Cross-linked Porous Gelatin Microparticles with Tunable Shape, Size, and Porosity.

Gelatin particles are relevant to many applications in the biomedical field due to their excellent biocompatibility and versatility. When prepared by double emulsion methods, porous microparticles with different architectures can be obtained. Controlling the shape, size, porosity, swelling, and stability against dissolution is fundamental toward their application under physiological conditions. We prepared porous gelatin microparticles from oil-in-water-in-oil emulsions, modifying the gelatin/surfactant ratio and the stirring speed. The effect on structural properties, including surface and inner porosities, was thoroughly assessed by multiple microscopy techniques (optical, electron, and confocal Raman). Selected samples were cross-linked with glutaraldehyde or glyceraldehyde, and their swelling properties and stability against dissolution were evaluated, while the influence of the cross-linking at the nanoscale was studied by scattering of X-rays. Depending on the preparation protocol, we obtained particles with different shapes (irregular or spherical), radii within ∼40 to 90 µm, and porosities up to 10 µm. The cross-linking extends the stability in water from a few minutes up to several days while the swelling ability and the mesh size at the nanoscale of the gelatin network are preserved. The analysis of the experimental results as a function of the preparation parameters demonstrates that microparticles with tunable features can be designed.

A bioinspired and sustainable route for the preparation of Ag-crosslinked alginate fibers decorated with silver nanoparticles.

Functional materials obtained through green and sustainable routes are attracting particular attention due to the need to reduce the environmental impact of the chemical industry. In this work we propose a bioinspired approach for the preparation of alginate fibers containing silver nanoparticles (AgNPs), to be used for antimicrobial purposes. We demonstrate that filiform polymeric structures with length of a few meters can be easily obtained by extruding an alginate solution in an aqueous Ag+-containing bath (i.e. wet spinning) and that treating the fibers with freshly-squeezed lemon juice leads to the formation of AgNPs homogeneously distributed within the polymeric network. Using mixtures of ascorbic and citric acid to mimic lemon juice composition we highlight the influence of the aforementioned molecules on the nanoparticles formation process as well as on the properties of the fibers. Varying the amount of citric and ascorbic acid used for the treatment allows to finely tune the thermal, morphological and water absorption properties of the fibers. This evidence, along with the possibility to easily monitor the preparation through FT-IR spectroscopy, endows the fibers with a high application potential in several fields such as wound healing, water/air purification and agriculture.

Bioinspired and bioderived nanomedicine for inflammatory bowel disease.

Due to its chronic nature and complex pathophysiology, inflammatory bowel disease (IBD) poses significant challenges for treatment. The long-term therapies for patients, often diagnosed between the ages of 20 and 40, call for innovative strategies to target inflammation, minimize systemic drug exposure, and improve patients' therapeutic outcomes. Among the plethora of strategies currently pursued, bioinspired and bioderived nano-based formulations have garnered interest for their safety and versatility in the management of IBD. Bioinspired nanomedicine can host and deliver not only small drug molecules but also biotherapeutics, be made gastroresistant and mucoadhesive or mucopenetrating and, for these reasons, are largely investigated for oral administration, while surprisingly less for rectal delivery, recommended first-line treatment approach for several IBD patients. The use of bioderived nanocarriers, mostly extracellular vesicles (EVs), endowed with unique homing abilities, is still in its infancy with respect to the arsenal of nanomedicine under investigation for IBD treatment. An emerging source of EVs suited for oral administration is ingesta, that is, plants or milk, thanks to their remarkable ability to resist the harsh environment of the upper gastrointestinal tract. Inspired by the unparalleled properties of natural biomaterials, sophisticated avenues for enhancing therapeutic efficacy and advancing precision medicine approaches in IBD care are taking shape, although bottlenecks arising either from the complexity of the nanomedicine designed or from the lack of a clear regulatory pathway still hinder a smooth and efficient translation to the clinics. This article is categorized under: Nanotechnology Approaches to Biology > Nanoscale Systems in Biology.

An Overview of Magnesium-Phosphate-Based Cements as Bone Repair Materials.

In the search for effective biomaterials for bone repair, magnesium phosphate cements (MPCs) are nowadays gaining importance as bone void fillers thanks to their many attractive features that overcome some of the limitations of the well-investigated calcium-phosphate-based cements. The goal of this review was to highlight the main properties and applications of MPCs in the orthopedic field, focusing on the different types of formulations that have been described in the literature, their main features, and the in vivo and in vitro response towards them. The presented results will be useful to showcase the potential of MPCs in the orthopedic field and will suggest novel strategies to further boost their clinical application.

