Sérgio Simões

Paclitaxel-loaded PLGA nanoparticles: preparation, physicochemical characterization and in vitro anti-tumoral activity.

The main objective of this study was to develop a polymeric drug delivery system for paclitaxel, intended to be intravenously administered, capable of improving the therapeutic index of the drug and devoid of the adverse effects of Cremophor EL. To achieve this goal paclitaxel (Ptx)-loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles (Ptx-PLGA-Nps) were prepared by the interfacial deposition method. The influence of different experimental parameters on the incorporation efficiency of paclitaxel in the nanoparticles was evaluated. Our results demonstrate that the incorporation efficiency of paclitaxel in nanoparticles was mostly affected by the method of preparation of the organic phase and also by the organic phase/aqueous phase ratio. Our data indicate that the methodology of preparation allowed the formation of spherical nanometric (<200 nm), homogeneous and negatively charged particles which are suitable for intravenous administration. The release behaviour of paclitaxel from the developed Nps exhibited a biphasic pattern characterised by an initial fast release during the first 24 h, followed by a slower and continuous release. The in vitro anti-tumoral activity of Ptx-PLGA-Nps developed in this work was assessed using a human small cell lung cancer cell line (NCI-H69 SCLC) and compared to the in vitro anti-tumoral activity of the commercial formulation Taxol. The influence of Cremophor EL on cell viability was also investigated. Exposure of NCI-H69 cells to 25 microg/ml Taxol resulted in a steep decrease in cell viability. Our results demonstrate that incorporation of Ptx in nanoparticles strongly enhances the cytotoxic effect of the drug as compared to Taxol, this effect being more relevant for prolonged incubation times.

Cationic lipid-DNA complexes in gene delivery : from biophysics to biological applications

Great expectations from the application of gene therapy approaches to human disease have been impaired by the unsatisfactory clinical progress observed. Among others, the use of an efficient carrier for nucleic acid-based medicines is considered to be a determinant factor for the successful application of this promising therapeutic strategy. The drawbacks associated with the use of viral vectors, namely those related with safety problems, have prompted investigators to develop alternative methods for gene delivery, cationic lipid-based systems being the most representative. This review focuses on the various parameters that are considered to be crucial to optimize the use of cationic lipid-DNA complexes for gene therapy purposes. Particular emphasis is devoted to the analysis of the different stages involved in the transfection process, from the biophysical aspects underlying the formation of the complexes to the different biological barriers that need to be surpassed for gene expression to occur.

Sterically Stabilized pH-sensitive Liposomes INTRACELLULAR DELIVERY OF AQUEOUS CONTENTS AND PROLONGED CIRCULATION IN VIVO

Liposomes that destabilize at mildly acidic pH are efficient tools for delivering water-soluble drugs into the cell cytoplasm. However, their use in vivo is limited because of their rapid uptake from circulation by the reticuloendothelial system. Lipid-anchored polyethylene glycol (PEG-PE) prolongs the circulation time of liposomes by steric stabilization. We have found that addition of PEG-PE to the membrane of pH-sensitive liposomes composed of cholesteryl hemisuccinate (CHEMS) and dioleoylphosphatidylethanolamine (DOPE) confers steric stability to these vesicles. This modification significantly decreases the pH-dependent release of a charged water-soluble fluorophore, calcein, from liposomes suspended in buffer or cell culture medium. However, the ability of such liposomes to release calcein intracellularly, measured by a novel flow cytometry technique involving dual fluorescence labeling, remains unaltered. As expected, the release of calcein from liposomes endocytosed by cells is inhibited upon pretreatment of the cells with NH4Cl, an inhibitor of endosome acidification. The unique properties of these liposomes were also demonstrated in vivo. The distribution kinetics of 111In-containing CHEMS/DOPE/PEG-PE liposomes injected intravenously into rats has pharmacokinetic parameters similar to control, non-pH-sensitive, sterically stabilized CHEMS/distearoylphosphatidylcholine/PEG-PE liposomes. In contrast, regular pH-sensitive liposomes lacking the PEG-PE component are cleared rapidly. Sterically stabilized pH-sensitive liposomes may therefore be useful for the intracellular delivery in vivo of highly negatively charged molecules such as genes, antisense oligonucleotides, and ribozymes for the treatment of various diseases.

Gene delivery by negatively charged ternary complexes of DNA, cationic liposomes and transferrin or fusigenic peptides.

