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The delivery of a dexamethasone formulation directly into the lung appears as an appropriate strategy to strengthen the systemic administration, reducing the dosage in the treatment of lung severe inflammations. For this purpose, a hyaluronic acid-dexamethasone formulation was developed, affording an inhalable reconstituted nanosuspension suitable to be aerosolized. The physico-chemical and biopharmaceutical properties of the formulation were tested: size, stability, loading of the spray-dried dry powder, reconstitution capability upon redispersion in aqueous media. Detailed structural insights on nanoparticles after reconstitution were obtained by light and X-ray scattering techniques. (1) The size of the nanoparticles, around 200 nm, is in the proper range for a possible engulfment by macrophages. (2) Their structure is of the core-shell type, hosting dexamethasone nanocrystals inside and carrying hyaluronic acid chains on the surface. This specific structure allows for nanosuspension stability and provides nanoparticles with muco-inert properties. (3) The nanosuspension can be efficiently aerosolized, allowing for a high drug fraction potentially reaching the deep lung. Thus, this formulation represents a promising tool for the lung administration via nebulization directly in the pipe of ventilators, to be used as such or as adjunct therapy for severe lung inflammation.
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Dexametasona/química , Ácido Hialurónico/química , Nanopartículas/química , Neumonía/tratamiento farmacológico , Administración por Inhalación , Aerosoles , Dexametasona/farmacología , Humanos , Ácido Hialurónico/farmacología , Nanopartículas/uso terapéuticoRESUMEN
Tamoxifen citrate (TMC), a non-steroidal antiestrogen drug used for the treatment of breast cancer, was loaded in a block copolymer of maltoheptaose-b-polystyrene (MH-b-PS) nanoparticles, a potential drug delivery system to optimize oral chemotherapy. The nanoparticles were obtained from self-assembly of MH-b-PS using the standard and reverse nanoprecipitation methods. The MH-b-PS@TMC nanoparticles were characterized by their physicochemical properties, morphology, drug loading and encapsulation efficiency, and release kinetic profile in simulated intestinal fluid (pH 7.4). Finally, their cytotoxicity towards the human breast carcinoma MCF-7 cell line was assessed. The standard nanoprecipitation method proved to be more efficient than reverse nanoprecipitation to produce nanoparticles with small size and narrow particle size distribution. Moreover, tamoxifen-loaded nanoparticles displayed spherical morphology, a positive zeta potential and high drug content (238.6 ± 6.8 µg mL-1) and encapsulation efficiency (80.9 ± 0.4 %). In vitro drug release kinetics showed a burst release at early time points, followed by a sustained release profile controlled by diffusion. MH-b-PS@TMC nanoparticles showed higher cytotoxicity towards MCF-7 cells than free tamoxifen citrate, confirming their effectiveness as a delivery system for administration of lipophilic anticancer drugs.
