Eder Lilia Romero

Drug delivery systems against leishmaniasis? Still an open question.

Background: Leishmania amastigotes live inside resident macrophages in different anatomic sites. Their hidden location is responsible for impairing the accession of therapeutic drugs. Drug delivery systems (DDSs) should allow the adverse effects caused by problematic routes of administration to be avoided as well as enhancing the antileishmanial activity and reducing the toxicity of the medication. However, after 30 years of research in the field, and since leishmaniasis is mostly a disease affecting the poorest populations, currently AmBisome® is the only DDS used against the visceral form, and most experimental development only relates to parenteral administration. Objective: We critically review the main DDSs designed against the different clinical forms of leishmaniasis. Methods: A literature search was performed on PubMed and through Google. Conclusions: On reviewing the experimental and clinical therapeutic performance of former and current DDSs and considering the main obstacles to be overcome, we discuss how nanomedicine can contribute to the development of new and more efficient strategies.

Nanotechnological approaches against Chagas disease

Over several thousand years, the flagellated Trypanosome cruzi-causative agent of Chagas disease-developed a complex life cycle between the reduviidae vectors and its human hosts. Due to their silent and hidden location, the intracellular amastigotes are mainly responsible for the nearly 50,000 annual deaths caused by the chronic chagasic cardiomyopathy. Chagas disease is the most important parasitic disease in the Americas, though treatments have not evolved towards a more efficient pharmacotherapy that (i) eradicates the scarce amastigotes present at the indeterminate/chronic form and (ii) employs less toxic drugs than benznidazole or nifurtimox. Nano-drug delivery systems (nanoDDS) represent useful means to selectively deliver the drug to intracellular targets. However, preclinical research in Chagas must be extended in order to improve the chances of a clinical implementation. The stages involved in this process are (i) selection of the appropriate drug for a specific parasite, (ii) development of a drug-loaded nanoDDS structure that displays the adequate pharmacokinetics, biodistribution and intracellular transit and (iii) selection of the right parasite form to target and the right stage of the disease for the treatment to be started. In this review we will critically overview the few research works published in the last 20years in the context of nanotechnology and Chagas diseases and highlight the gaps in knowledge towards the design of more efficient medicines to address this endemic.

Ethylendiamine core PAMAM dendrimers/siRNA complexes as in vitro silencing agents

We have screened the formation of complexes between ethylendiamine (EDA) core polyamidoamine (PAMAM) dendrimers (D) and a short interfering RNA (siRNA) as a function of three variables: the ionic strength of the medium (lacking or containing 150 mM NaCl), the D generation (G4, G5, G6 and G7) and the N/P ratio (nitrogen amines in D/phosphate in siRNA). It was observed that all D formed complexes with siRNA, being the size of the complexes strictly dependent on the ionic strength of the media. The strong electrostatic interactions occurring in NaCl lacking medium made siRNA-D complexes (siRNA-D) smaller than those obtained in NaCl containing medium (30-130 nm, +25 mV zeta potential vs. several microm-800 nm, 0 zeta potential, respectively). Not surprisingly, both the uptake and inhibition of EGFP expression in cell culture, resulted dependent on siRNA-D size. siRNA-D prepared in NaCl containing medium were poorly captured and presented a basal activity on phagocytic (J-774-EGFP) cells, being inactive on non-phagocytic cells (T98G-EGFP). However, the smaller siRNA-D prepared in NaCl lacking medium were massively captured, exhibiting the highest inhibition of EGFP expression at 50 nM siRNA (non-cytotoxic concentration). Remarkably, siRNA-G7 produced the highest inhibition of EGFP expression both in T98G-EGFP (35%) and J-774-EGFP (45%) cells, in spite of inducing a lower protection of siRNA against RNase A degradation. Taken together, our results showed that modifying the chemical structure of D is not the only way of achieving siRNA-D suitable for silencing activity. The simple use of a low ionic strength preparation media has been critical to get small siRNA-D that could be captured by cells and in particular, siRNA-G7 but not those formed by lower generation D, possessed structural constraints other than size that could favor its silencing activity.

Sunlight triggered photodynamic ultradeformable liposomes against Leishmania braziliensis are also leishmanicidal in the dark.

Being independent of artificial power sources, self administered sunlight triggered photodynamic therapy could be a suitable alternative treatment for cutaneous leishmaniasis, that avoids the need for injectables and the toxic side effects of pentavalent antimonials. In this work we have determined the in vitro leishmanicidal activity of sunlight triggered photodynamic ultradeformable liposomes (UDL). ZnPc is a hydrophobic Zn phthalocyanine that showed 20% anti-promastigote activity (APA) and 20% anti-amastigote activity (AA) against Leishmania braziliensis (strain 2903) after 15min sunlight irradiation (15J/cm(2)). However, when loaded in UDL as UDL-ZnPc (1.25μM ZnPc-1mM phospholipids) it elicited 100% APA and 80% AA at the same light dose. In the absence of host cell toxicity, UDL and UDL-ZnPc also showed non-photodynamic leishmanicidal activity. Confocal laser scanning microscopy of cryosectioned human skin mounted in non-occlusive Saarbrücken Penetration Model, showed that upon transcutaneous administration ZnPc penetrated nearly 10 folds deeper as UDL-ZnPc than if loaded in conventional liposomes (L-ZnPc). Quantitative determination of ZnPc confirmed that UDL-ZnPc penetrated homogeneously in the stratum corneum, carrying 7 folds higher amount of ZnPc 8 folds deeper than L-ZnPc. It is envisioned that the multiple leishmanicidal effects of UDL-ZnPc could play a synergistic role in prophylaxis or therapeutic at early stages of the infection.

