Susana Martins

Novel resveratrol nanodelivery systems based on lipid nanoparticles to enhance its oral bioavailability

Video abstract Video

Delivery of siRNA silencing P-gp in peptide-functionalized nanoparticles causes efflux modulation at the blood–brain barrier

AIMnExplore the use of transferrin-receptor peptide-functionalized nanoparticles (NPs) targeting blood-brain barrier (BBB) as siRNA carriers to silence P-glycoprotein (P-gp).nnnMATERIALS & METHODSnPermeability experiments were assessed through a developed BBB cell-based model; P-gp mRNA expression was evaluated in vitro; rhodamine 123 permeability was assessed after cell monolayer treatment with siRNA NPs.nnnRESULTSnBeyond their ability to improve siRNA permeability through the BBB by twofold, 96-h post-transfection, functionalized polymeric NPs successfully reduced P-gp mRNA expression up to 52%, compared with nonfunctionalized systems. Subsequently, the permeability of rhodamine 123 through the human BBB model increased up to 27%.nnnCONCLUSIONnDeveloped BBB-targeted NPs induced P-gp downregulation and consequent increase on P-gp substrate permeability, revealing their ability to modulate drug efflux at the BBB.

Development and Comparison of Different Nanoparticulate Polyelectrolyte Complexes as Insulin Carriers

xa0The overall objective of our research is to produce polyanion/chitosan nanoparticulate oral delivery systems for insulin. Specific objectives of the present study were to study dextran sulfate or alginate complexation with chitosan on mean particle size, insulin association efficiency, loading capacity and release profile. Nanoparticles were formed by ionotropic complexation and coacervation between polyanions (dextran sulfate and alginate) and chitosan. Diameter was evaluated with photon correlation spectroscopy, polymer interaction was confirmed by DSC and FTIR and particle morphology was assessed by SEM and TEM. Mean nanoparticle diameter ranged from 423 to 850xa0nm, insulin association efficiency from 63 to 94% and loading capacity from 5 to 13%. Dextran sulfate provided highest insulin association efficiency and retention of insulin in gastric simulated conditions. These nanoparticle systems show promise as insulin and potentially other therapeutic polypeptides carriers.

Solid lipid nanoparticles as intracellular drug transporters: an investigation of the uptake mechanism and pathway.

The aim of this work was to develop a systematic analysis of the cellular internalisation mechanism and pathway of solid lipid nanoparticles (SLN) internalisation. To evaluate if SLN show cell uptake and to understand the mechanism of internalisation, four human glioma cell lines (A172, U251, U373 and U87) and a human macrophage cell line (THP1) were used. For this purpose rhodamine 123 (R123) was loaded into SLN coated with polysorbate 60 and 80. Fluorescence microscopy and flow cell cytometry techniques were assessed to study internalisation of these systems within the cells. MTT studies were performed to evaluate the cytotoxicity of the R123-loaded SLN. To assess the SLN internalisation mechanism and intracellular pathway, excluding endocytosis mechanisms were applied. Our results revealed that R123-loaded SLN with mean size below 200 nm and slight negative surface charge (around -20 mV) have the ability to be internalised by gliomas in a higher amount than by macrophages. The mechanism of internalisation was found to be mainly through a clathrin-dependent endocytic pathway. In addition, the cytotoxicity of SLN was higher for gliomas than for macrophages. These results suggest that SLN can be a promising alternative in brain tumours treatment.

Brain delivery of camptothecin by means of solid lipid nanoparticles: Formulation design, in vitro and in vivo studies

For the purpose of brain delivery upon intravenous injection, formulations of camptothecin-loaded solid lipid nanoparticles (SLN), prepared by hot high pressure homogenisation, were designed. Incorporation of camptothecin in the hydrophobic and acidic environment of SLN matrix was chosen to stabilise the lactone ring, which is essential for its antitumour activity, and for avoiding premature loss of drug on the way to target camptothecin to the brain. A multivariate approach was used to assess the influence of the qualitative and quantitative composition on the physicochemical properties of camptothecin-loaded SLN in comparison to plain SLN. Mean particle sizes of ≤200 nm, homogenous size distributions and high encapsulation efficiencies (>90%) were achieved for the most suitable formulations. In vitro release studies in plasma, showed a prolonged release profile of camptothecin from SLN, confirming the physical stability of the particles under physiological pH. A higher affinity of the SLN to the porcine brain capillary endothelial cells (BCEC) was shown in comparison to macrophages. MTT studies in BCEC revealed a moderate decrease in the cell viability of camptothecin, when incorporated in SLN compared to free camptothecin in solution. In vivo studies in rats showed that fluorescently labelled SLN were detected in the brain after i.v. administration. This study indicates that the camptothecin-loaded SLN are a promising drug brain delivery system worth to explore further for brain tumour therapy.

