Porous polymer scaffolds are widely investigated as temporary implants in regenerative medicine to repair damaged tissues. While biocompatibility, degradability, mechanical properties comparable to the native tissues and controlled porosity are prerequisite for these scaffolds, their loading with pharmaceutical or biological active ingredients such as growth factors, in particular proteins, opens up new perspective for tissue engineering applications. This implies the development of scaffold loading strategies that minimize the risk of protein denaturation and allow to control their release profile. This work reports on a straightforward method for preparing bioactive dextran-based scaffolds from high internal phase emulsion (HIPE) templates containing poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) serving both as co-stabilizers for the emulsion and nanocarriers for drug or therapeutic protein models. Scaffold synthesis are achieved by photocuring of methacrylated dextran located in the external phase of a HIPE stabilized by the NPs in combination or not with a non-ionic surfactant. Fluorescent labelling of the NPs highlights their integration in the scaffold. The introduction of NPs, and even more so when combined with a surfactant, increases the stability and mechanical properties of the scaffolds. Cell viability tests demonstrate the non-toxic nature of these NPs-loaded scaffolds. The study of the release of a model protein from the scaffold, namely lysozyme, shows that its encapsulation in nanoparticles decreases the release rate and provides additional control over the release profile.

In view of inevitable recurrences despite resection, glioblastoma (GB) is still an unmet clinical need. Dealing with the stromal-cell derived factor 1-alpha (SDF-1 alpha)/CXCR4 axis as a hallmark of infiltrative GB tumors and with the resection cavity situation, the present study described the effects and relevance of a new engineered micro-nanostructured SF-HA-Hep aerogel sponges, made of silk fibroin (SF), hyaluronic acid (HA) and heparin (Hep) and loaded with SDF-1 alpha, to interfere with the GB ecosystem and residual GB cells, attracting and confining them in a controlled area before elimination. 70 pm-pore sponges were designed as an implantable scaffold to trap GB cells. They presented shape memory and fit brain cavities. Histological results after implantation in brain immunocompetent Fischer rats revealed that SFHA-Hep sponges are well tolerated for more than 3 months while moderately and reversibly colonized by immuno-inflammatory cells. The use of human U87MG GB cells overexpressing the CXCR4 receptor (U87MG-CXCR4 + ) and responding to SDF-1 alpha allowed demonstrating directional GB cell attraction and colonization of the device in vitro and in vivo in orthotopic resection cavities in Nude rats. Not modifying global survival, aerogel sponge implantation strongly shaped U87MG-CXCR4 + tumors in cavities in contrast to random infiltrative growth in controls. Overall, those results support the interest of SFHA-Hep sponges as modifiers of the GB ecosystem dynamics acting as "cell meeting rooms" and biocompatible niches whose properties deserve to be considered toward the development of new clinical procedures.

Small interfering RNA (siRNA) holds great potential to treat many difficult-to-treat diseases, but its delivery remains the central challenge. This study aimed at investigating the suitability of polymer-lipid hybrid nanomedicines (HNMeds) as novel siRNA delivery platforms for locoregional therapy of glioblastoma. Two HNMed formulations were developed from poly(lactic-co-glycolic acid) polymer and a cationic lipid: 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) or 3ß-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC-Chol). After characterization of the HNMeds, a model siRNA was complexed onto their surface to form HNMed/siRNA complexes. The physicochemical properties and siRNA binding ability of complexes were assessed over a range of nitrogen-to-phosphate (N/P) ratios to optimize the formulations. At the optimal N/P ratio of 10, complexes effectively bound siRNA and improved its protection from enzymatic degradation. Using the NIH3T3 mouse fibroblast cell line, DOTAP-based HNMeds were shown to possess higher cytocompatibility in vitro over the DC-Chol-based ones. As proof-of-concept, uptake and bioefficacy of formulations were also assessed in vitro on U87MG human glioblastoma cell line expressing luciferase gene. Complexes were able to deliver anti-luciferase siRNA and induce a remarkable suppression of gene expression. Noteworthy, the effect of DOTAP-based formulation was not only about three-times higher than DC-Chol-based one, but also comparable to lipofectamine model transfection reagent. These findings set the basis to exploit this nanosystem for silencing relevant GB-related genes in further in vitro and in vivo studies.

