Synthesis Strategies to Extend the Variety of Alginate-Based Hybrid Hydrogels for Cell Microencapsulation.

The production of hydrogel microspheres (MS) for cell immobilization, maintaining the favorable properties of alginate gels but presenting enhanced performance in terms of in vivo durability and physical properties, is desirable to extend the therapeutic potential of cell transplantation. A novel type of hydrogel MS was produced by straightforward functionalization of sodium alginate (Na-alg) with heterotelechelic poly(ethylene glycol) (PEG) derivatives equipped with either end thiol or 1,2-dithiolane moieties. Activation of the hydroxyl moieties of the alginate backbone in the form of imidazolide intermediate allowed for fast conjugation to PEG oligomers through a covalent carbamate linkage. Evaluation of the modified alginates for the preparation of MS combining fast ionic gelation ability of the alginate carboxylate groups and slow covalent cross-linking provided by the PEG-end functionalities highlighted the influence of the chemical composition of the PEG-grafting units on the physical characteristics of the MS. The mechanical properties of the MS (resistance and shape recovery) and durability of PEG-grafted alginates in physiological environment can be adjusted by varying the nature of the end functionalities and the length of the PEG chains. In vitro cell microencapsulation studies and preliminary in vivo assessment suggested the potential of these hydrogels for cell transplantation applications.

Contribution of polymeric materials to progress in xenotransplantation of microencapsulated cells: a review.

Cell microencapsulation and subsequent transplantation of the microencapsulated cells require multidisciplinary approaches. Physical, chemical, biological, engineering, and medical expertise has to be combined. Several natural and synthetic polymeric materials and different technologies have been reported for the preparation of hydrogels, which are suitable to protect cells by microencapsulation. However, owing to the frequent lack of adequate characterization of the hydrogels and their components as well as incomplete description of the technology, many results of in vitro and in vivo studies appear contradictory or cannot reliably be reproduced. This review addresses the state of the art in cell microencapsulation with special focus on microencapsulated cells intended for xenotransplantation cell therapies. The choice of materials, the design and fabrication of the microspheres, as well as the conditions to be met during the cell microencapsulation process, are summarized and discussed prior to presenting research results of in vitro and in vivo studies. Overall, this review will serve to sensitize medically educated specialists for materials and technological aspects of cell microencapsulation.

Cellular uptake and biocompatibility of bismuth ferrite harmonic advanced nanoparticles.

Bismuth Ferrite (BFO) nanoparticles (BFO-NP) display interesting optical (nonlinear response) and magnetic properties which make them amenable for bio-oriented diagnostic applications as intra- and extra membrane contrast agents. Due to the relatively recent availability of this material in well dispersed nanometric form, its biocompatibility was not known to date. In this study, we present a thorough assessment of the effects of in vitro exposure of human adenocarcinoma (A549), lung squamous carcinoma (NCI-H520), and acute monocytic leukemia (THP-1) cell lines to uncoated and poly(ethylene glycol)-coated BFO-NP in the form of cytotoxicity, haemolytic response and biocompatibility. Our results support the attractiveness of the functional-BFO towards biomedical applications focused on advanced diagnostic imaging. FROM THE CLINICAL EDITOR: Bismuth Ferrite nanoparticles (BFO-NP) have been recently successfully introduced as photodynamic tools and imaging probes. However, how these nanoparticles interact with various cells at the cellular level remains poorly understood. In this study, the authors performed in vitro experiments to assess the effects of uncoated and PEG-coated BFO-NP in the form of cytotoxicity, haemolytic response and biocompatibility.

Convenient synthesis of heterobifunctional poly(ethylene glycol) suitable for the functionalization of iron oxide nanoparticles for biomedical applications.

A straightforward route is proposed for the multi-gram scale synthesis of heterobifunctional poly(ethylene glycol) (PEG) oligomers containing combination of triethyloxysilane extremity for surface modification of metal oxides and amino or azido active end groups for further functionalization. The suitability of these PEG derivatives to be conjugated to nanomaterials was shown by pegylation of ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles (NPs), followed by functionalization with small peptide ligands for biomedical applications.

Novel trehalose-based excipients for stabilizing nebulized anti-SARS-CoV-2 antibody.