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Portadores de Fármacos/química , Sistemas de Liberación de Medicamentos , Glucanos , Nanopartículas/química , Poliestirenos , Tamoxifeno/administración & dosificación , Neoplasias de la Mama , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Fenómenos Químicos , Cromatografía Líquida de Alta Presión , Relación Dosis-Respuesta a Droga , Composición de Medicamentos , Liberación de Fármacos , Femenino , Glucanos/química , Humanos , Cinética , Modelos Teóricos , Estructura Molecular , Tamaño de la Partícula , Poliestirenos/química , Moduladores Selectivos de los Receptores de Estrógeno/administración & dosificación , Tamoxifeno/químicaRESUMEN
RATIONALE: Drug development efforts involving therapeutic peptides or proteins strongly lead optimization of drug delivery, drug stability, solubility and functionality. The key feature of controlled drug delivery is the use of biocompatible polymers able to interact via non-covalent bonds with an active principle through multiple functional groups. Here amide hydrogen/deuterium exchange (HDX) mass spectrometry was employed to localize insulin dynamics induced by interactions with three natural polysaccharides, i.e. chitosan (CH), sodium alginate (ALG) and chondroitin sulfate (CS). METHODS: LTQ-Orbitap continuous-labelling mass spectra were collected by diluting insulin stock solution (10 mM in 0.1% formic acid) to a final concentration of 0.1 mM in D2 O containing 1 mM deuterated ammonium acetate (final pH .6) (insulin:polysaccharide ratio 1:2, w/w). For peptide mapping, deuterated samples were quenched after 0.5, 30, 60, 120 minutes exchange by adding HCl (pH ) and digested with pepsin before LC-MS/MS analysis. RESULTS: Differences in the insulin backbone dynamics in the presence of the three polysaccharides were highlighted by monitoring peptic peptides at different time points. No significant differences were observed in the presence of CH, whereas the negatively charged ALG and CS were able to induce significant conformational variations at the B-chain level resulting in more protection against H/D exchange. The A-chain interacted only with CS reducing the protein mobility on a long time scale (120 min). HDX data evidenced heterogeneous insulin dynamics in the presence of ALG and CS. CONCLUSIONS: The studies reported here demonstrated the capabilities of mass spectrometry techniques and HDX methods to obtain useful information toward the flexibility and the behavior of native insulin in the presence of natural polysaccharides, and could provide insights to study the behavior of pharmaceutical formulations. Copyright © 2016 John Wiley & Sons, Ltd.
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Deuterio/análisis , Hidrógeno/análisis , Insulina/química , Espectrometría de Masas/métodos , Polisacáridos/química , Medición de Intercambio de Deuterio , HumanosAsunto(s)
Aptámeros de Nucleótidos/química , Quitosano/química , Fibronectinas/química , Osteoblastos/citología , Andamios del Tejido/química , Animales , Aptámeros de Nucleótidos/metabolismo , Adhesión Celular , Proliferación Celular , Fibronectinas/metabolismo , Espectrometría de Masas , Ratones , Osteoblastos/química , Osteoblastos/metabolismoRESUMEN
Effective strategies against the spread of respiratory viruses are needed, as tragically demonstrated during the COVID-19 pandemic. Apart from vaccines, other preventive or protective measures are necessary: one promising strategy involves the nasal delivery of preventive or protective agents, targeting the site of initial infection. Harnessing the immune system's ability to produce specific antibodies, a hyperimmune serum, collected from an individual vaccinated against SARS-CoV-2, was formulated as a dry powder for nasal administration. The selection of adequate excipients and process are key to maintaining protein stability and modulating the aerodynamic properties of the powders for reaching the desired respiratory regions. To this end, a hyperimmune serum was formulated with trehalose and mannitol as bulking agents during spray drying, then the ability of the redissolved immunoglobulins to bind Spike protein was verified by ELISA; foetal bovine serum was formulated in the same conditions as a reference. Moreover, a seroneutralization assay against SARS-CoV-2 pseudoviruses generated from different variants of concern was performed. The neutralizing ability of the serum was slightly reduced with respect to the starting serum when trehalose was used as a bulking agent. The powders were loaded in hypromellose capsules and aerosolized employing a nasal insufflator in an in vitro model of the nasal cavity connected to a Next Generation Impactor. The analysis of the powder distribution confirmed that all powders were inhalable and could target, at the same time, the upper and the lower airways. This is a preliminary proof-of-concept that this approach can constitute an effective strategy to provide broad coverage and protection against SARS-CoV-2, and in general against viruses affecting the airway. According to blood availability from donors, pools of hyperimmune sera could be rapidly formulated and administered, providing a simultaneous and timely neutralization of emerging viral variants.