Potential problems with the use of viral vectors for gene therapy necessitate the development of efficient nonviral vectors. The association of transferrin, or the pH-sensitive peptide GALA, with cationic liposomes composed of 1,2-dioleoyl-3-(trimethylammonium) propane and its equimolar mixture with dioleoylphosphatidylethanolamine, under conditions where the liposome/DNA complex is negatively charged, drastically increased luciferase expression from pCMVluc. The percentage of cells transfected, measured by β-galactosidase expression, was also increased by about 10-fold. The zeta potential of the ternary complexes was lower than that of the liposome/DNA complexes. Transfection activity of positively charged complexes was also enhanced by association with transferrin, GALA or the influenza hemagglutinin N terminal peptide HA-2, but to a smaller extent compared with the negatively charged complexes. The enhancement of gene delivery by transferrin or GALA was not affected significantly by the presence of serum and did not cause significant cytotoxicity. Our results indicate that negatively charged ternary complexes of cationic liposomes, DNA and transferrin, or fusigenic peptides, can facilitate efficient transfection of cultured cells, and that they may alleviate the drawbacks of the use of highly positively charged complexes for gene delivery in vivo.

Cationic liposomes for gene delivery

Cationic liposome–DNA complexes (lipoplexes) constitute a potentially viable alternative to viral vectors for the delivery of therapeutic genes. This review will focus on various parameters governing lipoplex biological activity, from their mode of formation to invivo behaviour. Particular emphasis is given to the mechanism of interaction of lipoplexes with cells, in an attempt to dissect the different barriers that need to be surpassed for efficient gene expression to occur. Aspects related to new trends in the formulation of lipid-based gene delivery systems aiming at overcoming some of their limitations will be covered. Finally, examples illustrating the potential of ca-tionic liposomes in clinical applications will be provided.

Mechanisms of gene transfer mediated by lipoplexes associated with targeting ligands or pH-sensitive peptides.

Association of a targeting ligand such as transferrin, or an endosome disrupting peptide such as GALA, with cationic liposome–DNA complexes (‘lipoplexes’) results in a significant enhancement of transfection of several cell types (Simões S et al, Gene Therapy 1998; 5: 955–964). Although these strategies can overcome some of the barriers to gene delivery by lipoplexes, the mechanisms by which they actually enhance tranfection is not known. In studies designed to establish the targeting specificity of transferrin, we found that apo-transferrin enhances transfection to the same extent as transferrin, indicating that internalization of the lipoplexes is mostly independent of transferrin receptors. These observations were reinforced by results obtained from competitive inhibition studies either by preincubating the cells with an excess of free ligand or with various ‘receptor-blocking’ lipoplexes. Transfection of cells in the presence of drugs that interfere with the endocytotic pathway provided additional insights into the mechanisms of gene delivery by transferrin- or GALA-lipoplexes. Our results indicate that transferrin-lipoplexes deliver transgenes by endocytosis primarily via a non-receptor-mediated mechanism, and that acidification of the endosomes is partially involved in this process.

Cationic Liposomes for Gene Delivery: From Biophysics to Biological Applications

The use of an efficient carrier for nucleic acid-based medicines is considered to be a determinant factor for the successful application of gene therapy. The drawbacks associated with the use of viral vectors, namely those related with safety problems, have prompted investigators to develop alternative methods for gene delivery, cationic lipid-based systems being the most representative. Despite extensive research in the last decade on the use of cationic liposomes as gene transfer vectors and the development of elegant strategies to enhance their biological activity, these systems are still far from being viable alternatives to the use of viral vectors in gene therapy. In this review considerations are made regarding the structure-activity relationships of cationic liposome/DNA complexes and the key formulation parameters influencing the features of lipoplexes are presented and discussed in terms of their effect on biological activity. Particular emphasis is given to the interaction of the lipoplexes with serum components as well as to novel strategies developed to circumvent difficulties that may emerge upon iv administration of the complexes. Finally, since the ability of the lipoplexes to be stored while preserving their transfection activity is a crucial issue for the repeated use of such carriers, approaches reported on the improvement of their physical stability are also reviewed.

Lipid-based nanoparticles for siRNA delivery in cancer therapy: paradigms and challenges.