Reconsidering the role of albumin towards amorphous calcium phosphate-based calciprotein particles formation and stability from a physico-chemical perspective.

The formation of calciprotein particles (CPPs) in serum is a physiological phenomenon fundamental to prevent the rise of ectopic calcifications. CPPs are colloidal hybrid particles made of amorphous calcium phosphate stabilized by a protein, fetuin-A. Since albumin is the most abundant protein present in serum, we aimed at understanding if it plays a synergic action together with fetuin-A towards CPPs formation and stability. CPPs were prepared using a constant fetuin-A concentration (5 µM) and different concentrations of albumin (0-606 µM). The stability of CPPs, their crystallization and sedimentation were followed in situ by combining turbidimetry, precipitation analysis and dynamic light scattering. The morphology was investigated by scanning electron microscopy and cryo-transmission electron microscopy, while crystallinity was inspected by infrared spectroscopy. The effect of albumin on the amount of formed CPPs was also studied, as well as the amount of protein adsorbed on CPPs. We found that albumin is not able to prolong the lifetime of the amorphous phase, but it is very effective in delaying the sedimentation of CPPs after crystallization. Albumin also significantly decreases the amount and size of CPPs when present in their synthetic medium, likely playing a fundamental role in our organism together with fetuin-A towards the stabilization of CPPs.

Effect of Atmospheric Pressure Plasma Jet Treatments on Magnesium Phosphate Cements: Performance, Characterization, and Applications.

Atmospheric pressure plasma treatments are nowadays gaining importance to improve the performance of biomaterials in the orthopedic field. Among those, magnesium phosphate-based cements (MPCs) have recently shown attractive features as bone repair materials. The effect of plasma treatments on such cements, which has not been investigated so far, could represent an innovative strategy to modify MPCs' physicochemical properties and to tune their interaction with cells. MPCs were prepared and treated for 5, 7.5, and 10 min with a cold atmospheric pressure plasma jet. The reactive nitrogen and oxygen species formed during the treatment were characterized. The surfaces of MPCs were studied in terms of the phase composition, morphology, and topography. After a preliminary test in simulated body fluid, the proliferation, adhesion, and osteogenic differentiation of human mesenchymal cells on MPCs were assessed. Plasma treatments induce modifications in the relative amounts of struvite, newberyite, and farringtonite on the surfaces on MPCs in a time-dependent fashion. Nonetheless, all investigated scaffolds show a good biocompatibility and cell adhesion, also supporting osteogenic differentiation of mesenchymal cells.

Polyenylphosphatidylcholine as bioactive excipient in tablets for the treatment of liver fibrosis.

Liver fibrosis is a condition characterized by the accumulation of extracellular matrix (ECM) arising from the myofibroblastic transdifferentiation of hepatic stellate cells (HSCs) occurring as the natural response to liver damage. To date, no pharmacological treatments have been specifically approved for liver fibrosis. We recently reported a beneficial effect of polyenylphosphatidylcholines (PPCs)-rich formulations in reverting fibrogenic features of HSCs. However, unsaturated phospholipids' properties pose a constant challenge to the development of tablets as preferred patient-centric dosage form. Profiting from the advantageous physical properties of the PPCs-rich Soluthin® S 80 M, we developed a tablet formulation incorporating 70% w/w of this bioactive lipid. Tablets were characterized via X-ray powder diffraction, thermogravimetry, and Raman confocal imaging, and passed the major compendial requirements. To mimic physiological absorption after oral intake, phospholipids extracted from tablets were reconstituted as protein-free chylomicron (PFC)-like emulsions and tested on the fibrogenic human HSC line LX-2 and on primary cirrhotic rat hepatic stellate cells (PRHSC). Lipids extracted from tablets and reconstituted in buffer or as PFC-like emulsions exerted the same antifibrotic effect on both activated LX-2 and PRHSCs as observed with plain S 80 M liposomes, showing that the manufacturing process did not interfere with the bioactivity of PPCs.

A study on biorelevant calciprotein particles: Effect of stabilizing agents on the formation and crystallization mechanisms.