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Imiquimod (IMQ) has been successfully formulated to date mainly as semi-solid lipophilic formulations for topical application. In this study, we investigated the solubility of IMQ in solvents suitable for developing innovative formulations in the form of powder obtained, for instance, by spray drying; thus, water, ethanol, methanol, acetone, acetonitrile, and dimethyl sulfoxide were tested at different temperatures. Temperature variations, stirring intensity, and the contact time between IMQ and the solvent greatly affected the evaluation of IMQ equilibrium solubility. The attainment of the solid-liquid equilibrium requires 13 days starting from solid IMQ and 2 days from a cooled-down supersaturated IMQ solution. A correlation between IMQ solubility and the solubility parameters of solvents was not found. IMQ solutions in water, ethanol, methanol, acetonitrile, and dimethyl sulfoxide were neither ideal nor regular. The Scatchard-Hildebrand equation does not apply to IMQ solutions because of association phenomena due to intermolecular hydrogen bonds and/or π-stacking, as supported by the hyperchromic effect that was very pronounced in highly polar solvents, such as water, with the increase in temperature. Finally, IMQ solubility values measured in acetone cannot be considered reliable due to the reaction with the solvent, leading to the formation of new molecules.
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TPGS (D-α-tocopheryl polyethylene glycol 1000 succinate) polymeric micelles show interesting properties for ocular administration thanks to their solubilization capability, nanometric size and tissue penetration ability. However, micelles formulations are generally characterized by low viscosity, poor adhesion and very short retention time at the administration site. Therefore, the idea behind this work is the preparation and characterization of a crosslinked film based on xanthan gum that contains TPGS micelles and is capable of controlling their release. The system was loaded with melatonin and cyclosporin A, neuroprotective compounds to be delivered to the posterior eye segment. Citric acid and heating at different times and temperatures were exploited as crosslinking approach, giving the possibility to tune swelling, micelles release and drug release. The biocompatibility of the platform was confirmed by HET-CAM assay. Ex vivo studies on isolated porcine ocular tissues, conducted using Franz cells and two-photon microscopy, demonstrated the potential of the xanthan gum-based platform and enlightened micelles penetration mechanism. Finally, the sterilization step was approached, and a process to simultaneously crosslink and sterilize the platform was developed.
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Administración Oftálmica , Preparaciones de Acción Retardada , Liberación de Fármacos , Micelas , Fármacos Neuroprotectores , Polisacáridos Bacterianos , Vitamina E , Polisacáridos Bacterianos/química , Animales , Porcinos , Fármacos Neuroprotectores/administración & dosificación , Fármacos Neuroprotectores/química , Fármacos Neuroprotectores/farmacocinética , Vitamina E/química , Vitamina E/administración & dosificación , Preparaciones de Acción Retardada/química , Ciclosporina/administración & dosificación , Ciclosporina/química , Melatonina/administración & dosificación , Melatonina/química , Melatonina/farmacología , Melatonina/farmacocinética , Esterilización , Reactivos de Enlaces Cruzados/química , Portadores de Fármacos/química , Ojo/efectos de los fármacos , Ojo/metabolismo , Sistemas de Liberación de Medicamentos/métodosRESUMEN
OBJECTIVES: The aim of the project was to develop and characterise powders containing a probiotic (Lactiplantibacillus plantarum [Lpb. plantarum], Lacticaseibacillus rhamnosus, or Lactobacillus acidophilus) to be administered to the lung for the containment of pathogen growth in patients with lung infections. METHODS: The optimised spray drying process for the powder manufacturing was able to preserve viability of the bacteria, which decreased of only one log unit and was maintained up to 30 days. RESULTS: Probiotic powders showed a high respirability (42%-50% of particles had a size < 5 µm) suitable for lung deposition and were proven safe on A549 and Calu-3 cells up to a concentration of 107 colony-forming units/mL. The Lpb. plantarum adhesion to both cell lines tested was at least 10%. Surprisingly, Lpb. plantarum powder was bactericidal at a concentration of 106 colony-forming units/mL on P. aeruginosa, whereas the other two strains were bacteriostatic. CONCLUSION: This work represents a promising starting point to consider a probiotic inhalation powder a value in keeping the growth of pathogenic microflora in check during the antibiotic inhalation therapy suspension in cystic fibrosis treatment regimen. This approach could also be advantageous for interfering competitively with pathogenic bacteria and promoting the restoration of the healthy microbiota.