Highly deformable and highly fluid vesicles as potential drug delivery systems: theoretical and practical considerations

Vesicles that are specifically designed to overcome the stratum corneum barrier in intact skin provide an efficient transdermal (systemic or local) drug delivery system. They can be classified into two main groups according to the mechanisms underlying their skin interaction. The first group comprises those possessing highly deformable bilayers, achieved by incorporating edge activators to the bilayers or by mixing with certain hydrophilic solutes. The vesicles of this group act as drug carriers that penetrate across hydrophilic pathways of the intact skin. The second group comprises those possessing highly fluid bilayers, owing to the presence of permeation enhancers. The vesicles of this group can act as carriers of drugs that permeate the skin after the barrier of the stratum corneum is altered because of synergistic action with the permeation enhancers contained in the vesicle structure. We have included a detailed overview of the different mechanisms of skin interaction and discussed the most promising preclinical applications of the last five years of Transfersomes® (IDEA AG, Munich, Germany), ethosomes, and invasomes as carriers of antitumoral and anti-inflammatory drugs applied by the topical route.

Increased brain radioactivity by intranasal 32P-labeled siRNA dendriplexes within in situ-forming mucoadhesive gels

Background Molecules taken up by olfactory and trigeminal nerve neurons directly access the brain by the nose-to-brain pathway. In situ-forming mucoadhesive gels would increase the residence time of intranasal material, favoring the nose-to-brain delivery. In this first approach, brain radioactivity after intranasal administration of 32P-small interference RNA (siRNA) complexed with poly(amidoamine) G7 dendrimers (siRNA dendriplexes) within in situ-forming mucoadhesive gels, was determined. Materials 32P-siRNA dendriplexes were incorporated into in situ-forming mucoadhesive gels prepared by blending thermosensitive poloxamer (23% w/w) with mucoadhesive chitosan (1% w/w, PxChi) or carbopol (0.25% w/w, PxBCP). Rheological properties, radiolabel release profile, and local toxicity in rat nasal mucosa were determined. The best-suited formulation was intranasally administered to rats, and blood absorption and brain distribution of radioactivity were measured. Results The gelation temperature of both formulations was 23°C. The PxChi liquid showed non-Newtonian pseudoplastic behavior of high consistency and difficult manipulation, and the gel retained 100% of radiolabel after 150 minutes. The PxCBP liquid showed a Newtonian behavior of low viscosity and easy manipulation, while in the gel phase showed apparent viscosity similar to that of the mucus but higher than that of aqueous solution. The gel released 35% of radiolabel and the released material showed silencing activity in vitro. Three intranasal doses of dendriplexes in PxCBP gel did not damage the rat nasal mucosa. A combination of 32P-siRNA complexation with dendrimers, incorporation of the dendriplexes into PxCBP gel, and administration of two intranasal doses was necessary to achieve higher brain radioactivity than that achieved by intravenous dendriplexes or intranasal naked siRNA. Conclusion The increased radioactivity within the olfactory bulb suggested that the combination above mentioned favored the mediation of a direct brain delivery.

In vitro phototoxicity of ultradeformable liposomes containing chloroaluminum phthalocyanine against New World Leishmania species.

The use of photodynamic therapy (PDT) against cutaneous leishmaniasis (CL) based on chloroaluminum phthalocyanine (ClAlPc) is a promissory alternative therapy. The main purpose of this article was to assess the internalization and in vitro phototoxic activities of ClAlPc encapsulated in ultradeformable liposomes (UDL-ClAlPc) in Leishmania parasites and mammalian cells. Cell internalization was determined by fluorescence microscopy, cell and parasite damage by standard MTT or direct microscopic analysis and a phototoxic index (PI) was calculated as the compound activity (IC(50)) at 0 J/cm(2)/IC(50) at 17 J/cm(2). Liposomal and free ClAlPc were internalized by infected and non-infected THP-1 cells and co-localized in the mitochondria. Treatment of UDL-ClAlPc was almost 10 times more photoactive than free ClAlPc on THP-1 cells and promastigotes and intracellular amastigotes of Leishmania chagasi and Leishmania panamensis. Liposomal compounds were active on non-irradiated and irradiated cells however PI higher than 50 were calculated. PI for amphotericin B referential drug were lower than 1.2. Empty liposomes tested at the same lipid concentration of active ClPcAl-liposomes were non-toxic. Upon photodynamic treatment a nonselective-parasite activity against intracellular amastigotes were observed and loss of membrane integrity resulting in a release of parasites was detected. Further studies oriented to evaluate both the state of infection after PDT and the effectiveness of UDL as delivery vehicles of ClAlPc in CL experimental models are required.