Nanotechnology application in cancer treatment is promising and is likely to quickly spread worldwide in the near future. To date, most scientific studies on nanomaterial development have focused on deepening the attitudes of end users and experts, leaving clinical practice implications unexplored. Neuro-oncology might be a promising field for the application of nanotechnologies, especially for malignant brain tumors with a low-survival rate such as glioblastoma (GBM). As to improving patients' quality of life and life expectancy, innovative treatments are worth being explored. Indeed, it is important to explore clinicians' intention to use experimental technologies in clinical practice. In the present study, we conducted an exploratory review of the literature about healthcare workers' knowledge and personal opinions toward nanomedicine. Our search (i) gives evidence for disagreement between self-reported and factual knowledge about nanomedicine and (ii) suggests the internet and television as main sources of information about current trends in nanomedicine applications, over scientific journals and formal education. Current models of risk assessment suggest time-saving cognitive and affective shortcuts, i.e., heuristics support both laypeople and experts in the decision-making process under uncertainty, whereas they might be a source of error. Whether the knowledge is poor, heuristics are more likely to occur and thus clinicians' opinions and perspectives toward new technologies might be biased.

A drawback in the development of treatments that can reach the retina is the presence of barriers in the eye that restrain compounds from reaching the target. Intravitreal injections hold promise for retinal delivery, but the natural defenses in the vitreous can rapidly degrade or eliminate therapeutic molecules. Injectable hydrogel implants, which act as a reservoir, can allow for long-term drug delivery with a single injection into the eye, but still suffer due to the fast clearance of the released drugs when traversing the vitreous and random diffusion that leads to lower pharmaceutic efficacy. A combination with HA-covered nanoparticles, which can be released from the gel and more readily pass through the vitreous to increase the delivery of therapeutic agents to the retina, represents an advanced and elegant way to overcome some of the limitations in eye drug delivery. In this article, we developed hybrid PLGA-Dotap NPs that, due to their hyaluronic acid coating, can improve in vivo distribution throughout the vitreous and delivery to retinal cells. Moreover, a hydrogel implant was developed to act as a depot for the hybrid NPs to better control and slow their release. These results are a first step to improve the treatment of retinal diseases by protecting and transporting the therapeutic treatment across the vitreous and to improve treatment options by creating a depot system for long-term treatments.

Amorphous calcium carbonate nanoparticles (ACC NPs) are promising multifunctional materials for healthcare applications. Due to their instability in aqueous media, pure ACC NPs for the biomedical field are increasingly synthesised in absolute ethanol, using the ammonia diffusion method (ADM). Although this method presents the advantage of providing stable ACC NPs without additives, it requires the use of pure ethanol as solvent. New insights into the formation mechanisms of ACC NPs in ethanol using gas diffusion are presented in this article. The optimisation of the process according to these findings can increase the mass concentration of ACC NPs by a factor of 3.5. As a result, the amount of ethanol required to produce a target mass of particles is significantly decreased, reducing the ecological impact of the process. The stabilisation of the resulting ACC NPs in aqueous media is achieved by a short-time process using phospholipids based on the ethanol injection method. By using the natural electrostatic affinity of negatively charged materials for the positive surface of ACC NPs in ethanol, we reduced the process time from 24 h to 2 minutes, compared with the closest state of the art, decreasing the operating time and corresponding energy consumption. The process does not require the use of synthetic PEGylated lipids for steric stabilisation. In addition, a natural egg-sourced phospholipid was identified as an efficient stabiliser for the first time. The upscaling of our process was successfully demonstrated using a 50 L reactor for bulk synthesis, as well as a continuous flow reactor for industrial continuous flow production.

Crystalline calcium carbonate (CaCO3) particles have opened exciting perspectives for the design of multifunctional drug carriers with precise control over their growth, size, shape, polymorph, and mesoporosity. These particles, with dimensions varying from 10 nm to several microns, are typically obtained by mixing salts of calcium and carbonate. In this chapter, we give an overview of the research progress on the synthesis and characterization of well-defined CaCO3 particles for biomedical applications, especially as delivery systems for various active substances.

