M. Luísa Corvo

Liposomal formulations of Cu,Zn-superoxide dismutase: physico-chemical characterization and activity assessment in an inflammation model

Different liposomal formulations of Cu,Zn-superoxide dismutase were studied. The effect of lipid composition, initial protein to lipid ratio and type of liposomes on the encapsulation parameters were studied. According to these parameters some liposomal formulations were selected for further tests: dehydration-rehydration vesicles (sDRV) and extruded vesicles (VET) made from PC:Chol:SA and PC:Chol:PI. After encapsulation in these vesicles, no significant loss of enzymatic activity of the enzyme was observed. Pharmacokinetic studies indicated that upon liposomal encapsulation of the enzyme, the terminal half-life of the enzyme after intravenous administration increased 5- to 10-fold. No acute toxicity was observed for the liposomal formulations. The therapeutic activity of the free and liposomal enzyme was assayed in Wistar rats with a chronic inflammation. All liposomal formulations tested showed a reduction of arthritis indexes: 25 to 50% of mean oedema regression was obtained with liposomal enzyme, while no regression with free enzyme and an increase of 50% of the oedema for the control animals were observed.

Biochemical changes in arthritic rats: dehydroascorbic and ascorbic acid levels

The objective of this work was to evaluate the use of vitamin C as a biomarker in the inflammatory phase of the rat adjuvant arthritis and to correlate it with other parameters used for disease evaluation. Paw swelling was used for physical evaluation and the levels of ascorbate and dehydroascorbate in the serum of male rats, before and after adjuvant arthritis induction, were quantified by a high-performance liquid chromatography method (HPLC). The optimised HPLC assay enabled the quantification of both forms of the vitamin in rat sera, with the same extraction method and using different detectors, instead of obtaining dehydroascorbate by subtraction of the total ascorbate measurement. This method was used to follow the severity of adjuvant arthritis and the results were correlated with other already established disease activity parameters. A decrease of ascorbic acid and dehydroascorbic acid was observed with the increase of right paw circumference during the course of adjuvant arthritis. The disease associated changes in the serum concentrations of ascorbic acid, from biosynthesis and from recycling, can be evaluated by the direct quantification of dehydroascorbic acid. This provides some evidence for the potential of the quantification of these biomarkers to study the disease activity, and as a tool for the establishment of therapeutic protocols, to evaluate the anti-inflammatory effect of new drugs or formulations.

New long circulating magnetoliposomes as contrast agents for detection of ischemia-reperfusion injuries by MRI

UNLABELLED New long circulating magnetoliposomes coated with polyethylene glycol (PEG), and loaded with PEG-coated 10nm superparamagnetic iron oxide nanoparticles (SPION), were developed. The magnetoliposomes relaxivities r1, r2 measured in a magnetic field of 7 T showed a minor effect on T1, but a major effect on T2. These nanosystems were used as a negative contrast agent for MRI in a nonclinical study to visualize, in a rat model of liver ischemia, ischemia-reperfusion injuries. Magnetic resonance micro-images (MRM) at 7 T were obtained for rat liver with and without magnetoliposomes administration and analyzed in comparison with liver biomarkers and histological results. These new long circulating magnetoliposomes enhanced the detection of lesions indicating their potential use as efficient MRI negative contrast agent for the detection of liver ischemia-reperfusion injuries. FROM THE CLINICAL EDITOR This paper describes the generation of PEGylated magnetoliposomes and demonstrates their feasibility as negative contrast agents in a liver ischemia-reperfusion rat model.

Formulation of oryzalin (ORZ) liposomes: In vitro studies and in vivo fate

Oryzalin (ORZ) is a dinitroaniline that has attracted increasing interest for the treatment of leishmaniasis. The possible use of ORZ as an antiparasitic agent is limited by low water solubility associated with an in vivo rapid clearance. The aim of this work was to overcome these unfavorable pharmaceutical limitations potentiating ORZ antileishmanial activity allowing a future clinical use. This was attained by incorporating ORZ in appropriate liposomes that act simultaneously as drug solvent and carrier delivering ORZ to the sites of Leishmania infection. The developed ORZ liposomal formulations efficiently incorporated and stabilised ORZ increasing its concentration in aqueous suspensions at least 150 times without the need of toxic solvents. The incorporation of ORZ in liposomes reduced the in vitro haemolytic activity and cytotoxicity observed for the free drug, while ORZ exhibits a stable association with liposomes during the first 24h after parenteral administration, significantly reducing ORZ blood clearance and elimination from the body. Simultaneously, an increased ORZ delivery was observed in the main organs of leishmanial infection with a 9-13-fold higher accumulation as compared to the free ORZ. These results support the idea that ORZ performance was strongly improved by the incorporation in liposomes. Moreover, ORZ liposomal formulations can be administrated in vivo in aqueous suspensions without the need of toxic solvents. It is expected an improvement in the therapeutic activity of liposomal ORZ that will be tested in future work.