COVID-19 is caused by the infection of the lungs by SARS-CoV-2. Monoclonal antibodies, such as sotrovimab, showed great efficiency in neutralizing the virus before its internalization by lung epithelial cells. However, parenteral routes are still the preferred route of administration, even for local infections, which requires injection of high doses of antibody to reach efficacious concentrations in the lungs. Lung administration of antibodies would be more relevant requiring lower doses, thus reducing the costs and the side effects. But aerosolization of therapeutic proteins is very challenging, as the different processes available are harsh and trigger protein aggregation and conformational changes. This decreases the efficiency of the treatment, and can increase its immunogenicity. To address those issues, we developed a series of new excipients composed of a trehalose core, a succinyl side chain and a hydrophobic carbon chain (from 8 to 16 carbons). Succinylation increased the solubility of the excipients, allowing their use at relevant concentrations for protein stabilization. In particular, the excipient with 16 carbons (C16TreSuc) used at 5.6 mM was able to preserve colloidal stability and antigen-binding ability of sotrovimab during the nebulization process. It could also be used as a cryoprotectant, allowing storage of sotrovimab in a lyophilized form during weeks. Finally, we demonstrated that C16TreSuc could be used as an excipient to stabilize antibodies for the treatment against COVID-19, by in vitro and in vivo assays. The presence of C16TreSuc during nebulization preserved the neutralization capacity of sotrovimab against SARS-CoV-2 in vitro; an increase of its efficacy was even observed, compared to the non-nebulized control. The in vivo study also showed the wide distribution of sotrovimab in mice lungs, after nebulization with 5.6 mM of excipient. This work brings a solution to stabilize therapeutic proteins during storage and nebulization, making pulmonary immunotherapy possible in the treatment of COVID-19 and other lung diseases.

Hierarchical Quatsome-RGD Nanoarchitectonic Surfaces for Enhanced Integrin-Mediated Cell Adhesion.

The synthesis and study of the tripeptide Arg-Gly-Asp (RGD), the binding site of different extracellular matrix proteins, e.g., fibronectin and vitronectin, has allowed the production of a wide range of cell adhesive surfaces. Although the surface density and spacing of the RGD peptide at the nanoscale have already shown a significant influence on cell adhesion, the impact of its hierarchical nanostructure is still rather unexplored. Accordingly, a versatile colloidal system named quatsomes, based on fluid nanovesicles formed by the self-assembling of cholesterol and surfactant molecules, has been devised as a novel template to achieve hierarchical nanostructures of the RGD peptide. To this end, RGD was anchored on the vesicle's fluid membrane of quatsomes, and the RGD-functionalized nanovesicles were covalently anchored to planar gold surfaces, forming a state of quasi-suspension, through a long poly(ethylene glycol) (PEG) chain with a thiol termination. An underlying self-assembled monolayer (SAM) of a shorter PEG was introduced for vesicle stabilization and to avoid unspecific cell adhesion. In comparison with substrates featuring a homogeneous distribution of RGD peptides, the resulting hierarchical nanoarchitectonic dramatically enhanced cell adhesion, despite lower overall RGD molecules on the surface. The new versatile platform was thoroughly characterized using a multitechnique approach, proving its enhanced performance. These findings open new methods for the hierarchical immobilization of biomolecules on surfaces using quatsomes as a robust and novel tissue engineering strategy.

Impact of Chemical Composition on the Nanostructure and Biological Activity of α-Galactosidase-Loaded Nanovesicles for Fabry Disease Treatment.

Fabry disease is a rare lysosomal storage disorder characterized by a deficiency of α-galactosidase A (GLA), a lysosomal hydrolase. The enzyme replacement therapy administering naked GLA shows several drawbacks including poor biodistribution, limited efficacy, and relatively high immunogenicity in Fabry patients. An attractive strategy to overcome these problems is the use of nanocarriers for encapsulating the enzyme. Nanoliposomes functionalized with RGD peptide have already emerged as a good platform to protect and deliver GLA to endothelial cells. However, low colloidal stability and limited enzyme entrapment efficiency could hinder the further pharmaceutical development and the clinical translation of these nanoformulations. Herein, the incorporation of the cationic miristalkonium chloride (MKC) surfactant to RGD nanovesicles is explored, comparing two different nanosystems-quatsomes and hybrid liposomes. In both systems, the positive surface charge introduced by MKC promotes electrostatic interactions between the enzyme and the nanovesicles, improving the loading capacity and colloidal stability. The presence of high MKC content in quatsomes practically abolishes GLA enzymatic activity, while low concentrations of the surfactant in hybrid liposomes stabilize the enzyme without compromising its activity. Moreover, hybrid liposomes show improved efficacy in cell cultures and a good in vitro/in vivo safety profile, ensuring their future preclinical and clinical development.

Beneficial Effects of Human Mesenchymal Stromal Cells on Porcine Hepatocyte Viability and Albumin Secretion.