Hyaluronic acid (HA) is a Glycosaminoglycan made of disaccharide units containing N-acetyl-D-glucosamine and glucuronic acid. Its molecular mass can reach 10 MDa and its physiological properties depend on its polymeric property, polyelectrolyte feature and viscous nature. HA is a ubiquitous compound found in almost all biological tissues and fluids. So far, HA grades are produced by biotechnology processes, while in the human organism it is a major component of the extracellular matrix (ECM) in brain tissue, synovial fluid, vitreous humor, cartilage and skin. Indeed, HA is capable of forming hydrogels, polymer crosslinked networks that are very hygroscopic. Based on these considerations, we propose an overview of HA-based scaffolds developed for brain cancer treatment, central and peripheral nervous systems, discuss their relevance and identify the most successful developed systems.

Polymeric, biodegradable, microspheres (MS) presenting a biomimetic surface of extracellular matrix (ECM) proteins are currently used for transporting cells and/or encapsulated proteins for regenerative medicine studies. They can be made of (lactic-co-glycolic acid) (PLGA) or of a more hydrophilic PLGA-P188 (Poloxamer188)-PLGA polymer allowing for the complete release of the therapeutic proteins. They promote stem cell adhesion, cell survival and differentiation after transplantation. Although the biological effectiveness of these microcarriers is established, a detailed understanding of the protein and cell interactions with the microcarrier surface remain unclear due to a lack of information of their surface properties. The aim of this study was to characterize the physicochemical properties of two polymeric MS systems and determine the effect of laminin and poly-d-lysine coated microcarriers on stem cell adhesion, survival and neuronal differentiation. The hydrophobicity and topography of PLGA MS promoted protein adsorption and the stem cells quickly adhered and spread on the surface of these microcarriers. In contrast less proteins adsorbed onto PLGA-P188-PLGA MS and although cells adhered to these microcarriers, they remained round and did not spread on their surface. Despite these early-stage differences, our results suggest that the nature of the MS does not strongly influence the long-term cell behavior. The cells exhibit the same cell number, differentiation profile and ability to secrete ECM molecules regardless of the type of microcarrier used. Likely the ECM molecules that form a microenvironment around both of these 3D microcarrier/cell constructs over time play a role in this converging cell behavior. We have thus furthered our understanding of the physicochemical properties of polymeric cell carriers affecting stem cell behavior to help tailor suitable microcarriers for neuroregenerative applications.

Chemokines such as stromal cell-derived factor-1α (SDF-1α) regulate the migration of cancer cells that can spread from their primary tumor site by migrating up an SDF-1α concentration gradient, facilitating their local invasion and metastasis. Therefore, the implantation of SDF-1α-releasing scaffolds can be a useful strategy to trap cancer cells expressing the CXCR4 receptor. In this work, SDF-1α was encapsulated into poly(lactic-co-glycolic acid) (PLGA)-based nanoparticles and subsequently electrospun with chitosan to produce nanofibrous scaffolds of average fiber diameter of 261 ± 45 nm, intended for trapping glioblastoma (GBM) cells. The encapsulated SDF-1α maintained its biological activity after the electrospinning process as assessed by its capacity to induce the migration of cancer cells. The scaffolds could also provide sustained release of SDF-1α for at least 5 weeks. Using NIH3T3 mouse fibroblasts, human Thp-1 macrophages, and rat primary astrocytes we showed that the scaffolds possessed high cytocompatibility in vitro. Furthermore, a 7-day follow-up of Fischer rats bearing implanted scaffolds demonstrated the absence of adverse effects in vivo. In addition, the nanofibrous structure of the scaffolds provided excellent anchoring sites to support the adhesion of human GBM cells by extension of their pseudopodia. The scaffolds also demonstrated slow degradation kinetics, which may be useful in maximizing the time window for trapping GBM cells. As surgical resection does not permit a complete removal of GBM tumors, our results support the future implantation of these scaffolds into the walls of the resection cavity to evaluate their capacity to attract and trap the residual GBM cells in the brain.

Common modes of transmission of an infectious disease include coughing, sneezing and exchange of body fluids. In a broad sense, what differentiates this definition of an infectious disease from a hereditary disease is that in the case of infectious diseases, a pathogen or causative organism is involved and that pathogen is transmitted or spread through humans, animals or vectors such as insects, while a hereditary disease is inherited from one's parents. Several drugs are available for the treatment of infectious diseases. The drugs are used alone, or more commonly, in combination, in what are known as "drug cocktails". Nanomedicine has the potential to address the challenges faced by therapeutics for infectious diseases, for example reformulating drugs to provide effective therapies in patient-convenient dosage forms and regimens, across the range of therapeutic interventions.

