Publications by authors named "Nuno R Ferreira"

12 Publications

  • Page 1 of 1

Cyclodextrin-based delivery systems for in vivo-tested anticancer therapies.

Drug Deliv Transl Res 2021 Feb;11(1):49-71

Department of Pharmaceutical Technology, Faculty of Pharmacy, University of Coimbra, Azinhaga Sta. Comba, 3000-548, Coimbra, Portugal.

Cyclodextrins (CDs) are naturally occurring macromolecules widely used as excipients on pharmaceutical formulations, evidencing a large spectrum of applications in the pharmaceutical industry. Their unique ability to act as molecular containers by entrapping a wide range of guest molecules in their internal cavity makes them a remarkable excipient to improve drug apparent solubility, stability, and bioavailability, and a valuable tool for the assembly of new drug delivery systems. These features are especially useful when it comes to chemotherapy, as most of the anticancer drugs present both low permeability and reduced water solubility. Therefore, guest-host inclusion complexes offer several potential advantages not only regarding the improvement of pharmaceutical formulations characteristics but also considering the reduction of drug toxic side effects. The combination of CDs with additional technologies and materials constitutes a potential strategy towards the development of advanced and multifunctional CD-based delivery systems. Paclitaxel, curcumin, camptothecin, doxorubicin, and cisplatin are among the most studied molecules with anticancer activities and have been successfully incorporated in such nanosystems. Exciting results using CDs and CD-based delivery systems have been obtained so far, paving the way towards the attainment of intelligent delivery systems to possibly address cancer therapeutics' unmet needs. In this review, a comprehensive exposition concerning in vivo-tested CD and CD-based delivery systems for anticancer therapy is undertaken. Additionally, the authors address the multivalent functionalities of CD-based delivery systems, namely the incorporation of active target ligands, stimuli-responsiveness components, surface functionalization, or further associations with other delivery systems, aiming at improved in vivo anticancer therapies. Graphical abstract.
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http://dx.doi.org/10.1007/s13346-020-00778-5DOI Listing
February 2021

Loading, release profile and accelerated stability assessment of monoterpenes-loaded solid lipid nanoparticles (SLN).

Pharm Dev Technol 2020 Sep 23;25(7):832-844. Epub 2020 Mar 23.

Faculty of Pharmacy, University of Coimbra, Coimbra, Portugal.

Glycerol monostearate solid lipid nanoparticles (SLN) were produced by hot high-pressure homogenization technique to load alpha-pinene, citral, geraniol or limonene. SLN were composed of 1 wt.% monoterpene, 4 wt.% of Imwitor 900K as a solid lipid and 2.5 wt.% of Poloxamer188 as a surfactant. Empty SLN consisted of 5 wt.% of Imwitor 900K and 2.5 wt.% of Poloxamer188. The mean particles size (Z-Ave) and polydispersity index (PDI) of SLN were analyzed by dynamic light scattering (DLS), while the zeta potential (ZP) of each formulation were measured by electrophoretic light scattering. LUMiSizer was applied to calculate the velocity distribution in the centrifugal field and instability index. Drug release profile from SLN was analyzed using Franz cell diffusion cells assayed by UV-Vis spectrophotometry, whereas the gas chromatography technique was applied to determine the encapsulation parameters of volatile monoterpenes. The matrix state, polymorphism and phase behavior of SLN were studied by X-ray diffraction (XRD, low and wide angles) and differential scanning calorimetry (DSC). Selected monoterpenes were successfully loaded in glycerol monostearate SLN. A burst release profile within the first 15 min was observed for all formulations, being the modified release profile dependent on the type of monoterpene and on the encapsulation efficiency.
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http://dx.doi.org/10.1080/10837450.2020.1744008DOI Listing
September 2020

Complex Polysaccharide-Based Nanocomposites for Oral Insulin Delivery.

Mar Drugs 2020 Jan 15;18(1). Epub 2020 Jan 15.

Department of Pharmaceutical technology, Faculty of Pharmacy of the University of Coimbra, 3000-548 Coimbra, Portugal.