HYPOTHESIS: Calciprotein particles (CPPs) are endogenous nanoparticles consisting of hybrid mineral-organic colloidal complexes made of calcium phosphates and Fetuin-A (Fet-A), a protein that in physiological conditions binds to amorphous calcium phosphate forming primary CPP (CPP1). CPP1 can crystallize resulting in hydroxyapatite-based secondary CPP (CPP2) that can eventually precipitate leading to vascular calcifications. The treatment of patients with molecules and ions that delay the amorphous-to-crystalline transition has shown promising results from a clinical perspective, but the study of their mechanism of action has not been thoroughly examined so far. EXPERIMENTS: This work describes the formation and crystallization mechanism of synthetic analogs of endogenous CPPs. The effect of different concentrations of Fet-A and of stabilizing agents (Mg2+, citrate and pyrophosphate) on the features and stability of CPPs was addressed by combining different characterization techniques such as turbidimetry, dynamic light scattering, infrared spectroscopy, and scanning electron microscopy. FINDINGS: The results show that the stabilizing agents display different action mechanisms and are effective to a different extent in preventing the formation of CPPs or delaying their crystallization. Such findings could be of interest to develop effective therapies for vascular calcifications and to deepen the understanding of amorphous calcium phosphate stabilization and its interaction with proteins.

Exploring the effect of Mg2+ substitution on amorphous calcium phosphate nanoparticles.

HYPOTHESIS: The study of Amorphous Calcium Phosphate (ACP) has become a hot topic due to its relevance in living organisms and as a material for biomedical applications. The preparation and characterization of Mg-substituted ACP nanoparticles (AMCP) with tunable Ca/Mg ratio is reported in the present study to address the effect of Mg2+ on their structure and stability. EXPERIMENTS: AMCPs particles were synthesized by precipitation of the precursors from aqueous solutions. The particles were analyzed in terms of morphology, crystallinity, and thermal stability, to get a complete overview of their physico-chemical characteristics. Computational methods were also employed to simulate the structure of ACP clusters at different levels of Mg2+ substitution. FINDINGS: Our results demonstrate that AMCP particles with tunable composition and crystallinity can be obtained. The analysis of the heat-induced crystallization of AMCP shows that particles' stability depends on the degree of Mg2+ substitution in the cluster, as confirmed by computational analyses. The presented results shed light on the effect of Mg2+ on ACP features at different structural levels and may be useful guidelines for the preparation and design of AMCP particles with a specific Ca/Mg ratio.

Effect of Biologically-Relevant Molecules on the Physico-Chemical Properties of Amorphous Magnesium-Calcium Phosphate Nanoparticles.

The recently-discovered endogenous formation of amorphous magnesium-calcium phosphate nanoparticles (AMCPs) in human distal small intestine occurs in a complex environment, which is rich in biologically-relevant molecules and macromolecules that can shape the properties and the stability of these inorganic particles. In this work, we selected as case studies four diverse molecules, which have different properties and are representative of intestinal luminal components, namely butyric acid, lactose, gluten and peptidoglycan. We prepared AMCPs in the presence of these four additives and we investigated their effect on the features of the particles in terms of morphology, porosity, chemical nature and incorporation/adsorption. The combined use of electron microscopy, infrared spectroscopy and thermal analysis showed that while the morphology and microstructure of the particles do not depend on the type of additive present during the synthesis, AMCPs are able to incorporate a significant amount of peptidoglycan, similarly to the process in which they are involved in vivo.

Exploring the interplay of mucin with biologically-relevant amorphous magnesium-calcium phosphate nanoparticles.

HYPOTHESIS: It has been recently shown that, in our organism, the secretions of Ca2+, Mg2+ and phosphate ions lead to the precipitation of amorphous magnesium-calcium phosphate nanoparticles (AMCPs) in the small intestine, where the glycoprotein mucin is one of the most abundant proteins, being the main component of the mucus hydrogel layer covering gut epithelium. Since AMCPs precipitate in vivo in a mucin-rich environment, we aim at studying the effect of this glycoprotein on the formation and features of endogenous-like AMCPs. EXPERIMENTS: AMCPs were synthesized from aqueous solution in the presence of different concentrations of mucin, and the obtained particles were characterised in terms of crystallinity, composition and morphology. Solid State NMR investigation was also performed in order to assess the interplay between mucin and AMCPs at a sub-nanometric level. FINDING: Results show that AMCPs form in the presence of mucin and the glycoprotein is efficiently incorporated in the amorphous particles. NMR indicates the existence of interactions between AMCPs and mucin, revealing how AMCPs in mucin-hybrid nanoparticles affect the features of both proteic and oligosaccharidic portions of the glycoprotein. Considering that the primary function of mucin is the protection of the intestine from pathogens, we speculate that the nature of the interaction between AMCPs and mucin described in the present work might be relevant to the immune system, suggesting a novel type of scenario which could be investigated by combining physico-chemical and biomedical approaches.

Modifying the crystallization of amorphous magnesium-calcium phosphate nanoparticles with proteins from Moringa oleifera seeds.