Inhibition of the PI3K/Akt/mTOR signaling pathway represents a potential issue for the treatment of cancer, including glioblastoma. As such, rapamycin that inhibits the mechanistic target of rapamycin (mTOR), the downstream effector of this signaling pathway, is of great interest. However, clinical development of rapamycin has floundered due to the lack of a suitable formulation of delivery systems. In the present study, a novel method for the formulation of safe rapamycin nanocarriers is investigated. A phase inversion process was adapted to prepare lipid nanocapsules (LNCs) loaded with the lipophilic and temperature sensitive rapamycin. Rapamycin-loaded LNCs (LNC-rapa) are ~110 nm in diameter with a low polydispersity index (<0.05) and the zeta potential of about −5 mV. The encapsulation efficiency, determined by spectrophotometry conjugated with filtration/exclusion, was found to be about 69%, which represents 0.6 wt% of loading capacity. Western blot analysis showed that LNC-rapa do not act synergistically with X-ray beam radiation in U87MG glioblastoma model in vitro. Nevertheless, it demonstrated the selective inhibition of the phosphorylation of mTORC1 signaling pathway on Ser2448 at a concentration of 1 μM rapamycin in serum-free medium. Interestingly, cells cultivated in normoxia (21% O2) seem to be more sensitive to mTOR inhibition by rapamycin than those cultivated in hypoxia (0.4% O2). Finally, we also established that mTOR phosphorylation inhibition by LNC-rapa induced a negative feedback through the activation of Akt phosphorylation. This phenomenon was more noticeable after stabilization of HIF-1α in hypoxia.

Extensive research and development work to promote preclinical and clinical use of the carrier has been documented in a decent number of literatures over the last decade. Proteins have the most dynamic and diverse role of any macromolecule in the body. They act as catalysts to biochemical reactions by forming receptors and channels in the membranes. Furthermore, proteins provide intracellular and extracellular scaffolding support and they transport molecules within the cells or from one organ to another. Proteins first emerged as a major class of pharmaceuticals in the 1980s, with a majority of them mainly developed for therapeutics and a small number for diagnostics and vaccines. Recombinant production of proteins is highly favoured over purification of proteins from their native source. Tremendous effort has been invested in cellular engineering to optimize various hosts for protein production and there are many examples in which proteins have been used in therapy successfully.

Polysaccharides have received a lot of attention in biomedical research for their high potential as scaffolds owing to their unique biological properties. Fibrillar scaffolds made of chitosan demonstrated high promise in tissue engineering, especially for skin. As far as bone regeneration is concerned, curdlan (1,3-β-glucan) is particularly interesting as it enhances bone growth by helping mesenchymal stem cell adhesion, by favoring their differentiation into osteoblasts and by limiting the osteoclastic activity. Therefore, we aim to combine both chitosan and curdlan polysaccharides in a new scaffold for bone regeneration. For that purpose, curdlan was electrospun as a blend with chitosan into a fibrillar scaffold. We show that this novel scaffold is biodegradable (8% at two weeks), exhibits a good swelling behavior (350%) and is non-cytotoxic in vitro. In addition, the benefit of incorporating curdlan in the scaffold was demonstrated in a scratch assay that evidences the ability of curdlan to express its immunomodulatory properties by enhancing cell migration. Thus, these innovative electrospun curdlan–chitosan scaffolds show great potential for bone tissue engineering.

Nanoparticles (NPs) based on biocompatible and biodegradable polymers such as poly(lactic-co-glycolic acid) (PLGA) and polycaprolactone (PCL) represent effective systems for systemic drug delivery. Upon injection into the blood circuit, the NP surface is rapidly modified due to adsorption of proteins that form a 'protein corona' (PC). The PC plays an important role in cellular targeting, uptake and NP bio-distribution. Hence, the study of interactions between NPs and serum proteins appears as key for biomedical applications and safety of NPs. In the present work, we report on the comparative protein fluorescence quenching extent, thermodynamics of protein binding and identification of proteins in the soft and hard corona layers of PLGA and PCL NPs. NPs were prepared via a single emulsion-solvent evaporation technique and characterized with respect to size, zeta potential, surface morphology and hydrophobicity. Protein fluorescence quenching experiments were performed against human serum albumin. The thermodynamics of serum protein binding onto the NPs was studied using isothermal titration calorimetry. Semi-quantitative analysis of proteins in the PC layers was conducted using gel electrophoresis and mass spectrometry using human serum. Our results demonstrated the influence of particle hydrophobicity on the thermodynamics of protein binding. Human serum proteins bind to a greater extent and with greater affinity to PCL NPs than PLGA NPs. Several proteins were detected in the hard and soft corona of the NPs, representing their unique proteome fingerprints. Some proteins were unique to the PCL NPs. We anticipate that our findings will assist with rational design of polymeric NPs for effective drug delivery applications.