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Lactobacillales , Probióticos , Infecciones por Pseudomonas , Humanos , Pseudomonas aeruginosa , Polvos , Antibacterianos/farmacologíaRESUMEN
The growing demand for personalized medicine requires innovation in drug manufacturing to combine versatility with automation. Here, three-dimensional (3D) printing was explored for the production of chitosan (CH)/alginate (ALG)-based hydrogels intended as active dressings for wound healing. ALG hydrogels were loaded with 0.75% w/v silver sulfadiazine (SSD), selected as a drug model commonly used for the therapeutic treatment of infected burn wounds, and four different 3D CH/ALG architectures were designed to modulate the release of this active compound. CH/ALG constructs were characterized by their water content, elasticity and porosity. ALG hydrogels (Young's modulus 0.582 ± 0.019 Mpa) were statistically different in terms of elasticity compared to CH (Young's modulus 0.365 ± 0.015 Mpa) but very similar in terms of swelling properties (water content in ALG: 93.18 ± 0.88% and in CH: 92.76 ± 1.17%). In vitro SSD release tests were performed by using vertical diffusion Franz cells, and statistically significant different behaviors in terms of the amount and kinetics of drugs released were observed as a function of the construct. Moreover, strong antimicrobial potency (100% of growth inhibition) against Staphylococcus aureus and Pseudomonas aeruginosa was demonstrated depending on the type of construct, offering a proof of concept that 3D printing techniques could be efficiently applied to the production of hydrogels for controlled drug delivery.
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The unique self-assembly properties of unimer micelles are exploited for the preparation of fluorescent nanocarriers embedding hydrophobic fluorophores. Unimer micelles are constituted by a (meth)acrylate copolymer with oligoethyleneglycol and perflurohexylethyl side chains (PEGMA90-co-FA10) in which the hydrophilic and hydrophobic comonomers are statistically distributed along the polymeric backbone. Thanks to hydrophobic interactions in water, the amphiphilic copolymer forms small nanoparticles (<10 nm), with tunable properties and functionality. An easy procedure for the encapsulation of a small hydrophobic molecule (C153 fluorophore) within unimer micelles is presented. UV-vis, fluorescence, and fluorescence anisotropy spectroscopic experimental data demonstrate that the fluorophore is effectively embedded in the nanocarriers. Moreover, the nanocarrier positively contributes to preserve the good emissive properties of the fluorophore in water. The efficacy of the dye-loaded nanocarrier as a fluorescent probe is tested in two-photon imaging of thick ex vivo porcine scleral tissue.
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In humans, the phosphorylated pathway (PP) converts the glycolytic intermediate D-3-phosphoglycerate (3-PG) into L-serine through the enzymes 3-phosphoglycerate dehydrogenase, phosphoserine aminotransferase (PSAT) and phosphoserine phosphatase. From the pathogenic point of view, the PP in the brain is particularly relevant, as genetic defects of any of the three enzymes are associated with a group of neurometabolic disorders known as serine deficiency disorders (SDDs). We recombinantly expressed and characterized eight variants of PSAT associated with SDDs and two non-SDD associated variants. We show that the pathogenetic mechanisms in SDDs are extremely diverse, including low affinity of the cofactor pyridoxal 5'-phosphate and thermal instability for S179L and G79W PSAT, loss of activity of the holo form for R342W PSAT, aggregation for D100A PSAT, increased Km for one of the substrates with invariant kcats for S43R PSAT, and a combination of increased Km and decreased kcat for C245R PSAT. Finally, we show that the flux through the in vitro reconstructed PP at physiological concentrations of substrates and enzymes is extremely sensitive to alterations of the functional properties of PSAT variants, confirming PSAT dysfunctions as a cause of SSDs.
