Recent Advances in Alginate-Based Hydrogels for Cell Transplantation Applications.

With its exceptional biocompatibility, alginate emerged as a highly promising biomaterial for a large range of applications in regenerative medicine. Whether in the form of microparticles, injectable hydrogels, rigid scaffolds, or bioinks, alginate provides a versatile platform for encapsulating cells and fostering an optimal environment to enhance cell viability. This review aims to highlight recent studies utilizing alginate in diverse formulations for cell transplantation, offering insights into its efficacy in treating various diseases and injuries within the field of regenerative medicine.

FAP Targeting of Photosensitizer-Loaded Polymersomes for Increased Light-Activated Cell Killing.

As current chemo- and photodynamic cancer therapies are associated with severe side effects due to a lack of specificity and to systemic toxicity, innovative solutions in terms of targeting and controlled functionality are in high demand. Here, we present the development of a polymersome nanocarrier equipped with targeting molecules and loaded with photosensitizers for efficient uptake and light-activated cell killing. Polymersomes were self-assembled in the presence of photosensitizers from a mixture of nonfunctionalized and functionalized PDMS-b-PMOXA diblock copolymers, the latter designed for coupling with targeting ligands. By encapsulation inside the polymersomes, the photosensitizer Rose Bengal was protected, and its uptake into cells was mediated by the nanocarrier. Inhibitor of fibroblast activation protein α (FAPi), a ligand for FAP, was attached to the polymersomes' surface and improved their uptake in MCF-7 breast cancer cells expressing relatively high levels of FAP on their surface. Once internalized by MCF-7, irradiation of Rose Bengal-loaded FAPi-polymersomes generated reactive oxygen species at levels high enough to induce cell death. By combining photosensitizer encapsulation and specific targeting, polymersomes represent ideal candidates as therapeutic nanocarriers in cancer treatment.

Nonclassical Nucleation and Crystallization of LiNbO3 Nanoparticles from the Aqueous Solvothermal Alkoxide Route.

The exact molecular reaction pathway and crystallization mechanisms of LiNbO3 nanoparticles under solvothermal conditions are derived through extensive time- and temperature-resolved experiments allowing to track all the transient molecular and solid species. Starting with a simple mixing of Li/Nb ethoxides, water addition is used to promote condensation after ligand exchange with different co-solvents including alcohols and glycols of variable carbon-chain length. A nonclassical nucleation scheme is first demonstrated after the identification of new octanuclear complexes with a {Li4Nb4O10} core whose solvophobic interactions mediate their aggregation, thus, resulting in a colloidal gel at room-temperature. Upon heating, a more or less frustrated aggregation-mediated crystallization process is then evidenced leading to LiNbO3 nanocrystals of adjustable mean size between 20 and 100 nm. Such a fine control can be attributed to the variable Nb-OR (R = alkoxy/glycoxy ligand) binding interactions at the surface of crystalline intermediates. Demonstration of such a nonclassical nucleation process and crystallization mechanism for LiNbO3 not only sheds light on the entire growth process of multifunctional nanomaterials with non-perovskite crystalline structures, but also opens new avenues for the identification of novel bimetallic oxoclusters involved in the formation of several mixed oxides from the aqueous alkoxide route.

Achieving high hybridization density at DNA biosensor surfaces using branched spacer and click chemistry.

The COVID-19 pandemic has highlighted the necessity to develop fast, highly sensitive and selective virus detection methods. Surface-based DNA-biosensors are interesting candidates for this purpose. Functionalization of solid substrates with DNA must be precisely controlled to achieve the required accuracy and sensitivity. In particular, achieving high hybridization density at the sensing surface is a prerequisite to reach a low limit of detection. We herein describe a strategy based on peptides as anchoring units to immobilize DNA probes at the surface of borosilicate slides. While the coating pathway involves copper-catalyzed click chemistry, a copper-free variation is also reported. The resulting biochips display a high hybridization density (2.9 pmol per cm2) with their targeted gene sequences.

Modulating the Surface Properties of Lithium Niobate Nanoparticles by Multifunctional Coatings Using Water-in-Oil Microemulsions.

