António J. Almeida

Lymphatic Uptake of Pulmonary Delivered Radiolabelled Solid Lipid Nanoparticles

Lymphatic drainage plays an important role in the uptake of particulates in the respiratory system, being also associated to the spreading of lung cancer through metastasis development. In recent years solid lipid nanoparticles (SLN) have been proposed as carriers of anti-tumoural drugs, for their low toxicity and surface characteristics make them suitable for either imaging (gamma-scintigraphy) or therapy upon encapsulation of cytotoxic drugs. Assessment of inhaled radiolabelled SLN biodistribution is described in the present work. Methods : Nanoparticles (200 nm) were radiolabelled with 99m Tc using the lipophilic chelator d, l -hexamehylpropyleneamine oxime (HMPAO). Biodistribution studies were carried out following aerosolisation and administration of a 99m Tc-HMPAO-SLN suspension to a group of adult male Wistar rats. A 60 min dynamic image acquisition was performed in a gamma-camera, followed by static image collection at 30 min intervals up to 4 h postinhalation. Radiation counting was performed in organ samples, collected after the animals were sacrificed. Results : The data show an important and significant uptake of the radiolabelled SLN into the lymphatics after inhalation, and a high rate of distribution in periaortic, axillar and inguinal lymph nodes. Conclusion Results indicate that SLN could be effective colloidal carriers for lymphoscintigraphy or therapy upon pulmonary delivery.

Peptide-loaded solid lipid nanoparticles (SLN) : influence of production parameters

Solid lipid nanoparticles (SLN) are an alternative to particulate carriers made of biodegradable polyesters. The SLN have been sought as vehicles for drug molecules, and their production often uses physiological lipids or lipid molecules with an history of safe use in human medicine. However, little has been studied regarding the incorporation of peptides into SLN. This report describes the first studies on the incorporation of lysozyme, as a model peptide, in SLN. Previous to nanoparticle preparation, lysozyme was solubilised, until saturation, into the melted lipid phase. Production was carried out by a cold homogenisation process. The entrapment efficiency was dependent on the initial solubility of the peptide in the lipid phase of the final preparation. The influence of formulation parameters (e.g. type of lipid, time of exposure to different temperatures, pressure and the number of homogenisation cycles) on the integrity and activity of the enzyme, was also assessed. The lysozyme molecule remained intact throughout the process without loosing its activity, as shown by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and the rate of lysis of Micrococcus lysolideikticus, respectively. This study shows that some proteins are able to endure the harsh procedures of formulation by high pressure homogenisation, making possible the use of SLN as antigen carriers for vaccine delivery.

Immune Response to Nasal Delivery of Antigenically Intact Tetanus Toxoid Associated with Poly(l-lactic acid) Microspheres in Rats, Rabbits and Guinea-pigs

Abstract— Tetanus toxoid was adsorbed onto poly(l‐lactic acid) microspheres. Analyses by sodium dodecyl sulphate‐polyacrylamide gel electrophoresis and Western blotting suggest that the formulation procedure does not affect the stability or the antigenic properties of the protein. After nasal administration to guinea‐pigs, the resulting preparation enhanced the immune response to the tetanus toxoid when compared with the free antigen. The increase in systemic immunoglobulin G titre was almost immediate in the group treated with the adsorbed tetanus toxoid (time zero, 140; week 2, 1550; week 4, 2760), reaching 36000 two weeks after the booster (week 7), whereas the free antigen produced an immune response similar to that found in non‐treated animals. In a parallel experiment, latex particles of a similar size to poly(l‐lactic acid) microspheres, administered to both rats and rabbits, were detected in the blood stream. These findings with tetanus toxoid demonstrate the use of microsphere delivery systems for nasal application of antigens. These had a profound effect on the immune response, and indicate possibilities for overcoming some of the barriers to drug absorption in general.

Nasal Delivery of Vaccines

Only relatively recently the significance of inducing not only systemic immunity but also significant local immunity at susceptible mucosal surfaces has become appreciated. A new field of mucosal immunity has been established as information accumulates on mucosal-associated lymphoid tissue (MALT) and on its role in both local and systemic immune responses. This review describes the formation of vaccines to be delivered to one of MALT components, i.e. the nasal-associated lymphoid tissue (NALT), which bears some similarities with the Peyers patches of the intestine. The association of antigens with adjuvants and particulate carriers such as microparticles, nanoparticles and liposomes is emphasised.

Microcomposites theophylline/hydrogenated palm oil from a PGSS process for controlled drug delivery systems

Abstract New controlled-release carriers of theophylline prepared with hydrogenated palm oil (HPO) were obtained by Particles from gas saturated solutions (PGSS) at different working pressures. Pressure had no significant effect on the mean diameter of the particles (of about 3.0 μm). However, spherical morphology with a regular surface was preferentially obtained at higher expansion pressures. HPLC analysis revealed a low theophylline encapsulation in the HPO matrix and showed that considerable amounts of theophylline were located at the particles’ surface. Dissolution studies showed that theophylline released from the HPO matrix follows Higuchis model for simple diffusional processes. However, successful correlation with experiment was achieved only with the Brophy and Deasy long-time correction to the Higuchi equation.

The size of solid lipid nanoparticles: an interpretation from experimental design.