HYPOTHESIS: Endogenous Amorphous Magnesium-Calcium Phosphates (AMCPs) form in the human body and, besides their biomedical implications, the development of effective stabilization strategies is an open challenge. An interesting approach consists of stabilizing amorphous phosphates with macromolecules that have beneficial effects from a nutritional/medical point of view, for a potential application of the hybrid particles in nutraceutics or drug delivery. EXPERIMENTAL: We investigated the effect of proteins extracted from Moringa oleifera seeds (MO) on the features of synthetic analogs of AMCPs and on their crystallization pathway. The stability of the amorphous phase was studied using infrared spectroscopy and X-ray diffraction. To unravel the effect of the protein on the nano-scale structure of the inorganic particles, we also studied how MO affects the features of the amorphous phase using thermal analysis, small angle X-ray scattering and confocal Raman microscopy. FINDINGS: We observed that MO markedly delays the transition from amorphous to crystalline phosphate in a concentration-dependent fashion. Interestingly, MO not only enhances the lifetime of the amorphous phase, but also influences the type and amount of crystalline material formed. The results are relevant from both a fundamental and an applied perspective, paving the way for the use of these hybrids in the field of nutraceutics and drug delivery.

Unravelling the Effect of Citrate on the Features and Biocompatibility of Magnesium Phosphate-Based Bone Cements.

In the framework of new materials for orthopedic applications, Magnesium Phosphate-based Cements (MPCs) are currently the focus of active research in biomedicine, given their promising features; in this field, the loading of MPCs with active molecules to be released in the proximity of newly forming bone could represent an innovative approach to enhance the in vivo performances of the biomaterial. In this work, we describe the preparation and characterization of MPCs containing citrate, an ion naturally present in bone which presents beneficial effects when released in the proximity of newly forming bone tissue. The cements were characterized in terms of handling properties, setting time, mechanical properties, crystallinity, and microstructure, so as to unravel the effect of citrate concentration on the features of the material. Upon incubation in aqueous media, we demonstrated that citrate could be successfully released from the cements, while contributing to the alkalinization of the surroundings. The cytotoxicity of the materials toward human fibroblasts was also tested, revealing the importance of a fine modulation of released citrate to guarantee the biocompatibility of the material.

The importance of being amorphous: calcium and magnesium phosphates in the human body.

This article focuses on the relevance of amorphous calcium (and magnesium) phosphates in living organisms. Although crystalline calcium phosphate (CaP)-based materials are known to constitute the major inorganic constituents of human hard tissues, amorphous CaP-based structures, often in combination with magnesium, are frequently employed by Nature to build up components of our body and guarantee their proper functioning. After a brief description of amorphous calcium phosphate (ACP) formation mechanism and structure, this paper is focused on the stabilization strategies that can be used to enhance the lifetime of the poorly stable amorphous phase. The various locations of our body in which ACP (pure or in combination with Mg2+) can be found (i.e. bone, enamel, small intestine, calciprotein particles and casein micelles) are highlighted, showing how the amorphous nature of ACP is often of paramount importance for the achievement of a specific physiological function. The last section is devoted to ACP-based biomaterials, focusing on how these materials differ from their crystalline counterparts in terms of biological response.

Formation and properties of amorphous magnesium-calcium phosphate particles in a simulated intestinal fluid.

HYPOTHESIS: The endogenous self-assembly of amorphous magnesium-calcium phosphate (AMCP) nanoparticles in human small intestine is an intriguing and newly-discovered process involved in immune-surveillance mechanisms. The study of nano and microparticles formation in complex media mimicking in vivo conditions contributes to unravel the features of endogenous AMCPs and, from a physico-chemical perspective, to shed light on the effect of biorelevant molecules on the precipitation of AMCPs. EXPERIMENTS: Endogenous-like AMCPs have been synthesized in a commercial simulated intestinal fluid (SIF), which contains biorelevant molecules such as lecithin and taurocholate. The properties of these particles were compared to the features of AMCPs synthesized in water. The stability of the amorphous phase as a function of time, as well as AMCPs' morphology, have been investigated. In particular, the effect of the organic molecules present in the SIF was examined in terms of incorporation in the nano and micro particles and on their nanoscale structure. FINDINGS: Taurocholate and lecithin, present in the SIF, enhance stability of amorphous phase against particles crystallization, and lead to the formation of smaller AMCP aggregates with a rougher surface. They are also incorporated in the inorganic phase, and their self-assembled structure leads to the formation of core-shell nanoparticles.

Tuning the properties of magnesium phosphate-based bone cements: Effect of powder to liquid ratio and aqueous solution concentration.