Regulatory Aspects of Oncologicals: Nanosystems Main Challenges

The design and development of engineered nanosystems for disease diagnosis, prevention and treatment has been boosted by significant advances observed in distinct areas. This has been combined in order to attain efficient and safe products. Even if some of those nanoproducts have successfully reached the market, the consensus underlying the nanomedicines-related regulatory requirements has still to be fully acquired by part of the scientific community and thus academia and pharmaceutical companies could be facing considerable obstacles during the research and development life cycle of these medicinal products. However, considerable progress has been made in the last years, reflecting the recognition by Regulatory Authorities of specific features associated to the nanosystems-based medicines. As a consequence, the regulatory environment for those innovative medicinal nanosystems has been increasingly challenged by key issues, which has been an opportunity to provide clearer guidance for their development.

Therapeutic activity of superoxide dismutase-containing enzymosomes on rat liver ischaemia-reperfusion injury followed by magnetic resonance microscopy

&NA; Liver ischaemia‐reperfusion injury (IRI) may occur during hepatic surgery and is unavoidable in liver transplantation. Superoxide dismutase enzymosomes (SOD‐enzymosomes), liposomes where SOD is at the liposomal surface expressing enzymatic activity in intact form without the need of liposomal disruption, were developed with the aim of having a better insight into its antioxidant therapeutic outcome in IRI. We also aimed at validating magnetic resonance microscopy (MRM) at 7 T as a tool to follow IRI. SOD‐enzymosomes were characterized and tested in a rat ischaemia‐reperfusion model and the therapeutic outcome was compared with conventional long circulating SOD liposomes and free SOD using biochemical liver injury biomarkers, histology and MRM. MRM results correlated with those obtained using classical biochemical biomarkers of liver injury and liver histology. Moreover, MRM images suggested that the therapeutic efficacy of both SOD liposomal formulations used was related to prevention of peripheral biliary ductular damage and disrupted vascular architecture. Therefore, MRM at 7 T is a useful technique to follow IRI. SOD‐enzymosomes were more effective than conventional liposomes in reducing liver ischaemia‐reperfusion injury and this may be due to a short therapeutic window. Graphical abstract Figure. No caption available.

Gene Silencing using siRNA for Preventing Liver Ischaemia-Reperfusion Injury

BACKGROUND Ischaemia-reperfusion injury (IRI), a major complication occurring during organ transplantation, involves an initial ischemia insult, due to loss of blood supply, followed by an inflammation-mediated reperfusion injury. A variety of molecular targets and pathways involved in liver IRI have been identified. Gene silencing through RNA interference (RNAi) by means of small interference RNA (siRNA) targeting mediators of IRI is a promising therapeutic approach. OBJECTIVE This study aims at reviewing the use of siRNAs as therapeutic agents to prevent IRI during liver transplantation. METHOD We review the crucial choice of siRNA targets and the advantages and problems of the use of siRNAs. RESULTS We propose possible targets for siRNA therapy during liver IRI. Moreover, we discuss how drug delivery systems, namely liposomes, may improve siRNA therapy by increasing siRNA stability in vivo and avoiding siRNA off-target effects. CONCLUSION siRNA therapeutic potential to preclude liver IRI can be improved by a better knowledge of what molecules to target and by using more efficient delivery strategies.

Current aspects of breast cancer therapy and diagnosis based on a nanocarrier approach

Breast cancer is the most frequently diagnosed cancer and the leading cause of cancer death in women worldwide. Chemotherapy against breast carcinoma cells using nanocarriers directed to tumors is known as targeted therapy, and it provides a more specific therapy against cancer cells at the molecular level. Therefore, the use of nanocarriers is an advantageous therapeutic approach when compared to other cancer therapeutics. Over the years, it has become an enabling technology with potential for personalized cancer therapy, in which cancer cell detection, diagnosis, and therapy could be tailored to each individual tumors molecular profile because specific biomarkers could be used to predict disease progression and clinical outcomes. Here, we review nanosystems containing anticancer agents that are used for metastatic breast cancer in clinical practice, clinical trials, and research orientations. We also provide an overview of biomarkers used in metastatic breast cancer allowing for improved prognosis and therapy.

