M.M. Cruz

Formulation of L-asparaginase-loaded poly(lactide-co-glycolide) nanoparticles : influence of polymer properties on enzyme loading, activity and in vitro release

This paper describes the preparation and characterisation of poly(lactide-co-glycolide) (PLG) nanoparticles containing the enzyme L-asparaginase. L-Asparaginase was encapsulated in PLG nanospheres using a water-in-oil-in-water solvent evaporation technique. The effect of the copolymer molecular weight and the presence of carboxyl-end groups in the copolymer chain on the physicochemical and in vitro release properties of the nanoparticles was investigated. Results indicated that size, encapsulation efficiency and in vitro release properties (enzymatic activity retention and protein quantification) of the nanoparticles were affected by the PLG molecular weight. As expected, nanoparticles made of high-molecular-weight PLG had a larger size, a higher loading and la slower release rate than those made od a low-molecular-weight PLG. Nevertheless, the most relevant factor affecting the entrapment and release of L-asparaginase from PLG nanoparticles was the presence of free carboxyl-end groups in the PLG chain. The nanoparticles made of PLG with free carboxyl-end groups had a high protein loading (4.86%, w/w) and provided a continuous delivery of the active enzyme for 20 days. However, the enzyme loading was lower (2.65%, w/v) and no active enzyme was detected in the release medium after a 14-day incubation period when nanoparticles were made of PLG with carboxyl-end groups esterified. These results give evidence of the potential of PLG nanospheres for the continuous delivery of L-asparaginase for extended periods of time and show the effect of the PLG chain end-groups in the amount and activity of the enzyme loaded into the nanospheres.

Developments on drug delivery systems for the treatment of mycobacterial infections.

The clinical management of tuberculosis and other mycobacterial diseases with antimycobacterial chemotherapy remains a difficult task. The classical treatment protocols are long-lasting; the drugs reach mycobacteria-infected macrophages in low amounts and/or do not persist long enough to develop the desired antimycobacterial effect; and the available agents induce severe toxic effects. Nanotechnology has provided a huge improvement to pharmacology through the designing of drug delivery systems able to target phagocytic cells infected by intracellular pathogens, such as mycobacteria. Liposomes and nanoparticles of polymeric nature represent two of the most efficient drug carrier systems that after in vivo administration are endocytosed by phagocytic cells and then release the carried agents into these cells. This article reviews the relevant publications describing the effectiveness of the association of antimycobacterial agents with liposomes or nanoparticles for the treatment of mycobacterioses, particularly for Mycobacterium tuberculosis and M. avium infections. The increased therapeutic index of antimycobacterial drugs; the reduction of dosing frequency; and the improvement of solubility of hydrophobic agents, allowing the administration of higher doses, have been demonstrated in experimental infections. These advantages may lead to new therapeutic protocols that will improve patient compliance and, consequently, lead to a more successful control of mycobacterial infections. The potential therapeutic advantages resulting from the use of non-invasive administration routes for nanoparticulate systems are also discussed.

Design and characterization of enzymosomes with surface-exposed superoxide dismutase

Superoxide dismutase (SOD) was chemically modified by covalent linkage of fatty acid chains to the accessible epsilon-amino groups of the enzyme. This acylation method gave rise to a different enzyme entity (Ac-SOD) as evidenced by different physicochemical properties such as octanol/water partition coefficient and isoelectric point (pI) as compared to SOD. Ac-SOD was incorporated in conventional and long-circulating liposomes (LCL) and characterized in terms of incorporation efficiency, protein to lipid ratio (Prot/Lip), enzymatic activity retention and zeta potential. The observation that Ac-SOD liposomes present enzymatic activity on their external surface indicates that these formulations can act independent of rate and extent of enzyme release as required in case of SOD liposomes. The decrease of superficial charge of liposomal formulations containing Ac-SOD, as compared to SOD liposomes, may be related to the negatively charged enzyme molecules localized on the liposome surface. The comparative characterization of Ac-SOD and SOD liposomal formulations evidenced that the two enzyme forms differ substantially regarding their intraliposomal location: SOD tends to be localized in the internal aqueous spaces, whereas Ac-SOD is expected to be localized in the lipid bilayers of the liposomes, partially buried into the outer surface and exposed to the external medium. These liposomal structures with surface-exposed SOD were designated as Ac-SOD enzymosomes. The properties of these enzymosomes may influence the therapeutic effect, as the release of the enzyme from extravasated vesicles is no longer a necessary requirement for achieving dismutating activity within the inflamed target site.