Topical and mucosal liposomes for vaccine delivery

Mucosal (and in minor extent transcutanous) stimulation can induce local or distant mucosa secretory IgA. Liposomes and other vesicles as mucosal and transcutaneous adjuvants are attractive alternatives to parenteral vaccination. Liposomes can be massively produced under good manufacturing practices and stored for long periods, at high antigen/vesicle mass ratios. However, their uptake by antigen-presenting cells (APC) at the inductive sites remains as a major challenge. As neurotoxicity is a major concern in intranasal delivery, complexes between archaeosomes and calcium as well as cationic liposomes complexed with plasmids encoding for antigenic proteins could safely elicit secretory and systemic antigen-specific immune responses. Oral bilosomes generate intense immune responses that remain to be tested against challenge, but the admixing with toxins or derivatives is mandatory to reduce the amount of antigen. Most of the current experimental designs, however, underestimate the mucus blanket 100- to 1000-fold thicker than a 100-nm diameter liposome, which has first to be penetrated to access the underlying M cells. Overall, designing mucoadhesive chemoenzymatic resistant liposomes, or selectively targeted to M cells, has produced less relevant results than tailoring the liposomes to make them mucus penetrating. Opposing, the nearly 10 µm thickness stratum corneum interposed between liposomes and underlying APC can be surpassed by ultradeformable liposomes (UDL), with lipid matrices that penetrate up to the limit with the viable epidermis. UDL made of phospholipids and detergents, proved to be better transfection agents than conventional liposomes and niosomes, without the toxicity of ethosomes, in the absence of classical immunomodulators.

Uptake and intracellular traffic of siRNA dendriplexes in glioblastoma cells and macrophages

Background Gene silencing using small interfering RNA (siRNA) is a promising new therapeutic approach for glioblastoma. The endocytic uptake and delivery of siRNA to intracellular compartments could be enhanced by complexation with polyamidoamine dendrimers. In the present work, the uptake mechanisms and intracellular traffic of siRNA/generation 7 dendrimer complexes (siRNA dendriplexes) were screened in T98G glioblastoma and J774 macrophages. Methods The effect of a set of chemical inhibitors of endocytosis on the uptake and silencing capacity of dendriplexes was determined by flow cytometry. Colocalization of fluorescent dendriplexes with endocytic markers and occurrence of intracellular dissociation were assessed by confocal laser scanning microscopy. Results Uptake of siRNA dendriplexes by T98G cells was reduced by methyl-β-cyclodextrin, and genistein, and cytochalasine D, silencing activity was reduced by genistein; dendriplexes colocalized with cholera toxin subunit B. Therefore, caveolin-dependent endocytosis was involved both in the uptake and silencing activity of siRNA dendriplexes. On the other hand, uptake of siRNA dendriplexes by J774 cells was reduced by methyl-β-cyclodextrin, genistein, chlorpromazine, chloroquine, cytochalasine D, and nocodazole, the silencing activity was not affected by chlorpromazine, genistein or chloroquine, and dendriplexes colocalized with transferrin and cholera toxin subunit B. Thus, both clathrin-dependent and caveolin-dependent endocytosis mediated the uptake and silencing activity of the siRNA dendriplexes. SiRNA dendriplexes were internalized at higher rates by T98G but induced lower silencing than in J774 cells. SiRNA dendriplexes showed relatively slow dissociation kinetics, and their escape towards the cytosol was not mediated by acidification independently of the uptake pathway. Conclusion The extent of cellular uptake of siRNA dendriplexes was inversely related to their silencing activity. The higher silencing activity of siRNA dendriplexes in J774 cells could be ascribed to the contribution of clathrin-dependent and caveolin-dependent endocytosis vs only caveolin-dependent endocytosis in T98G cells.

Ultradeformable archaeosomes as new topical adjuvants.

UNLABELLED Ultradeformable archaeosomes (UDA) are vesicles made of soybean phosphatidylcholine (SPC), sodium cholate (NaChol) and polar lipids from Halorubrum tebenquichense (3:1:3 wt/wt). Although ultradeformable liposomes (UDL, made of SPC and NaChol at 6:1 wt/wt) and UDA were neither captured nor caused cytotoxicity on keratinocytes, UDA was avidly captured by macrophages, their viability being reduced by 0.4-1.6 mg/mL phospholipids by 25 to 60%. Instead, UDL were poorly captured and caused no toxicity. Balb/C mice immunized by the topical route with four doses of ovalbumin (OVA)-loaded UDA, at 75 μg OVA/600 μg phospholipids (125 nm mean size and -42 mV zeta potential), induced IgG titers tenfold to 100-fold higher than those immunized with OVA-loaded UDL at the same dosage. Both matrices penetrate to the same skin depth (nearly 10 μm after 1 hour on excised human skin), being the higher topical adjuvancy and higher phagocytic uptake of UDA related to its glycolipid content. FROM THE CLINICAL EDITOR This work summarizes key findings related to the development of ultradeformable archaeosomes as vehicles utilized in transdermal delivery systems with improved skin penetration.