Polyelectrolyte nanocomposites rarely reach a stable state and aggregation often occurs. Here, we report the synthesis of nanocomposites for the oral delivery of insulin composed of alginate, dextran sulfate, poly-(ethylene glycol) 4000, poloxamer 188, chitosan, and bovine serum albumin. The nanocomposites were obtained by Ca-induced gelation of alginate followed by an electrostatic-interaction process among the polyelectrolytes. Chitosan seemed to be essential for the final size of the nanocomposites and there was an optimal content that led to the synthesis of nanocomposites of 400-600 nm hydrodynamic size. The enhanced stability of the synthesized nanocomposites was assessed with LUMiSizer after synthesis. Nanocomposite stability over time and under variations of ionic strength and pH were assessed with dynamic light scattering. The rounded shapes of nanocomposites were confirmed by scanning electron microscopy. After loading with insulin, analysis by HPLC revealed complete drug release under physiologically simulated conditions.
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http://dx.doi.org/10.3390/md18010055DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7024366PMC
January 2020

Effect of Polysaccharide Sources on the Physicochemical Properties of Bromelain-Chitosan Nanoparticles.

Polymers (Basel) 2019 Oct 15;11(10). Epub 2019 Oct 15.

Department of Pharmaceutical Technology, Faculty of Pharmacy, University of Coimbra (FFUC), Pólo das Ciências da Saúde, 3000 Coimbra, Portugal.

Bromelain, a set of proteolytic enzymes potential pharmaceutical applications, was encapsulated in chitosan nanoparticles to enhance enzyme stability, and the effect of different chitosan sources was evaluated. Chitosan types (i.e., low molecular weight chitosan, chitosan oligosaccharide lactate, and chitosan from shrimp shells) produced nanoparticles with different physicochemical properties, however in all cases, particle size and zeta potential decreased, and polydispersity index increased after bromelain addition. Bromelain encapsulation was higher than 84% and 79% for protein content and enzymatic activity, respectively, with low molecular weight chitosan presenting the highest encapsulation efficiency. Nanoparticle suspension was also tested for accelerated stability and rheological behavior. For the chitosan-bromelain nanoparticles, an instability index below 0.3 was recorded and, in general, the loading of bromelain in chitosan nanoparticles decreased the cohesiveness of the final suspension.
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http://dx.doi.org/10.3390/polym11101681DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6835720PMC
October 2019

Evaluation of the Influence of Process Parameters on the Properties of Resveratrol-Loaded NLC Using 2 Full Factorial Design.

Antioxidants (Basel) 2019 Aug 3;8(8). Epub 2019 Aug 3.

Department of Pharmaceutical Technology, Faculty of Pharmacy, University of Coimbra, 3000-548 Coimbra, Portugal.

Resveratrol (RSV) is a natural antioxidant commonly found in grapes, berries, and nuts that has shown promising results in the treatment of a variety of degenerative and age-related diseases. Despite the proven beneficial results on reduction of reactive oxidant species (ROS) and on inflammatory process, RSV shows various limitations including low long-term stability, aqueous solubility, and bioavailability, restricting its applications in the medical-pharmaceutical area. To overcome these limitations, it has been applied in pharmaceutical formulations as nanostructured lipid carriers (NLC). Thus, the present study focuses on the optimization of the production process of NLC. NLC was produced by high shear homogenization (HSH) and ultrasound method (US) using Compritol ATO C888 as solid lipid and Miglyol 812 as liquid lipid. In order to obtain an optimized formulation, we used a 2 full factorial design with triplicate of central point investigating the effects of the production process parameters; shear intensity and homogenization time, on the mean particle size (PS) and polydispersity index (PDI). Instability index, encapsulation efficiency, and production yield were also evaluated. As the PS and PDI values obtained with 6 min of shear at 19,000 rpm and 10 min of shear and 24,000 rpm were similar, the instability index (<0.1) was also used to select the optimal parameters. Based on the results of the experimental design and instability index, it was concluded that the shear rate of 19,000 rpm and the shear time of 6 min are the optimal parameters for RSV-loaded NLC production. Factorial design contributed therefore to optimize the variables of the NLC production process from a small number of experiments.
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http://dx.doi.org/10.3390/antiox8080272DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6719996PMC
August 2019

Development and Optimization of Alpha-Pinene-Loaded Solid Lipid Nanoparticles (SLN) Using Experimental Factorial Design and Dispersion Analysis.

Molecules 2019 Jul 24;24(15). Epub 2019 Jul 24.

Department of Pharmaceutical Technology, Faculty of Pharmacy, University of Coimbra (FFUC), Pólo das Ciências da Saúde, Azinhaga de Santa Comba, 3000-548 Coimbra, Portugal.