Therapeutic Efficacy of Liposomal Rifabutin in a Mycobacterium avium Model of Infection

ABSTRACT Liposomal formulations of rifabutin were developed, and the effects of some parameters on the incorporation efficiency were studied. The antimycobacterial activity of rifabutin incorporated into liposomes prepared with phosphatidylcholine and phosphatidylserine (molar ratio, 7:3) was evaluated in a murine model of infection with a virulentMycobacterium avium strain (strain P1581) and was compared with that of free rifabutin. The influences of the size of the liposomal rifabutin formulation, the administered doses, and the treatment schedules on the evolution of infection were studied. Two types of treatment schedules were assayed: therapeutic and prophylactic. The therapeutic treatment started 2 weeks after infection, while the prophylactic treatment began 1 day before the experimental infection with mycobacteria. Incorporation of rifabutin in liposomes resulted in a significant enhancement of activity againstM. avium infection compared to that of rifabutin in the free form in both schedules. These results demonstrate that liposomal formulations of antibiotics such as rifabutin may be effective for the treatment or prophylaxis of infectious diseases.

Liposomal Superoxide Dismutases and Their Use in the Treatment of Experimental Arthritis

It has long been suggested that superoxide dismutase (SOD) be used for antioxidant therapy on the basis of its ability to catalyze the dismutation of superoxide radicals involved in the pathogenesis of several inflammatory disorders such as rheumatoid arthritis. However, the administration of SOD in free form has some disadvantages, most importantly, the low accumulation of SOD in inflamed areas due to its reduced half-life in the bloodstream and its rapid renal excretion. To overcome this, SOD can be incorporated either in highly loaded conventional liposomes (SA-liposomes) or long circulating liposomes (PEG-liposomes). After an appropriate formulation of SOD in SA-liposomes, the therapeutic effect is strongly increased, as indicated by a reduction of about 40% of inflammation edema compared with treatment with nonencapsulated enzyme. Compared with SA-liposomes, PEG-liposomes show superior therapeutic activity. A second approach consists of the construction of a hydrophobic SOD derivative (Ac-SOD) that can be partially inserted within the lipid matrix of liposomes and that expresses enzymatic activity to the external medium. This hydrophobic enzyme, Ac-SOD, associated with liposomes (so called Ac-SOD-enzymosomes), is able to exert its therapeutic activity while circulating in the organism, regardless of the integrity of the liposomes. Ac-SOD-enzymosomes have a more rapid antiinflammatory effect than SOD liposomes, confirming that the release of Ac-SOD from liposomes is no longer required to achieve dismutation. Different methodologies for the preparation of SOD and Ac-SOD liposomal formulations (conventional and long circulating) have been established and are described in detail here.

What Makes Umbilical Cord Tissue-Derived Mesenchymal Stromal Cells Superior Immunomodulators When Compared to Bone Marrow Derived Mesenchymal Stromal Cells?

MSCs derived from the umbilical cord tissue, termed UCX, were investigated for their immunomodulatory properties and compared to bone marrow-derived MSCs (BM-MSCs), the gold-standard in immunotherapy. Immunogenicity and immunosuppression were assessed by mixed lymphocyte reactions, suppression of lymphocyte proliferation and induction of regulatory T cells. Results showed that UCX were less immunogenic and showed higher immunosuppression activity than BM-MSCs. Further, UCX did not need prior activation or priming to exert their immunomodulatory effects. This was further corroborated in vivo in a model of acute inflammation. To elucidate the potency differences observed between UCX and BM-MSCs, gene expression related to immune modulation was analysed in both cell types. Several gene expression profile differences were found between UCX and BM-MSCs, namely decreased expression of HLA-DRA, HO-1, IGFBP1, 4 and 6, ILR1, IL6R and PTGES and increased expression of CD200, CD273, CD274, IL1B, IL-8, LIF and TGFB2. The latter were confirmed at the protein expression level. Overall, these results show that UCX seem to be naturally more potent immunosuppressors and less immunogenic than BM-MSCs. We propose that these differences may be due to increased levels of immunomodulatory surface proteins such as CD200, CD273, CD274 and cytokines such as IL1β, IL-8, LIF and TGFβ2.