This study aimed to investigate the role of different factors affecting the size of solid lipid nanoparticles (SLN), prepared by the emulsification-solvent evaporation method. A double factorial design was conducted so as to cover a wide range of sizes, highlighting zones with different behaviour with respect to changes in the controlled variables: lipid concentration, solvent:lipid ratio and emulsifier concentration. The solvent:lipid ratio constituted the main factor influencing particle size. Increasing the amount of solvent induced a decrease in the size. This was a general trend, essentially independent from solvent and lipid type. The amount of emulsifier had a non-trivial impact on size, depending on whether systems were located below, above or close to the optimal surface coverage. The amount of lipid had a limited influence upon particle size, being more relevant for lower lipid concentrations. An optimal formulation was selected for intermediate levels of the three variables. Sonication reduced both particle size and polydispersity. These particles were also tested as drug carriers using simvastatin as a model of lipophilic drug. SLN were able to entrap a high amount of simvastatin, with little effect upon size and zeta potential, constituting a promising carrier for lipophilic drugs.

The enhancement of the immune response against S. equi antigens through the intranasal administration of poly-epsilon-caprolactone-based nanoparticles

Strangles is a bacterial infection of the Equidae family that affects the nasopharynx and draining lymph nodes, caused by Streptococcus equi subspecies equi. This agent is responsible for 30% of all worldwide equine infections and is quite sensitive to penicillin and other antibiotics. However, prevention is still the best option because the current antibiotic therapy and vaccination is often ineffective. As S. equi induces very strong systemic and mucosal responses in convalescent horses, an effective and economic strangles vaccine is still a priority. In this study the humoral, cellular and mucosal immune responses to S. equi antigens encapsulated or adsorbed onto poly-epsilon-caprolactone nanospheres were evaluated in mice. Particles were produced by a double (w/o/w) emulsion solvent evaporation technique and contained mucoadhesive polymers (alginate or chitosan) and absorption enhancers (spermine, oleic acid). Their intranasal administration, particularly those constituted by the mucoadhesive polymers, increased the immunogenicity and mucosal immune responses (SIgA) to the antigen. The inclusion of cholera toxin B subunit in the formulations successfully further activated the paths leading to Th1 and Th2 cells. Therefore, those PCL nanospheres are potential carriers for the delivery of S.equi antigens to protect animals against strangles.

Poly(methyl methacrylate) particulate carriers in drug delivery

Poly(methyl methacrylate) (PMMA) is one of the most widely explored biomedical materials because of its biocompatibility, and recent publications have shown an increasing interest in its applications as a drug carrier. PMMA-based particulate carriers (PMMAP) can be prepared either by polymerization methods or from pre-formed polymer-based techniques. Potential biomedical application of these particles includes their use as adjuvant for vaccines and carrier of many drugs as antibiotics and antioxidants via different routes of administration. Release of drugs from PMMAP occurs typically in a biphasic way with an incomplete drug release. To improve release profiles, recent strategies are focusing on increasing polymer hydrophilicity by synthesizing functionalized PMMA microspheres or by formulating PMMA composites with hydrophilic polymers. This review examines the current status of preparation techniques, drug release kinetics, biomedical applications and toxicity of these nano/micro PMMA-based particulate carriers.

Lipid nanoparticles containing oryzalin for the treatment of leishmaniasis

Oryzalin is a dinitroaniline drug that has attracted recent interest for the treatment of leishmaniasis. Its use as an antiparasitic therapeutic agent is limited by the low water solubility associated with an in vivo rapid clearance, leading to the administration of larger and possibly toxic doses in in vivo studies, and the use of solvents that may lead to undesirable side effects. In the present work oryzalin-containing lipid nanoparticles were produced by a emulsion-solvent evaporation technique using a composition suitable for parenteral administration, i.e., tripalmitin (solid lipid) and a complex mixture of three emulsifying agents (soya lecithin, Tween® 20 and sodium deoxycholate). Physicochemical characterization included the determination of mean particle size, polydispersity index, zeta potential, encapsulation efficiency and DSC studies. Final formulations revealed values of <140 nm (PI<0.2) and zeta potential of ≈-35 mV, as well as encapsulation efficiency >75%. The effects of various processing parameters, such as lipid and surfactant and composition and concentration, as well as the stability during the harsh procedures of autoclaving (121°C/15 min) and freeze-drying were also evaluated. Formulations revealed to be stable throughout freeze-drying and moist-heath sterilization without significant variations on physicochemical properties and no significant oryzalin losses. The use of a complex surfactant mixture proved crucial for preserving formulation stability. Particularly, lecithin appears as a key component in the stabilization of tripalmitin-based oryzalin-containing lipid nanoparticles. Finally, cell viability studies demonstrated that the incorporation of oryzalin in nanoparticles decreases cytotoxicity, thus suggesting this strategy may improve tolerability and therapeutic index of dinitroanilines.

Preclinical evaluation of a pulmonary delivered paclitaxel-loaded lipid nanocarrier antitumor effect

UNLABELLED Lung cancer remains a leading cause of death due to the low efficacy of chemotherapy, mainly related to the administration route used. Therefore, alternative administration routes are needed. Paclitaxel (PTX) is an insoluble anticancer drug active against solid tumors, such as those found in lung cancer, that has stimulated an intense research effort over recent years. Solid lipid nanoparticles (SLNs) are potential carriers for poorly soluble drugs, being biodegradable systems that served as alternatives to the usual colloidal carriers. That system was used to deliver PTX to the lungs and seem to fulfill the requirements for an optimum particulate carrier. Furthermore, PTX-loaded SLN pulmonary administration provided a target administration, which is expected to avoid high concentration of the drug at nontarget tissues, reducing toxicity, and increasing the drugs therapeutic index. The rationale of this study was to deliver a colloidal system to the lung lymphatics through a pulmonary route for cancer therapy. FROM THE CLINICAL EDITOR Paclitaxel-loaded solid lipid nanoparticles were used to target tumors in a murine lung cancer model enabling high PTX concentration in the target with reduced systemic toxicity and increased therapeutic index.