The encapsulation of bicyclic monoterpene α-pinene into solid lipid nanoparticles (SLN) is reported using experimental factorial design, followed by high-end dispersion analyzer LUMiSizer. This equipment allows the characterization of the α-pinene-loaded SLN instability phenomena (e.g., sedimentation, flotation or coagulation), as well as the determination of the velocity distribution in the centrifugal field and the particle size distribution. In this work, SLN were produced by hot high-pressure homogenization technique. The influence of the independent variables, surfactant and lipid ratio on the physicochemical properties of SLN, such as mean particle size (Z-Ave), polydispersity index (PDI) and zeta potential (ZP), was estimated using a 2-factorial design. The Z-Ave and PDI were analyzed by dynamic light scattering, while ZP measurements were recorded by electrophoretic light scattering. Based on the obtained results, the optimal SLN dispersion was composed of 1 wt.% of α-pinene, 4 wt.% of solid lipid (Imwitor 900 K) and 2.5 wt.% of surfactant (Poloxamer 188), depicting 136.7 nm of Z-Ave, 0.170 of PDI and 0 mV of ZP. Furthermore, LUMISizer has been successfully used in the stability analysis of α-pinene-loaded SLN.
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http://dx.doi.org/10.3390/molecules24152683DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6696006PMC
July 2019

Anti-inflammatory and anti-cancer activity of citral: Optimization of citral-loaded solid lipid nanoparticles (SLN) using experimental factorial design and LUMiSizer®.

Int J Pharm 2018 Dec 29;553(1-2):428-440. Epub 2018 Oct 29.

Department of Pharmaceutical Technology, Faculty of Pharmacy, University of Coimbra (FFUC), Pólo das Ciências da Saúde, Azinhaga de Santa Comba, 3000-548 Coimbra, Portugal. Electronic address:

Essential oils containing monoterpenes are widely used in pharmaceuticals and cosmetic products on account of their wide range of bioactive properties (including anti-cancer activity). Two monoterpenes (citral and geraniol) were firstly tested for their anti-inflammatory activity in a RAW 264.7 cell line, demonstrating citral to have enhanced capacity to inhibit NO production (ca. 84% for citral and 52% for geraniol at the lowest tested concentration of 5 µg/ml). As citral showed higher NO inhibitory activity than geraniol, to measure the level of cytotoxicity of citral, AlamarBlue reduction assay was run in two cell models (non-tumoral HaCaT and tumoral A431). Citral exhibited a strong cytotoxic effect in both cell lines, i.e. cell viability lower that 10% after 24 h exposure at 100 µg/ml of monoterpene. An optimized solid lipid nanoparticles (SLNs) formulation for citral was further developed by design of experiments (2 factorial design), followed by accelerated stability testing (LUMiSizer®). An optimal SLN composed of 1 wt% of citral, 4 wt% of lipid and 2.5 wt% surfactant were successfully produced by hot high pressure homogenization (hot HPH) showing a mean particle size (Z-Ave) of 97.7 nm and polydispersity index of 0.249. The produced formulations were analyzed in a high-end dispersion analyzer LUMiSizer® to characterize any demixing phenomena, demonstrating to be long-term stable at room temperature (25 °C), exhibiting very low instability indices (0.032 after production and 0.042 after one month of storage).
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http://dx.doi.org/10.1016/j.ijpharm.2018.10.065DOI Listing
December 2018

biomass increases dendritic arborization of newly-generated neurons in mouse hippocampal dentate gyrus.

Oncotarget 2018 Aug 31;9(68):32929-32942. Epub 2018 Aug 31.

CNC-Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal.

Brain cognitive reserve refers to the ability of the brain to manage different challenges that arise throughout life, making it resilient to neuropathology. Hippocampal adult neurogenesis has been considered to be a relevant contributor for brain cognitive reserve and brain plasticity. (CV), a common healthful mushroom, has been receiving increasing attention by its antitumoral, anti-inflammatory, antioxidant, antibacterial, and immunomodulatory properties, including in the hippocampus. Herein, we evaluated whether CV biomass oral administration for 2.5 months enhances hippocampal neurogenic reserve under normal/physiological conditions, by quantifying hippocampal dentate gyrus (DG) granular cell layer (GCL) and subgranular zone (SGZ) volumes, proliferation, number and dendritic complexity features of hippocampal newly-generated neurons. We also analyzed β-catenin levels in DG newly-generated immature neurons, because it plays a major role in neurogenesis. Although no differences were observed in the volume of GCL and SGZ layers, in proliferation and in the number of newly-generated neurons of controls and CV-administered mice, we found that CV administration promotes a significant increase in dendritic length and branching and total dendritic volume of immature neurons, suggesting a positive effect of oral CV administration in the hippocampal neurogenic reserve. We also observed that β-catenin levels are increased both in the nucleus and cytoplasm of DG immature neurons, suggesting that Wnt/β-catenin signalling may play an important role in the CV positive effect on the differentiation of these cells. These data unveil a so far unexplored neurogenic potential of CV supplementation, which emerges as a possible preventive strategy for different neurological conditions.
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http://dx.doi.org/10.18632/oncotarget.25978DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6152478PMC
August 2018