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Encéfalo , Transaminasas , Humanos , Transaminasas/genética , Fosfato de Piridoxal , Serina/genéticaRESUMEN
Nasal vaccination has been shown to provide optimal protection against respiratory pathogens. However, mucosal vaccination requires the implementation of specific immunization strategies to improve its effectiveness. Nanotechnology appears a key approach to improve the effectiveness of mucosal vaccines, since several nanomaterials provide mucoadhesion, enhance mucosal permeability, control antigen release and possess adjuvant properties. Mycoplasma hyopneumoniae is the main causative agent of enzootic pneumonia in pigs, a respiratory disease responsible for considerable economic losses in the pig farming worldwide. The present work developed, characterized, and tested in vivo an innovative dry powder nasal vaccine, obtained from the deposition on a solid carrier of an inactivated antigen and a chitosan-coated nanoemulsion, as an adjuvant. The nanoemulsion was obtained through a low-energy emulsification technique, a method that allowed to achieve nano droplets in the order of 200 nm. The oil phase selected was alpha-tocopherol, sunflower oil, and poly(ethylene glycol) hydroxystearate used as non-ionic tensioactive. The aqueous phase contained chitosan, which provides a positive charge to the emulsion, conferring mucoadhesive properties and favoring interactions with inactivated M. hyopneumoniae. Finally, the nanoemulsion was layered with a mild and scalable process onto a suitable solid carrier (i.e., lactose, mannitol, or calcium carbonate) to be transformed into a solid dosage form for administration as dry powder. In the experimental study, the nasal vaccine formulation with calcium carbonate was administered to piglets and compared to intramuscular administration of a commercial vaccine and of the dry powder without antigen, aimed at evaluating the ability of IN vaccination to elicit an in vivo local immune response and a systemic immune response. Intranasal vaccination was characterized by a significantly higher immune response in the nasal mucosa at 7 days post-vaccination, elicited comparable levels of Mycoplasma-specific IFN-γ secreting cells and comparable, if not higher, responsiveness of B cells expressing IgA and IgG in peripheral blood mononuclear cells, with those detected upon a conventional intramuscular immunization. In conclusion, this study illustrates a simple and effective strategy for the development of a dry powder vaccine formulation for nasal administration which could be used as alternative to current parenteral commercial vaccines.
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The nebulization of alpha-1 antitrypsin (AAT) for its administration to the lung could be an interesting alternative to parenteral infusion for patients suffering from AAT genetic deficiency (AATD). In the case of protein therapeutics, the effect of the nebulization mode and rate on protein conformation and activity must be carefully considered. In this paper two types of nebulizers, i.e., a jet and a mesh vibrating system, were used to nebulize a commercial preparation of AAT for infusion and compared. The aerosolization performance, in terms of mass distribution, respirable fraction, and drug delivery efficiency, as well as the activity and aggregation state of AAT upon in vitro nebulization were investigated. The two nebulizers demonstrated equivalent aerosolization performances, but the mesh nebulizer provided a higher efficiency in the delivery of the dose. The activity of the protein was acceptably preserved by both nebulizers and no aggregation or changes in its conformation were identified. This suggests that nebulization of AAT represents a suitable administration strategy ready to be translated to the clinical practice for delivering the protein directly to the lungs in AATD patients, either as a support therapy to parenteral administration or for subjects with a precocious diagnosis, to prevent the onset of pulmonary symptoms.
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Sistemas de Liberación de Medicamentos , Nebulizadores y Vaporizadores , Humanos , Aerosoles , Pulmón/metabolismoRESUMEN
This work illustrates the development of a dry inhalation powder of cyclosporine-A for the prevention of rejection after lung transplantation and for the treatment of COVID-19. The influence of excipients on the spray-dried powder's critical quality attributes was explored. The best-performing powder in terms of dissolution time and respirability was obtained starting from a concentration of ethanol of 45% (v/v) in the feedstock solution and 20% (w/w) of mannitol. This powder showed a faster dissolution profile (Weibull dissolution time of 59.5 min) than the poorly soluble raw material (169.0 min). The powder exhibited a fine particle fraction of 66.5% and an MMAD of 2.97 µm. The inhalable powder, when tested on A549 and THP-1, did not show cytotoxic effects up to a concentration of 10 µg/mL. Furthermore, the CsA inhalation powder showed efficiency in reducing IL-6 when tested on A549/THP-1 co-culture. A reduction in the replication of SARS-CoV-2 on Vero E6 cells was observed when the CsA powder was tested adopting the post-infection or simultaneous treatment. This formulation could represent a therapeutic strategy for the prevention of lung rejection, but is also a viable approach for the inhibition of SARS-CoV-2 replication and the COVID-19 pulmonary inflammatory process.