Porcine hepatocytes transplanted during acute liver failure might support metabolic functions until the diseased liver recovers its function. Here, we isolated high numbers of viable pig hepatocytes and evaluated hepatocyte functionality after encapsulation. We further investigated whether coculture and coencapsulation of hepatocytes with human multipotent mesenchymal stromal cells (MSC) are beneficial on hepatocyte function. Livers from 10 kg pigs (n = 9) were harvested, and hepatocytes were isolated from liver suspensions for microencapsulation using alginate and poly(ethylene-glycol)- (PEG-) grafted alginate hydrogels, either alone or in combination with MSC. Viability, albumin secretion, and diazepam catabolism of hepatocytes were measured for one week. 9.2 ± 3.6 × 109 hepatocytes with 95.2 ± 3.1% viability were obtained after isolation. At day 3, free hepatocytes displayed 99% viability, whereas microencapsulation in alginate and PEG-grafted alginate decreased viability to 62% and 48%, respectively. Albumin secretion and diazepam catabolism occurred in free and microencapsulated hepatocytes. Coencapsulation of hepatocytes with MSC significantly improved viability and albumin secretion at days 4 and 8 (p < 0.05). Coculture with MSC significantly increased and prolonged albumin secretion. In conclusion, we established a protocol for isolation and microencapsulation of high numbers of viable pig hepatocytes and demonstrated that the presence of MSC is beneficial for the viability and function of porcine hepatocytes.

Multi-harmonic Imaging in the Second Near-Infrared Window of Nanoparticle-Labeled Stem Cells as a Monitoring Tool in Tissue Depth.

In order to assess the therapeutic potential of cell-based strategies, it is of paramount importance to elaborate and validate tools for monitoring the behavior of injected cells in terms of tissue dissemination and engraftment properties. Here, we apply bismuth ferrite harmonic nanoparticles (BFO HNPs) to in vitro expanded human skeletal muscle-derived stem cells (hMuStem cells), an attractive therapeutic avenue for patients suffering from Duchenne muscular dystrophy (DMD). We demonstrate the possibility of stem cell labeling with HNPs. We also show that the simultaneous acquisition of second- and third-harmonic generation (SHG and THG) from BFO HNPs helps separate their response from tissue background, with a net increase in imaging selectivity, which could be particularly important in pathologic context that is defined by a highly remodelling tissue. We demonstrate the possibility of identifying <100 nm HNPs in depth of muscle tissue at more than 1 mm from the surface, taking full advantage of the extended imaging penetration depth allowed by multiphoton microscopy in the second near-infrared window (NIR-II). Based on this successful assessment, we monitor over 14 days any modification on proliferation and morphology features of hMuStem cells upon exposure to PEG-coated BFO HNPs at different concentrations, revealing their high biocompatibility. Successively, we succeed in detecting individual HNP-labeled hMuStem cells in skeletal muscle tissue after their intramuscular injection.

Drug screening boosted by hyperpolarized long-lived states in NMR.

Transverse and longitudinal relaxation times (T1ρ and T1) have been widely exploited in NMR to probe the binding of ligands and putative drugs to target proteins. We have shown recently that long-lived states (LLS) can be more sensitive to ligand binding. LLS can be excited if the ligand comprises at least two coupled spins. Herein we broaden the scope of ligand screening by LLS to arbitrary ligands by covalent attachment of a functional group, which comprises a pair of coupled protons that are isolated from neighboring magnetic nuclei. The resulting functionalized ligands have longitudinal relaxation times T1((1)H) that are sufficiently long to allow the powerful combination of LLS with dissolution dynamic nuclear polarization (D-DNP). Hyperpolarized weak "spy ligands" can be displaced by high-affinity competitors. Hyperpolarized LLS allow one to decrease both protein and ligand concentrations to micromolar levels and to significantly increase sample throughput.

Deep UV generation and direct DNA photo-interaction by harmonic nanoparticles in labelled samples.

A biophotonics approach based on the nonlinear optical process of second harmonic generation is presented and demonstrated on malignant human cell lines labelled by harmonic nanoparticles. The method enables independent imaging and therapeutic action, selecting each modality by simply tuning the excitation laser wavelength from infrared to visible. In particular, the generation of deep ultraviolet radiation at 270 nm allows direct interaction with nuclear DNA in the absence of photosensitizing molecules.

Harmonic nanocrystals for biolabeling: a survey of optical properties and biocompatibility.

Nonlinear optical nanocrystals have been recently introduced as a promising alternative to fluorescent probes for multiphoton microscopy. We present for the first time a complete survey of the properties of five nanomaterials (KNbO(3), LiNbO(3), BaTiO(3), KTP, and ZnO), describing their preparation and stabilization and providing quantitative estimations of their nonlinear optical response. In the light of their prospective use as biological and clinical markers, we assess their biocompatibility on human healthy and cancerous cell lines. Finally, we demonstrate the great potential for cell imaging of these inherently nonlinear probes in terms of optical contrast, wavelength flexibility, and signal photostability.

Targeted pH-dependent fluorescent activity-based cathepsin probes.

Bifunctional, pH-activatable BODIPY dyes were developed and incorporated in mannose cluster-containing activity-based probes for cysteine proteases. Mannose receptor-dependent uptake of the probes in dendritic cells, followed by trafficking to acidic cellular compartments resulted in fluorescence as seen by live-cell imaging, and subsequent cathepsin inhibition.