Optimization of linalool-loaded solid lipid nanoparticles using experimental factorial design and long-term stability studies with a new centrifugal sedimentation method.

Int J Pharm 2018 Oct 31;549(1-2):261-270. Epub 2018 Jul 31.

Department of Pharmaceutical Technology, Faculty of Pharmacy, University of Coimbra, Portugal; REQUIMTE/LAQV, Group of Pharmaceutical Technology, Faculty of Pharmacy, University of Coimbra, Portugal. Electronic address:

Linalool (CHO), also known as 3, 7-dimethyl-1, 6-octadien-3-ol, is the most common acyclic monoterpene tertiary alcohol present in essential oils of several aromatic plant species. Previous studies indicate that linalool is a valuable compound with a wide range of therapeutic properties. The promising therapeutic effects of linalool are however limited by its poor water solubility and volatility. Recently, the encapsulation of linalool in drug delivery systems, such as solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC) has demonstrated to overcome linalool physicochemical limitations The present study aimed the production and optimization of linalool encapsulation in SLN applying the experimental full factorial design. The estimation of the long-term stability of the produced linalool-loaded SLN was carried out using a new centrifugal sedimentation method, LUMiSizer®. SLN dispersions were produced by the hot high pressure homogenization (HPH) method. The influence of the independent variables, surfactant and lipid concentrations on linalool-loaded SLN particle size, polydispersity index (PI) and zeta potential (ZP) was evaluated by a 2 factorial design composed of 2 variables which were set at 2-levels each. For each of the three dependent variables, analysis of the variance (ANOVA) was performed using a 95% confidence interval. The concentration of surfactant, as well as, the interaction between the different concentrations of lipid and surfactant, hada statistically significant effect on the particle size and PI. Experimental factorial design has been successfully employed to develop an optimal SLN dispersion, requiring a minimum of performed experiments. Based on the obtained results, the optimal linalool-loaded SLN dispersion was composed of 1% (w/v) linalool 2% (w/v) of solid lipid and 5% (w/v) of surfactant. Furthermore, the stability analysis revealed that the produced linalool-loaded SLN dispersions have limited storage stability which can be easily overcome through the assembly of a polymeric coating on the SLN surface. LUMiSizer® has been successfully used in the kinetic analysis of linalool-SLN during accelerated storage time.
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http://dx.doi.org/10.1016/j.ijpharm.2018.07.068DOI Listing
October 2018

Simultaneous measurements of ascorbate and glutamate in vivo in the rat brain using carbon fiber nanocomposite sensors and microbiosensor arrays.

Bioelectrochemistry 2018 Jun 31;121:142-150. Epub 2018 Jan 31.

Faculty of Pharmacy, University of Coimbra, 3000-548 Coimbra, Portugal; Center for Neuroscience and Cell Biology, University of Coimbra, 3004-517 Coimbra, Portugal. Electronic address:

Nanocomposite sensors consisting of carbon fiber microelectrodes modified with Nafion® and carbon nanotubes, and ceramic-based microelectrode biosensor arrays were used to measure ascorbate and glutamate in the brain with high spatial, temporal and chemical resolution. Nanocomposite sensors displayed electrocatalytic properties towards ascorbate oxidation, translated into a negative shift from +0.20V to -0.05V vs. Ag/AgCl, as well as a significant increase (10-fold) of electroactive surface area. The estimated average basal concentration of ascorbate in vivo in the CA1, CA3 and dentate gyrus (DG) sub regions of the hippocampus were 276±60μM (n=10), 183±30μM (n=10) and 133±42μM (n=10), respectively. The glutamate microbiosensor arrays showed a high sensitivity of 5.3±0.8pAμM (n=18), and LOD of 204±32nM (n=10), and t response time of 0.9±0.02s (n=6) and high selectivity against major interferents. The simultaneous and real-time measurements of glutamate and ascorbate in the hippocampus of anesthetized rats following local stimulus with KCl or glutamate revealed a dynamic interaction between the two neurochemicals.
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http://dx.doi.org/10.1016/j.bioelechem.2018.01.009DOI Listing
June 2018

Coupling of ascorbate and nitric oxide dynamics in vivo in the rat hippocampus upon glutamatergic neuronal stimulation: a novel functional interplay.

