M.C. Pedroso de Lima

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.

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.

In Situ Forming Chitosan Hydrogels Prepared via Ionic/Covalent Co-Cross-Linking

In situ forming chitosan hydrogels have been prepared via coupled ionic and covalent cross-linking. Thus, different amounts of genipin (0.05, 0.10, 0.15, and 0.20% (w/w)), used as a chemical cross-linker, were added to a solution of chitosan that was previously neutralized with a glycerol-phosphate complex (ionic cross-linker). In this way, it was possible to overcome the pH barrier of the chitosan solution, to preserve its thermosensitive character, and to enhance the extent of cross-linking in the matrix simultaneously. To investigate the contributions of the ionic cross-linking and the chemical cross-linking, separately, we prepared the hydrogels without the addition of either genipin or the glycerol-phosphate complex. The addition of genipin to the neutralized solution disturbs the ionic cross-linking process and the chemical cross-linking becomes the dominant process. Moreover, the genipin concentration was used to modulate the network structure and performance. The more promising formulations were fully characterized, in a hydrated state, with respect to any equilibrium swelling, the development of internal structure, the occurrence of in vitro degradability and cytotoxicity, and the creation of in vivo injectability. Each of the hydrogel systems exhibited a notably high equilibrium water content, arising from the fact that their internal structure (examined by conventional SEM, and environmental SEM) was highly porous with interconnecting pores. The porosity and the pore size distribution were quantified by mercury intrusion porosimetry. Although all gels became degraded in the presence of lysozyme, their degradation rate greatly depended on the genipin load. Through in vitro viability tests, the hydrogel-based formulations were shown to be nontoxic. The in vivo injection of a co-cross-linking formulation revealed that the gel was rapidly formed and localized at the injection site, remaining in position for at least 1 week.

siRNA delivery by a transferrin-associated lipid-based vector: a non-viral strategy to mediate gene silencing

RNA interference provides a powerful technology for specific gene silencing. Therapeutic applications of small interfering RNA (siRNA) however require efficient vehicles for stable complexation, protection, and extra‐ and intracellular delivery of these nucleic acids. Here, we evaluated the potential of transferrin (Tf)‐associated liposomes for siRNA complexation and gene silencing.

Transfection of human macrophages by lipoplexes via the combined use of transferrin and pH‐sensitive peptides

The crucial function of macrophages in a variety of biological processes and pathologies render these cells important targets for gene therapeutic interventions. Commonly used synthetic gene delivery vectors have not been successful in transfecting these non‐dividing cells. A combination strategy involving cationic liposomes to condense and carry DNA, transferrin to facilitate cellular uptake, and the pH‐sensitive peptide GALA to promote endosome destabilization, resulted in significant expression of a luciferase gene. Transfection of macrophages was dependent on the degree of differentiation of the cells. The quaternary complexes of cationic liposomes, DNA, transferrin, and GALA exhibited a net negative charge, which may obviate a limitation of cationic synthetic vectors in vivo. The lack of cytotoxicity and the expected lack of immunogenicity of these complexes may render them useful for gene delivery to macrophages in vivo. J. Leukoc. Biol. 65: 270–279; 1999.

Evaluation of lipid-based reagents to mediate intracellular gene delivery

We characterized different cationic lipid-based gene delivery systems consisting of both liposomes and nonliposomal structures, in terms of their in vitro transfection activity, resistance to the presence of serum, protective effect against nuclease degradation and stability under different storage conditions. The effect of lipid/DNA charge ratio of the resulting complexes on these properties was also evaluated. Our results indicate that the highest levels of transfection activity were observed for complexes prepared from nonliposomal structures composed of FuGENE 6. However, their DNA protective effect was shown to be lower than that observed for cationic liposome formulations when prepared at the optimal (+/-) charge ratio. Our results suggest that lipoplexes are resistant to serum up to 30% when prepared at a 2:1 lipid/DNA charge ratio. However, when they were prepared at higher (+/-) charge ratios, they become sensitive to serum for even lower concentrations (10%). Replacement of dioleoyl-phosphatidylethanolamine (DOPE) by cholesterol enhanced the resistance of the complexes to the inhibitory effect of serum. This different biological activity in the presence of serum was attributed to different extents of binding of serum proteins to the complexes, as evaluated by the immunoblotting assay. Studies on the stability under storage show that lipoplexes maintain most of their biological activity when stored at -80 degrees C, following their fast freezing in liquid nitrogen.