Magnetism in Ar-implanted ZnO

ZnO single crystals were implanted with Ar ions with an energy of 100 keV and different fluences. Ferromagnetic behaviour is observed at room temperature after implantation. This behaviour is suppressed after consecutive annealings at 400 and 500 °C. Although trace transition metal impurities have been identified in the virgin samples, it is shown that they cannot account for the observed magnetic behaviour that is assigned to the presence of implantation-induced lattice defects.

Hyperthermia studies of ferrite nanoparticles synthesized in the presence of cotton

MFe2O4 (M = Co, Fe, Mn) compounds were synthesized using hydrothermal treatment in the presence of medicinal cotton. Two sets of nanoparticles were produced for each composition and subsequently characterized by XRD, TEM and SEM. The nanoparticles obtained from the solution display the expected spinel structure and different mean sizes (below 16 nm); the nanoparticles embedded in cotton were subjected to a calcination process for cotton elimination. Regarding these calcinated samples, the spinel structure was maintained for CoFe2O4, a mixture of phases was identified for the M = Mn sample and, in the case of iron, the magnetite phase was converted to hematite (α-Fe2O3). After cotton elimination the samples exhibit a morphology which evidences the role of cotton as a template. To evaluate the quality of the nanoparticles for hyperthermia, SQUID magnetometry and Mossbauer spectroscopy were used to perform the magnetic characterization of all products, and the specific loss power (SLP) was determined by induction heating measurements. All the ferrite NP obtained by hydrothermal synthesis in the presence of cotton display good hyperthermia performance. MnFe2O4 nanoparticles exhibit the highest SLP value, 90 W g−1, followed by Fe3−xO4, and CoFe2O4. In the case of CoFe2O4, the specific loss power of the NP obtained after cotton elimination is enhanced by 50% which is explained by the NP morphology adopted from the cotton template during the synthesis.

Synthesis and characterization of magnetoliposomes for MRI contrast enhancement.

This work assesses the characteristics of magnetoliposomes of soybean phosphatidylcholine (SPC):cholesterol (Chol) loaded with superparamagnetic iron oxide nanoparticles (IONPs) stabilized with tetramethylammonium hydroxide (TMAOH) and their capacity to enhance magnetic resonance imaging (MRI) contrast. Magnetoliposomes of SPC were used for comparative studies. IONPs and magnetoliposomes were characterized using transmission electron microscopy, dynamic light scattering, SQUID magnetometry, FTIR and MRI. The saturation magnetization at 10K was ~0.06 Am(2)/kg for SPC:Chol magnetoliposomes with 7 g iron oxide/mol of lipid and ~0.05 Am(2)/kg for SPC magnetoliposomes with 21 g iron oxide/mol of lipid. As these values are associated with the number of incorporated magnetic IONPs, the saturation magnetization is 1.2 times higher for magnetoliposomes of SPC:Chol as compared with magnetoliposomes of SPC alone. The behavior of temperature dependence in both cases is typical of superparamagnetic particles. FTIR spectra evidence the increase of magnetoliposome membrane ordering with the presence of Chol. Principal component analysis (PCA) applied to FTIR spectra evidenced a clear distinction between scores for SPC:Chol, and SPC magnetoliposomes and for SPC empty liposomes. PCA applied to FTIR data differentiate magnetoliposomes from empty liposomes. MR images of aqueous phantoms obtained with and without magnetoliposomes, clearly evidence their effect on T2 image weighting.

Influence of oxygen stoichiometry on the electronic properties of La4Ni3O10±δ

Some different oxygen content La4Ni3O10+δ (δ=0.02,0.12,−0.22) compounds were prepared in order to study the influence of Ni3+ content on the electronic properties. A metal–metal transition was detected at 140 K for the as-prepared (La4Ni3O10.02) and reduced (La4Ni3O9.78) compounds. From the electrical conductivity, Seebeck coefficient and magnetic studies of the different samples, oxygen stoichiometry was found to be a critical parameter in defining the existence of a less conductive low temperature state. The results are interpreted assuming the existence of charge order in the nickel oxide planes of the layered perovskite-type structure.