Brain Res Bull 2015 May 14;114:13-9. Epub 2015 Mar 14.

Center for Neuroscience, Cell Biology, University of Coimbra, 3004 517 Coimbra, Portugal; Faculty of Pharmacy, University of Coimbra, 3000 548 Coimbra, Portugal. Electronic address:

Background: Ascorbate and neuronal-derived nitric oxide (NO) play regulatory roles in the brain that tare dependent on their compartmentalization and diffusion. Glutamatergic activation triggers both ascorbate fluxes toward extracellular medium and NO production. The information on the profiles of change in time and space upon glutamatergic activation is scarce and yet this knowledge is important for the understanding of ascorbate and NO functions in vivo, in particular in the case of a coupled interaction between both dynamics.

Hypothesis: NO produced upon NMDA receptor activation is a modulator of ascorbate release to the extracellular space.

Methods: In this work, carbon fiber microelectrodes for simultaneous measurements of these substances in the hippocampus were used to collect information about ascorbate and NO dynamic profiles in real time.

Results: Glutamate stimulation evoked transient ascorbate and NO signals with high degree of spatial and temporal correlation between them. Combined experiments encompassing direct stimulus with NO and inhibitors of glutamate uptake and nNOS provided additional evidence supporting the modulator role of NO in the release of ascorbate to the extracellular space.

Conclusions: The coupling between NO and ascorbate upon glutamatergic activation points to a functional impact on the activities of both compounds and, although the precise molecular mechanism needs to be clarified, such a coupling lays the foundations for new regulatory mechanisms in the brain.
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http://dx.doi.org/10.1016/j.brainresbull.2015.03.002DOI Listing
May 2015

The redox interplay between nitrite and nitric oxide: From the gut to the brain.

Redox Biol 2013 May 9;1:276-84. Epub 2013 May 9.

Department of Pharmacy and Center for Neurosciences and Cell Biology, University of Coimbra, Health Sciences Campus, Azinhaga de Santa Comba, 3000-548 Coimbra, Portugal.

The reversible redox conversion of nitrite and nitric oxide ((•)NO) in a physiological setting is now widely accepted. Nitrite has long been identified as a stable intermediate of (•)NO oxidation but several lines of evidence support the reduction of nitrite to nitric oxide in vivo. In the gut, this notion implies that nitrate from dietary sources fuels the longstanding production of nitrite in the oral cavity followed by univalent reduction to (•)NO in the stomach. Once formed, (•)NO boosts a network of reactions, including the production of higher nitrogen oxides that may have a physiological impact via the post-translational modification of proteins and lipids. Dietary compounds, such as polyphenols, and different prandial states (secreting specific gastric mediators) modulate the outcome of these reactions. The gut has unusual characteristics that modulate nitrite and (•)NO redox interplay: (1) wide range of pH (neutral vs acidic) and oxygen tension (c.a. 70 Torr in the stomach and nearly anoxic in the colon), (2) variable lumen content and (3) highly developed enteric nervous system (sensitive to (•)NO and dietary compounds, such as glutamate). The redox interplay of nitrite and (•)NO might also participate in the regulation of brain homeostasis upon neuronal glutamatergic stimulation in a process facilitated by ascorbate and a localized and transient decrease of oxygen tension. In a way reminiscent of that occurring in the stomach, a nitrite/(•)NO/ascorbate redox interplay in the brain at glutamatergic synapses, contributing to local (•)NO increase, may impact on (•)NO-mediated process. We here discuss the implications of the redox conversion of nitrite to (•)NO in the gut, how nitrite-derived (•)NO may signal from the digestive to the central nervous system, influencing brain function, as well as a putative ascorbate-driven nitrite/NO pathway occurring in the brain.
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http://dx.doi.org/10.1016/j.redox.2013.04.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3757698PMC
May 2013