Tf-lipoplexes for neuronal siRNA delivery: A promising system to mediate gene silencing in the CNS

Although RNAi-based gene silencing holds a great potential for treatment of neurological disorders, its application to the CNS has been restricted by low levels of tissue distribution and cellular uptake. In this work we report that cationic lipid-based vectors can enhance siRNA delivery to neurons both in vitro and in vivo. DOTAP:Chol liposomes associated with transferrin (Tf) and complexed with siRNAs (Tf-lipoplexes) were delivered to primary cultures of luciferase-expressing cortical neurons. Confocal microscopy studies revealed efficient cellular uptake of Cy3-labelled siRNAs after Tf-lipoplex delivery, which was reduced but not completely inhibited by blocking the Tf-receptor with excess Tf. Gene silencing was also evaluated after delivery of anti-luciferase or anti-c-Jun siRNAs. Our results demonstrate that Tf-lipoplexes achieve up to 50% luciferase and c-Jun knockdown, 48 h after transfection, without significant cytotoxicity. Similar results were observed in vivo, where a 40% reduction of luciferase activity was found in the striatum of luciferase mice. In addition, fluorescence microscopy studies showed extensive local distribution and internalization of Tf-lipoplex-associated Cy3-siRNAs without tissue toxicity. Overall, our results demonstrate that Tf-lipoplexes can mediate efficient gene silencing in neuronal cells, both in vitro an in vivo, which may prove useful in therapeutic approaches to neuronal protection and repair.

Association of albumin or protamine to lipoplexes: enhancement of transfection and resistance to serum

The successful application of gene therapy depends on the availability of carriers to efficiently deliver genetic material into target cells. Such efficacy is strongly related to key parameters including serum resistance and protection of DNA.

Tf-lipoplex-mediated NGF gene transfer to the CNS: neuronal protection and recovery in an excitotoxic model of brain injury

The development of efficient systems for in vivo gene transfer to the central nervous system (CNS) may provide a useful therapeutic strategy for the alleviation of several neurological disorders. In this study, we evaluated the feasibility of nonviral gene therapy to the CNS mediated by cationic liposomes. We present evidence of the successful delivery and expression of both a reporter and a therapeutic gene in the rodent brain, as evaluated by immunohistochemical assays. Our results indicate that transferrin-associated cationic liposome/DNA complexes (Tf-lipoplexes) allow a significant enhancement of transfection activity as compared to plain complexes, and that 8/1 (+/−) Tf-lipoplexes constitute the best formulation to mediate in vivo gene transfer. We demonstrated that Tf-lipoplex-mediated nerve growth factor transgene expression attenuates the morphological damages of the kainic acid-induced lesion as assessed by 2,3,5-triphenyltetrazolium chloride (TTC) vital staining. These findings suggest the usefulness of these lipid-based vectors in mediating the delivery of therapeutic genes to the CNS.

Vanadium compounds as therapeutic agents: Some chemical and biochemical studies

The behaviour of three vanadium(V) systems, namely the pyridinone (V(V)-dmpp), the salicylaldehyde (V(V)-salDPA) and the pyrimidinone (V(V)-MHCPE) complexes, is studied in aqueous solutions, under aerobic and physiological conditions using (51)V NMR, EPR and UV-Visible (UV-Vis) spectroscopies. The speciations for the V(V)-dmpp and V(V)-salDPA have been previously reported. In this work, the system V(V)-MHCPE is studied by pH-potentiometry and (51)V NMR. The results indicate that, at pH ca. 7, the main species present are (V(V)O(2))L(2) and (V(V)O(2))LH(-1) (L=MHCPE(-)) and hydrolysis products, similar to those observed in aqueous solutions of V(V)-dmpp. The latter species is protonated as the pH decreases, originating (V(V)O(2))L and (V(V)O(2))LH. All the V(V)-species studied are stable in aqueous media with different compositions and at physiological pH, including the cell culture medium. The compounds were screened for their potential cytotoxic activity in two different cell lines. The toxic effects were found to be incubation time and concentration dependent and specific for each compound and type of cells. The HeLa tumor cells seem to be more sensitive to drug effects than the 3T3-L1 fibroblasts. According to the IC(50) values and the results on reversibility to drug effects, the V(V)-species resulting from the V(V)-MHCPE system show higher toxicity in the tumor cells than in non-tumor cells, which may indicate potential antitumor activity.