalline and colloidal TiO(2), both characterized by powder X-ray diffraction, were used to obtain the adducts 1 and 2, respectively. Adducts 1 and 2 appeared constituted by nanoparticles of about 50nm and 90nm grouped in clusters of about 0.2microm and 2.5microm, respectively. Higher amounts of drugs were adsorbed on adduct 2 (7.2+/-0.3%) with respect to adduct 1 (4.0+/-0.3%). In vitro studies demonstrate that the adducts were able to release the drug in the pH range of 6-9, whereas they remained essentially stable in the pH range of 0-5. In vivo studies indicate that after oral administration to male Wistar rats, the microparticles of adduct 2 were able to prolong the presence of risedronate in the bloodstream during an 8h period, resulting in a relative bioavailability almost doubled with respect to the free drug. This behaviour allows envisioning an improvement of the risedronate therapeutic effects and/or a reduction of its frequency of administration with consequent reduction of gastro-oesophageal injuries typically induced by oral administration of bisphosphonates.

Studying the in vitro behavior of cationic solid lipid nanoparticles as a nonviral vector

Several advanced in vitro studies have proved the broad potential of cationic solid lipid nanoparticles (cSLNs) as synthetic nucleic acid vectors that have been proposed as an alternative to liposomes. Certainly, results regarding their transfection performances [1] are encouraging but are sometimes not as good as expected, even though the carrier cytotoxicity is always quite low. It is hoped that in the coming years these carriers may allow for higher efficiency over delivery of gene materials and that the development of upscalable and reproducible production of stable systems can be harnessed successfully. Nanovectors already play a very important role in pharmaceutical applications for the delivery of drugs or other biologically active materials. Over the past couple of decades, the incredible number of in vitro studies in chemical and biological engineering on nanocarrier systems provide several important aspects that relate to optimization of particle structures and characterize the mechanisms by which these particles interact with cells, and describing their behavior at the cellular, biochemical and molecular level.

Development and characterization of PLGA nanoparticles as delivery systems of a prodrug of zidovudine obtained by its conjugation with ursodeoxycholic acid.

Abstract Context: Zidovudine (AZT) is employed against AIDS and hepatitis; its use is limited by active efflux transporters (AETs) that induce multidrug resistance for intracellular therapies and hamper AZT to reach the brain. Ursodeoxycholic acid (UDCA) conjugation with AZT (prodrug UDCA–AZT) allows to elude the AET systems. Objective: To investigate the effect of the Pluronic F68 coating on the loading, release and stability of poly(D,L lactide-co-glicolide) nanoparticles (NPs) embedded with UDCA–AZT. Materials and methods: The mean diameter of the NP prepared by nanoprecipitation or emulsion/solvent evaporation methods was determined using both photon correlation spectroscopy and sedimentation field–flow fractionation; particle morphology was detected by scanning electron microscope. The stability of the free and encapsulated UDCA–AZT was evaluated in rat liver homogenates by high-performance liquid chromatography analysis. Results and discussion: The mean diameter of the NPs was found to be ∼600 nm with a relatively high polydispersity. The NPs obtained by emulsion/solvent evaporation were not able to control the prodrug release, differently from NPs obtained by nanoprecipitation. The presence of the Pluronic coating did not substantially modify the kinetics of the drug release, or the extent of the burst effect that were instead only influenced by the preparation parameters. UDCA–AZT incorporated in the NPs was more stable in the rat liver homogenates than the free prodrug and no influence of the Pluronic coating was observed. Conclusions: Considering the different potential applications of nanoparticles coated and uncoated with Pluronic (brain and macrophage targeting, respectively), both of these nanoparticle systems could be useful in the therapies against HIV.

Flow cytometry and live confocal analysis for the evaluation of the uptake and intracellular distribution of FITC-ODN into HaCaT cells

In this study, the mechanism of the internalization and the cellular distribution of 59 fluorescein conjugated PS-ODN (FITC-ODN) after transfection with different mixed lipidic vesicles/oligo complexes (lipoplexes) have been investigated. Mixed lipidic vesicles were prepared with one of the most used cationic lipid (DOTAP) and different amounts of a cholic acid (UDCA) to release the oligo into HaCaT cells. Using flow cytometry, the cellular uptake of the oligo was studied with and without different inhibitors able to block selectively the different pathways involved in the internalization mechanism. The intracellular distribution of the oligo was analyzed by confocal laser scanning microscopy (CLSM), treating the cells with the lipoplexes and directly observing without any fixing procedure. To better carry out the colocalization studies, fluorescent-labeled markers, specific for the different cellular compartments, were coincubated with 59 fluorescein-conjugated 29-mer phosphorotioate oligonucleotide (FITC-ODN). The different lipidic vesicles affect the internalization mechanism of FITC-ODN. After using the inhibitors, the uptake of complexes involved a different internalization mechanism. The live CLSM analysis demonstrated that, after 1 hour from the complex incubation, the oligo was transferred into cells and localized into the endosomes; after 24 hours, the oligo was intracellularly localized close to the nuclear structure in a punctuate pattern. However, the results from fusion experiments showed also a binding of a quite low amount of oligo with the cell membranes.