The use of magnesium phosphate-based cements (MPCs) in the biomedical field has recently come under investigation in the scientific community, as these materials display many intriguing properties in the replacement and/or integration of calcium phosphate-based bone cements; however, the diverse preparation conditions reported in the literature make it difficult to evaluate how the modification of a specific parameter in the preparation of the paste affects the final properties of the material. In this paper, we prepared and characterized MPCs by mixing a tri-magnesium phosphate powder with a solution of di-ammonium hydrogen phosphate, so to form struvite as a final setting product. The powder to liquid ratio and the concentration of the aqueous solution were systematically varied, and their effect on the properties of the final product was studied. The handling properties of the pastes were investigated, as well as the crystallinity and the microstructure; the porosity and compressive strength of the final materials were also assessed. The multi-technique approach allowed us to relate the amount of formed struvite with the properties of the material, and to identify the preparation conditions to be used to obtain a cement with desired features.

Enhanced formation of hydroxyapatites in gelatin/imogolite macroporous hydrogels.

HYPOTHESIS: Gelatin is widely investigated for the fabrication of synthetic scaffolds in bone tissue engineering. Practical limitations to its use are mainly due to the fast dissolution rate in physiological conditions and to the lack of pores with suitable dimensions for cell permeation. The aim of this work is to exploit imogolite clays as nucleation sites for the growth of calcium phosphates in gelatin-based hydrogels and to take advantage of a cryogenic treatment to obtain pores of ∼100µm. EXPERIMENTS: We evaluated the effect of imogolites and a biocompatible cross-linker on the gelatin network in terms of morphology, thermal and rheological behavior. The hydrogels were cryogenically-treated and characterized to investigate the modification of the polymer network, both at the micro- and nano-scale. The samples were mineralized to investigate the effect of imogolites on the formation of calcium phosphates. FINDINGS: The interaction between gelatin, imogolite and cross-linker leads to the modification of the hydrogel structure at the micro-scale, while minor effects are detected at the nano-scale. The cryogenic procedure is successful in generating pores with the desired size, while the presence of imogolites in the hydrogel promotes hydroxyapatites formation. These results demonstrate that imogolites can be effectively employed as functional fillers in polymer-based scaffolds.

Effect of pH and Mg2+ on Amorphous Magnesium-Calcium Phosphate (AMCP) stability.

HYPOTHESIS: Amorphous Magnesium-Calcium Phosphate (AMCP) particles in the distal small intestine have been shown to have a fundamental role in mammals' immune-surveillance mechanisms. Their formation in the gut lumen and their stability against crystallization are expected to depend upon physiological conditions such as pH and [Mg2+]. Knowing the influence of these parameters on AMCP stability would allow to predict the presence and the activity of the particles in physiological or pathological conditions. EXPERIMENTS: We performed the synthesis of AMCP particles at physiological temperature, in phosphate buffer at variable pH from ∼7.0 to 7.4. The stability of the particles was then tested by dispersing them in different conditions of [Mg2+], pH and concentration, so to mimic different biological conditions. The particles were characterized in terms of morphology, crystallinity, chemical composition and porosity. FINDINGS: The characterization showed that we managed to prepare AMCPs with features matching those of the endogenous particles. Both the lifetime of the amorphous phase and the nature of the formed crystalline material were found to depend upon [Mg2+], pH and concentration. This article paves the way for the comprehension of possible dysfunctions of the gut immune-surveillance mechanisms due to imbalances of these physico-chemical parameters.

Transitioning the production of lipidic mesophase-based delivery systems from lab-scale to robust industrial manufacturing following a risk-based quality by design approach augmented by artificial intelligence.

Lipidic mesophase drug carriers have demonstrated the capacity to host and effectively deliver a wide range of active pharmaceutical ingredients, yet they have not been as extensively commercialized as other lipid-based products, such as liposomal delivery systems. Indeed, scientists are primarily focused on investigating the physics of these systems, especially in biological environments. Meanwhile, the production methods remain less advanced, and researchers are still uncertain about how the manufacturing process might affect the quality of formulations. Bringing these products to the market will require an industrial translation process. In this scenario, we have developed a robust strategy to produce lipidic mesophase-based drug delivery systems using a dual-syringe setup. We identified four critical process parameters in the newly developed method (dual-syringe method), in comparison to eight in the standard production method (gold standard), and we defined their optimal limits following a Quality by Design approach. The robustness and versatility of the proposed method were assessed experimentally by incorporating drugs with diverse physicochemical properties and augmented by machine learning which, by predicting the drug release from lipidic mesophases, reduces the formulation development time and costs.