Brain targeting effect of camptothecin-loaded solid lipid nanoparticles in rat after intravenous administration

This study intended to investigate the ability of solid lipid nanoparticles (SLN) to deliver camptothecin into the brain parenchyma after crossing the blood-brain barrier. For that purpose, camptothecin-loaded SLN with mean size below 200 nm, low polydispersity index (<0.25), negative surface charge (-20 mV), and high camptothecin association efficiency (>94%) were produced. Synchrotron small and wide angle X-ray scattering (SAXS/WAXS) analysis indicates that SLN maintain their physical stability in contact with DMPC membrane, whereas SLN change the lamellar structure of DMPC into a cubic phase, which is associated with efficient release of the incorporated drugs. Cytotoxicity studies against glioma and macrophage human cell lines revealed that camptothecin-loaded SLN induced cell death with the lowest maximal inhibitory concentration (IC50) values, revealing higher antitumour activity of camptothecin-loaded SLN against gliomas. Furthermore, in vivo biodistribution studies of intravenous camptothecin-loaded SLN performed in rats proved the positive role of SLN on the brain targeting since significant higher brain accumulation of camptothecin was observed, compared to non-encapsulated drug. Pharmacokinetic studies further demonstrated lower deposition of camptothecin in peripheral organs, when encapsulated into SLN, with consequent decrease in potential side toxicological effects. These results confirmed the potential of camptothecin-loaded SLN for antitumour brain treatments.

Lipid nanoparticles for topical and transdermal application for alopecia treatment: development, physicochemical characterization, and in vitro release and penetration studies

Alopecia is a dermatological disorder, commonly known as hair loss, which affects up to half of the Caucasian male population by middle age, and almost all (95%) Caucasian men by old age. Considering that alopecia affects so many people and that there is currently no scientifically proven treatment with few side effects, new drug-delivery systems able to improve alopecia therapy are urgently required. With this purpose in mind, the present study aimed to develop lipid nanoparticles (nanostructured lipid carriers) with the ability to incorporate and deliver anti-alopecia active compounds (minoxidil and finasteride) into the dermis and hair follicles. Lipid nanoparticles, prepared by ultrasonication method, showed mean particle sizes around 200 nm, which is sufficient for reaching the dermis and hair follicles, and zeta potential values around −30 mV, which indicates good physical stability. Over 28 days of storage, no significant variations in these parameters were observed, which indicates that all nanoformulations are stable in storage over that period. Cryo-scanning electron microscope measurements showed that all the lipid nanoparticles exhibited a spherical shape and a smooth surface regardless of their composition. Differential scanning calorimetry studies allowed the determination of phase transition temperatures and confirmed the recrystallization of the lipid nanoparticles (recrystallization index between 11% and 86%). A high loading efficiency was achieved for finasteride (between 70% and 90%), while less than 30% was achieved for minoxidil nanoparticles, over 28 days. Controlled release assays in physiological conditions demonstrated that nanoparticles loaded with minoxidil yielded a prolonged release, as desired. Penetration assays through pig ear skin demonstrated that nanoparticles loaded with minoxidil and finasteride had low levels of penetration. These results suggest that the proposed novel formulation presents several good characteristics indicating their suitability for dermal delivery of anti-alopecia active compounds.

siRNA as a tool to improve the treatment of brain diseases: Mechanism, targets and delivery

As the population ages, brain pathologies such as neurodegenerative diseases and brain cancer increase their incidence, being the need to find successful treatments of upmost importance. Drug delivery to the central nervous system (CNS) is required in order to reach diseases causes and treat them. However, biological barriers, mainly blood-brain barrier (BBB), are the key obstacles that prevent the effectiveness of possible treatments due to their ability to strongly limit the perfusion of compounds into the brain. Over the past decades, new approaches towards overcoming BBB and its efflux transporters had been proposed. One of these approaches here reviewed is through small interfering RNA (siRNA), which is capable to specifically target one gene and silence it in a post-transcriptional way. There are different possible functional proteins at the BBB, as the ones responsible for transport or just for its tightness, which could be a siRNA target. As important as the effective silence is the way to delivery siRNA to its anatomical site of action. This is where nanotechnology-based systems may help, by protecting siRNA circulation and providing cell/tissue-targeting and intracellular siRNA delivery. After an initial overview on incidence of brain diseases and basic features of the CNS, BBB and its efflux pumps, this review focuses on recent strategies to reach brain based on siRNA, and how to specifically target these approaches in order to treat brain diseases.