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Alpha 1-antitrypsin is one of the first protein therapeutics introduced on the market more than 30 years ago, and to date, it is indicated only for the treatment of the severe forms of a genetic condition known as alpha-1 antitrypsin deficiency. The only approved preparations are derived from plasma, posing potential problems associated with its limited supply and high processing costs. Moreover, augmentation therapy with alpha-1 antitrypsin is still limited to intravenous infusions, a cumbersome regimen for patients. Here, we review the recent literature on its possible future developments, focusing on i) the recombinant alternatives to the plasma-derived protein, ii) novel formulations, and iii) novel administration routes. Regulatory issues and the still unclear noncanonical functions of alpha-1 antitrypsin, possibly associated with the glycosylation pattern found only in the plasma-derived protein, have hindered the introduction of new products. However, potentially new therapeutic indications other than the treatment of alpha-1 antitrypsin deficiency might open the way to new sources and new formulations.
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Enfisema Pulmonar , Deficiencia de alfa 1-Antitripsina , Pruebas Diagnósticas de Rutina , Glicosilación , Humanos , alfa 1-Antitripsina , Deficiencia de alfa 1-Antitripsina/tratamiento farmacológicoRESUMEN
Rifaximin is a locally acting antibiotic practically insoluble in water. It presents several crystal phases characterized by different degrees of hydration. The aim of this work is to investigate the dissolution behaviour of rifaximin α, ß, and amorphous forms in relation to their relative thermodynamic stability to contribute to clarifying possible solvent- or humidity-mediated conversion patterns. Kinetic and intrinsic solubility were investigated along with particle size distribution, specific surface area, and external morphology. The solution and moisture mediated conversion from metastable α and amorphous forms to stable ß form were elucidated by coupling intrinsic dissolution test with chemometric analysis as well as by dynamic vapour sorption measurements. The dissolution behaviour of the α form stems mainly from the transition to ß form that occurs upon exposition to relative humidity higher than 40%. The α form converted more rapidly than the amorphous form due to the smaller supersaturation ratio. It can be concluded that, due to its marked tendency to transform into ß form, the dissolution test for the α form, even if conducted according to compendial procedures, needs to be accompanied by a panel of further tests that allow to uniquely identify the solid phase under investigation.
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Alpha-1 antitrypsin (AAT) deficiency is a genetic disorder associated with pulmonary emphysema and bronchiectasis. Its management currently consists of weekly infusions of plasma-purified human AAT, which poses several issues regarding plasma supplies, possible pathogen transmission, purification costs, and parenteral administration. Here, we investigated an alternative administration strategy for augmentation therapy by combining recombinant expression of AAT in bacteria and the production of a respirable powder by spray drying. The same formulation approach was then applied to plasma-derived AAT for comparison. Purified, active, and endotoxin-free recombinant AAT was produced at high yields and formulated using L-leucine and mannitol as excipients after identifying compromise conditions for protein activity and good aerodynamic performances. An oxygen-free atmosphere, both during formulation and powder storage, slowed down methionine-specific oxidation and AAT inactivation. This work is the first peer-reviewed report of AAT formulated as a dry powder, which could represent an alternative to current treatments.