Development of New Contrast Agents for Imaging Function and Metabolism by Magnetic Resonance Imaging

Liposomes are interesting nanosystems with a wide range of medical application. One particular application is their ability to enhance contrast in magnetic resonance images; when properly loaded with magnetic/superparamagnetic nanoparticles, this means to act as contrast agents. The design of liposomes loaded with magnetic particles, magnetoliposomes, presents a large number of possibilities depending on the application from image function to metabolism. More interesting is its double function application as theranostics (diagnostics and therapy). The synthesis, characterization, and possible medical applications of two types of magnetoliposomes are reviewed. Their performance will be compared, in particular, their efficiency as contrast agents for magnetic resonance imaging, measured by their relaxivities r1 and r2 relating to their particular composition. One of the magnetoliposomes had 1,2-diacyl-sn-glycero-3-phosphocholine (soy) as the main phospholipid component, with and without cholesterol, varying its phospholipid to cholesterol molar ratios. The other formulation is a long-circulating liposome composed of 1,2-diacyl-sn-glycero-3-phosphocholine (egg), cholesterol, and 1,2-distearoyl-sn-glycerol-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]. Both nanosystems were loaded with superparamagnetic iron oxide nanoparticles with different sizes and coatings.

Abstract C233: Limiting tumor invasion with multifunctional nanoparticle targeting the tumor microenvironment.

Combining multi-targeted strategies with in vivo stable drug retention and increased intracellular delivery is a promising strategy to treat solid tumors. We have evaluated the therapeutic impact of targeting two different populations within the tumor microenvironment (endothelial and cancer cells), using the same F3 peptide-targeted sterically-stabilized pH-sensitive liposome containing doxorubicin. We have demonstrated that it targets the nucleolin receptor on a cell- and ligand-specific manner and has the ability to promote intracellular and triggered drug release into breast cancer cells (hormone-dependent and triple negative subtypes). We have further evidenced that the overexpression of nucleolin in several cancer cell lines of diverse histological origins. We have demonstrated that the unique combination of targeting specificity and intracellular triggered release of a therapeutic agent, doxorubicin, has resulted in enhanced cytotoxic activity in those specific cell lines, when compared to the non-targeted counterpart. Active tumor accumulation of the radioactive lipid and drug tracers of targeted pH-sensitive liposomes, performed on Balb/c nude female mice bearing breast tumors implanted orthotopically, was registered for a time-point as short as 4 h and reaching 48% of the injected dose/g of tumor. In compliance to these data was the doxorubicin tumor-to-heart ratio of 80.8 for F3-targeted pH-sensitive liposomes vs 3.3 and 12.2 for the non-targeted and targeted non-pH-sensitive liposomes, respectively, 24 h post-injection. These data suggested a decrease on the potential dose-cumulative toxicity to the heart, namely relative to the non-targeted non-pH-sensitive counterpart. In mice treated with the F3-targeted pH-sensitive nanoparticle, significant decrease of the viable rim area (50%, accompanied by a strong necrosis) and microvascular density (80%) was observed. Moreover, suppression of invasion to surrounding healthy tissues was depicted, which may increase the probability of tumors falling in the category of “negative margins”, with reduced risk of relapse. The clinical potential of such strategy was reinforced by the successful ex-vivo targeting studies, where specific cellular association was observed in tumor cells harvested from tumors of patients submitted to mastectomy and overexpressing the nucleolin receptor, independent of age, proliferation index, oestrogen, progesterone and HER2 receptor expression. Overall, this work has demonstrated that the combination of tumor and vascular targeting with intracellular triggered release of the encapsulated drug is crucial for the successful therapeutic outcome of a nanotechnology-based strategy against a solid tumor, and should be considered for clinical evaluation. In addition, we have provided evidence of specific targeting to tumor cells of diverse histological origins with the same nanotechnology-based platform, which may be a breakthrough in the development of anticancer therapies, especially when considering tumor cell heterogeneity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C233.