Glioblastoma (GBM) is the most frequent and aggressive primary tumor of the brain and averages a life expectancy in diagnosed patients of only 15 months. Hence, more effective therapies against this malignancy are urgently needed. Several diseases, including cancer, are featured by high levels of reactive oxygen species (ROS), which are possible GBM hallmarks to target or benefit from. Therefore, the covalent linkage of drugs to ROS-responsive molecules can be exploited aiming for a selective drug release within relevant pathological environments. In this work, we designed a new ROS-responsive prodrug by using Melphalan (MPH) covalently coupled with methoxy polyethylene glycol (mPEG) through a ROS-cleavable group thioketal (TK), demonstrating the capacity to self-assembly into nanosized micelles. Full chemical-physical characterization was conducted on the polymeric-prodrug and proper controls, along with in vitro cytotoxicity assayed on different GBM cell lines and "healthy" astrocyte cells confirming the absence of any cytotoxicity of the prodrug on healthy cells (i.e. astrocytes). These results were compared with the non-ROS responsive counterpart, underlining the anti-tumoral activity of ROS-responsive compared to the non-ROS-responsive prodrug on GBM cells expressing high levels of ROS. On the other hand, the combination treatment with this ROS-responsive prodrug and X-ray irradiation on human GBM cells resulted in an increase of the antitumoral effect, and this might be connected to radiotherapy. Hence, these results represent a starting point for a rationale design of innovative and tailored ROS-responsive prodrugs to be used in GBM therapy and in combination with radiotherapy.

Protein-coated polymer-based microparticles are attractive supports for cell delivery, but the interplay between microparticle properties, protein coating, and cell response is poorly understood. The interest in alternative microparticle formulations increases the need for a better understanding of how functional protein coatings form on different microparticles. In this work, microparticle formulations based on biodegradable polymers [poly (lactic-co-glycolic acid) (PLGA) and the triblock copolymer PLGA-poloxamer-PLGA] were prepared via an emulsion-based process. To explore the impact that the use of a surfactant has on the properties of the microparticles, the emulsion was stabilized by using either a surfactant, poly(vinyl alcohol), or an organic solvent, propylene glycol. Four different types of microparticles were prepared through combinations of the two types of polymers and the two types of stabilizers. The coating of microparticles with proteins/polypeptides such as fibronectin and poly-d-lysine has been demonstrated before and is an integral step for their application as microcarriers, e.g., for cell delivery; however, the impact of the microparticles’ surface chemical properties on the formation (prevalence and distribution) of the mixed polypeptide coatings and the influence on subsequent cell attachment remain to be elucidated. Using a colocalization analysis approach on ToF-SIMS images of protein-coated microparticles, we show that the use of propyleneglycol over PVA as well as the substitution of PLGA by the triblock copolymer resulted in enhanced protein adsorption. Furthermore, if propyleneglycol is used, the substitution of PLGA with the triblock copolymer leads to increased stem cell adhesion.

This work aims to design biocompatible aerogel sponges that can host and control the release of stromal cellderived factor-1 alpha (SDF-1 alpha or CXCL12), a key protein for applications ranging from regenerative medicine to cancer therapy (notably for neural tissues). Miscibility of silk fibroin (SF) and hyaluronic acid (HA) was investigated by means of fluorescence and scanning electron microscopy to identify processing conditions. Series of freeze-dried sponges were prepared by associating and cross-linking within the same 3D structure, HA, SF, poly-c-lysine (PLL) and heparin (hep). Aerogel sponges presented high swelling degree and porosity (similar to 90 %), adequate mean pore diameter (ca. 60 mu m) and connectivity for welcoming cells, and a soft texture close to that of the brain (6-13 kPa Young's Modulus). Addition of SF yielded sponges with slower biodegradation. SF-HA and SF-HA-hep sponges retained 75 % and 93 % of the SDF-1 alpha respectively after 7 days and were found to be cytocompatible in vitro.