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The search for best performing carriers for dry powder inhalers is getting a great deal of interest to overcome the limitations posed by lactose. The aerosolization of adhesive mixtures between a carrier and a micronized drug is strongly influenced by the carrier solid-state properties. This work aimed at crystallizing kinetically stable D-mannitol polymorphs and at investigating their aerosolization performance when used in adhesive mixtures with two model drugs (salbutamol sulphate, SS, and budesonide, BUD) using a median and median/high resistance inhaler. A further goal was to assess in vitro the cytocompatibility of the produced polymer-doped mannitol polymorphs toward two lung epithelial cell lines. Kinetically stable (up to 12 months under accelerate conditions) α, and δ mannitol forms were crystallized in the presence of 2% w/w PVA and 1% w/w PVP respectively. These solid phases were compared with the ß form and lactose as references. The solid-state properties of crystallized mannitol significantly affected aerosolization behavior, with the δ form affording the worst fine particle fraction with both the hydrophilic (9.3 and 6.5%) and the lipophilic (19.6 and 32%) model drugs, while α and ß forms behaved in the same manner (11-13% for SS; 53-58% for BUD) and better than lactose (8 and 13% for SS; 26 and 39% for BUD). Recrystallized mannitol, but also PVA and PVP, proved to be safe excipients toward lung cell lines. We concluded that, also for mannitol, the physicochemical properties stemming from different crystal structures represent a tool for modulating carrier-drug interaction and, in turn, aerosolization performance.
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Nanotherapeutics based on biocompatible lipid matrices allow for enhanced solubility of poorly soluble compounds in the treatment of ophthalmic diseases, overcoming the anatomical and physiological barriers present in the eye, which, despite the ease of access, remains strongly protected. Micro-/nanoemulsions, solid lipid nanoparticles (SLN) or nanostructured lipid carriers (NLC) combine liquid and/or solid lipids with surfactants, improving drug stability and ocular bioavailability. Current research and development approaches based on try-and-error methodologies are unable to easily fine-tune nanoparticle populations in order to overcome the numerous constraints of ocular administration routes, which is believed to hamper easy approval from regulatory agencies for these systems. The predictable quality and specifications of the product can be achieved through quality-by-design (QbD) implementation in both research and industrial environments, in contrast to the current quality-by-testing (QbT) framework. Mathematical modelling of the expected final nanoparticle characteristics by variation of operator-controllable variables of the process can be achieved through adequate statistical design-of-experiments (DoE) application. This multivariate approach allows for optimisation of drug delivery platforms, reducing research costs and time, while maximising the understanding of the production process. This review aims to highlight the latest efforts in implementing the design of experiments to produce optimised lipid-based nanocarriers intended for ophthalmic administration. A useful background and an overview of the different possible approaches are presented, serving as a starting point to introduce the design of experiments in current nanoparticle research.
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Three-dimensional (3D) printing has gained popularity in tissue engineering and in the field of cartilage regeneration. This is due to its potential to generate scaffolds with spatial variation of cell distribution or mechanical properties, built with a variety of materials that can mimic complex tissue architecture. In the present study, horse articular chondrocytes were cultured for 2 and 4 weeks in 3D-printed chitosan (CH)-based scaffolds prepared with or without hyaluronic acid and in the presence of fetal bovine serum (FBS) or platelet lysate (PL). These 3D culture systems were analyzed in terms of their capability to maintain chondrocyte differentiation in vitro. This was achieved by evaluating cell morphology, immunohistochemistry (IHC), gene expression of relevant cartilage markers (collagen type II, aggrecan, and Sox9), and specific markers of dedifferentiated phenotype (collagen type I, Runx2). The morphological, histochemical, immunohistochemical, and molecular results demonstrated that the 3D CH scaffold is sufficiently porous to be colonized by primary chondrocytes. Thereby, it provides an optimal environment for the colonization and synthetic activity of chondrocytes during a long culture period where a higher rate of dedifferentiation can be generally observed. Enrichment with hyaluronic acid provides an optimal microenvironment for a more stable maintenance of the chondrocyte phenotype. The use of 3D CH scaffolds causes a further increase in the gene expression of most relevant ECM components when PL is added as a substitute for FBS in the medium. This indicates that the latter system enables a better maintenance of the chondrocyte phenotype, thereby highlighting a fair balance between proliferation and differentiation.