Multivariate design for the evaluation of lipid and surfactant composition effect for optimisation of lipid nanoparticles

Physicochemical properties of lipid nanoparticles (LN), such as size, size distribution and surface charge, have a major influence both, on in vitro stability and delivery of the incorporated drug in vivo. With the purpose of understanding how these properties are influenced by variations of LN composition (e.g. lipid and surfactant type and concentration) 2(2) factorial designs with centre point were applied for several types of lipids and surfactants in the present study. Tested factors and levels were the type and concentration of lipid (cetyl palmitate, Dynasan 114 and Witepsol E85) at the concentrations of 5%, 10% and 15%, in combination with type and concentration of surfactant (polysorbate 20, 40, 60 and 80 and poloxamer 188 and 407) at concentrations of 0.8%, 1.2% and 2.0%. Responses measured within the design space were the mean size and polydispersity index (photon correlation spectroscopy), content of microparticles (optical single particle sizing), macroscopic appearance, pH and zeta potential on the day of production, 1 and 2 years after production. Multivariate evaluation and modelling were performed starting with a principal component analysis (PCA) and followed by partial least square regression analysis (PLS) to assess both qualitative and quantitative influence of the investigated factors in the LN. Our study showed that both, lipid and surfactant concentration and the type of surfactant are crucial parameters for the particle size of the LN prepared by high pressure homogenisation (HPH). For LN stability during 2 years both, lipid and surfactant types and concentrations were identified as the most relevant parameters. Among the surfactants most suitable for producing LN with small sizes were the polysorbates and the lipid yielding best storage stability was cetyl palmitate. Furthermore, the models allowed the prediction of the mean size of LN that could be achieved with a certain lipid/surfactant combination and concentration. The obtained results are considered useful for future design of stable LN formulations without the need of extensive empirical testing of formulation parameters within the given HPH technology.

Development and validation of a simple reversed-phase HPLC method for the determination of camptothecin in animal organs following administration in solid lipid nanoparticles

A simple, sensitive and specific high-performance liquid chromatography (HPLC) assay for the quantification of camptothecin (CPT), a potent anticancer candidate, incorporated into solid lipid nanoparticles (SLN) in several rat organs (brain, heart, kidneys liver, lung, spleen) and serum was developed and validated. The sample pre-treatment involved organs homogenisation followed by CPT extraction. The samples were injected onto an analytical reversed-phase (RP) Mediterranea™ Sea18 column maintained at 30°C. The chromatographic separation was achieved by gradient elution consisting of triethyamine buffer pH 5.5 and acetonitrile at a flow rate of 1.2 mL/min in 16 min of run time and retention time of 9.8 min (lactone). Fluorescence detection was used at the excitation and emission of 360 and 440 nm, respectively. The calibration curves in the different organs, serum and in PB3 were linear (r(2)>0.9999) over CPT concentrations ranging from 1 to 200 ng/mL or 0.5 to 200 ng/mL (n=6), respectively. The method was shown to be specific, accurate (between 94.4±4.5% and 108.9±0.6%) and precise at the intra-day and inter-day levels as reflected by the coefficient of variation (CV<6.3%) at three different concentrations (10, 50 and 100 ng/mL) in all matrices. Stability studies showed that CPT was stable in all matrices after 24h of incubation at room temperature (RT), after 24 h in the autosampler or after three freeze/thaw cycles. The mean recoveries of CPT in suspension, loaded into SLN and in a physical mixture with SLN at three concentrations of 10, 50 and 200 ng/mL were higher than 86.4%. The detection limit (DL) was ≤0.2 ng/mL and the quantification limit (QL) was ≤0.5 ng/mL. The method developed is reliable, precise and accurate and can be used in the determination of CPT amount in rat organ samples after i.v. administration of CPT in suspension, in physical mixture with SLN and incorporated in SLN.