RNA interference (RNAi) is a specific gene-silencing mechanism that can be mediated by the delivery of chemical synthesized small-interfering RNA (siRNA). RNAi might constitute a novel therapeutic approach for cancer treatment because researchers can easily design siRNA molecules to inhibit, specifically and potently, the expression of any protein involved in tumor initiation and progression. Despite all the potential of siRNA as a novel class of drugs, the limited cellular uptake, low biological stability, and unfavorable pharmacokinetics of siRNAs have limited their application in the clinic. Indeed, blood nucleases easily degrade naked siRNAs, and the kidneys rapidly eliminate these molecules. Furthermore, at the level of target cells, the negative charge and hydrophilicity of siRNAs strongly impair their cellular internalization. Therefore, the translation of siRNA to the clinical setting is highly dependent on the development of an appropriate delivery system, able to ameliorate siRNA pharmacokinetic and biodistribution properties. In this regard, major advances have been achieved with lipid-based nanocarriers sterically stabilized by poly(ethylene glycol) (PEG), such as the stabilized nucleic acid lipid particles (SNALP). However, PEG has not solved all the major problems associated with siRNA delivery. In this Account, the major problems associated with PEGylated lipid-based nanoparticles, and the different strategies to overcome them are discussed. Although PEG has revolutionized the field of nanocarriers, cumulative experience has revealed that upon repeated administration, PEGylated liposomes lose their ability to circulate over long periods in the bloodstream, a phenomenon known as accelerated blood clearance. In addition, PEGylation impairs the internalization of the siRNA into the target cell and its subsequent escape from the endocytic pathway, which reduces biological activity. An interesting approach to overcome such limitations relies on the design of novel exchangeable PEG-derivatized lipids. After systemic administration, these lipids can be released from the nanoparticle surface. Moreover, the design and synthesis of novel cationic lipids that are more fusogenic and the use of internalizing targeting ligands have contributed to the emergence of novel lipid-based nanoparticles with remarkable transfection efficiency.

On the mechanisms of internalization and intracellular delivery mediated by pH-sensitive liposomes

We investigated the molecular mechanisms by which pH-sensitive liposomes surpass the cytoplasmic and endosomal membranes to deliver their aqueous contents into the cytoplasm. Various liposome formulations were evaluated for their efficacy to mediate intracellular delivery of encapsulated material, including a novel sterically stabilized pH-sensitive formulation ((DOPE:CHEMS:DSPE-PEG(2000) (6:4:0.3)) that was previously developed in our laboratories. In an attempt to fully characterize the nature of liposome-cell interactions different approaches based on a dual-labeling fluorescence assay were used. Our results indicate that the efficacy of interaction of pH-sensitive liposomes, both plain and sterically stabilized, with cells is strongly determined by the inclusion of DOPE in their composition, independently of the type of the amphiphilic stabilizer used. In fact, DOPE-containing liposomes shown to be non-pH sensitive by biophysical assays, mediated cytoplasmic delivery of their contents as efficiently as well known pH-sensitive formulations (e.g. DOPE:CHEMS). However, among the different formulations studied, DOPE:CHEMS liposomes were those exhibiting the highest extent of cell association. Moreover, our results with cells pretreated with metabolic inhibitors or lysosomotropic agents clearly indicate that DOPE-containing liposomes are internalized essentially by endocytosis and that acidification of the endosomes is not the only mechanism involved in the destabilization of the liposomes inside the cell.

Mechanisms of Singlet‐Oxygen and Superoxide‐Ion Generation by Porphyrins and Bacteriochlorins and their Implications in Photodynamic Therapy

New halogenated and sulfonated bacteriochlorins and their analogous porphyrins are employed as photosensitizers of singlet oxygen and the superoxide ion. The mechanisms of energy and electron transfer are clarified and the rates are measured. The intermediacy of a charge-transfer (CT) complex is proved for bacteriochlorins, but excluded for porphyrins. The energies of the intermediates and the rates of their interconversions are measured, and are used to obtain the efficiencies of all the processes. The mechanism of formation of the hydroxyl radical in the presence of bacteriochlorins is proposed to involve a photocatalytic step. The usefulness of these photosensitizers in the photodynamic therapy (PDT) of cancer is assessed, and the following recommendations are given for the design of more effective PDT protocols employing such photosensitizers: 1) light doses should be given over a more extended period of time when the photosensitizers form CT complexes with molecular oxygen, and 2) Fe(2+) may improve the efficiency of such photosensitizers if co-located in the same cell organelle assisting with an in vivo Fenton reaction.