Inorganic nanoparticles (NPs) have emerged as promising tools in biomedical applications, owing to their inherent physicochemical properties and their ease of functionalization. In all potential applications, the surface functionalization strategy is a key step to ensure that NPs are able to overcome the barriers encountered in physiological media, while introducing specific reactive moieties to enable post-functionalization. Silanization appears as a versatile NP-coating strategy, due to the biocompatibility and stability of silica, thus justifying the need for robust and well controlled silanization protocols. Herein, we describe a procedure for the silica coating of harmonic metal oxide NPs (LiNbO3, LNO) using a water-in-oil microemulsion (W/O ME) approach. Through optimized ME conditions, the silanization of LNO NPs was achieved by the condensation of silica precursors (TEOS, APTES derivatives) on the oxide surface, resulting in the formation of coated NPs displaying carboxyl (LNO@COOH) or azide (LNO@N3) reactive moieties. LNO@COOH NPs were further conjugated to an unnatural azido-containing small peptide to obtain silica-coated LNO NPs (LNO@Talys), displaying both azide and carboxyl moieties, which are well suited for biomedical applications due to the orthogonality of their surface functional groups, their colloidal stability in aqueous medium, and their anti-fouling properties.

A DNA biosensors-based microfluidic platform for attomolar real-time detection of unamplified SARS-CoV-2 virus.

The emergence of the coronavirus 2019 (COVID-19) arose the need for rapid, accurate and massive virus detection methods to control the spread of infectious diseases. In this work, a device, deployable in non-medical environments, has been developed for the detection of non-amplified SARS-CoV-2 RNA. A SARS-CoV-2 specific probe was designed and covalently immobilized at the surface of glass slides to fabricate a DNA biosensor. The resulting system was integrated in a microfluidic platform, in which viral RNA was extracted from non-treated human saliva, before hybridizing at the surface of the sensor. The formed DNA/RNA duplex was detected in presence of SYBR Green I using an opto-electronic system, based on a high-power LED and a photo multiplier tube, which convert the emitted fluorescence into an electrical signal that can be processed in less than 10 min. The limit of detection of the resulting microfluidic platform reached six copies of viral RNA per microliter of sample (equal to 10 aM) and satisfied the safety margin. The absence of non-specific adsorption and the selectivity for SARS-CoV-2 RNA were established. In addition, the designed device could be applicable for the detection of a variety of viruses by simple modification of the immobilized probe.

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.

Core-Shell Structured Chitosan-Polyethylenimine Nanoparticles for Gene Delivery: Improved Stability, Cellular Uptake, and Transfection Efficiency.

The delivery of nucleic acids relies on vectors that condense and encapsulate their cargo. Especially nonviral gene delivery systems are of increasing interest. However, low transgene expression levels and limited tolerability of these systems remain a challenge. The improvement of nucleic acid delivery using depolymerized chitosan-polyethylenimine DNA complexes (dCS-PEI/DNA) is investigated. The secore complexes are further combined with chitosan-based shells and functionalized with polyethylene glycol (PEG) and cell penetrating peptides. This modular approach allows to evaluate the effect of functional shell components on physicochemical particle characteristics and biological effects. The optimized ternary complex combines a core-dCS-linear PEI/DNA complex with a shell consisting of dCS-PEG-COOH, which results in improved nucleic acid encapsulation, cellular uptake and transfection potency in human hepatoma HuH-7cells and murine primary hepatocytes. Effects on transgene expression are confirmed in wild-type mice following retrograde intrabiliary infusion. After administration of only 100 ng complexed DNA, ternary complexes induced a high reporter gene signal for three days. It is concluded that ternary coreshell structured nanoparticles comprising functionalized chitosan can be used for in vitro andin vivo gene delivery.

Photoresponsive Nanocarriers Based on Lithium Niobate Nanoparticles for Harmonic Imaging and On-Demand Release of Anticancer Chemotherapeutics.

Nanoparticle-based drug delivery systems have the potential for increasing the efficiency of chemotherapeutics by enhancing the drug accumulation at specific target sites, thereby reducing adverse side effects and mitigating patient acquired resistance. In particular, photo-responsive nanomaterials have attracted much interest due to their ability to release molecular cargos on demand upon light irradiation. In some settings, they can also provide complementary information by optical imaging on the (sub)cellular scale. We herein present a system based on lithium niobate harmonic nanoparticles (LNO HNPs) for the decoupled multi-harmonic cell imaging and near-infrared light-triggered delivery of an erlotinib derivative (ELA) for the treatment of epidermal growth factor receptor (EGFR)-overexpressing carcinomas. The ELA cargo was covalently conjugated to the surface of silica-coated LNO HNPs through a coumarinyl photo-cleavable linker, achieving a surface loading of the active molecule of 27 nmol/mg NPs. The resulting nanoconjugates (LNO-CM-ELA NPs) were successfully imaged upon pulsed laser excitation at 1250 nm in EGFR-overexpressing human prostate cancer cells DU145 by detecting the second harmonic emission at 625 nm, in the tissue transparency window. Tuning the laser at 790 nm resulted in the uncaging of the ELA cargo as a result of the second harmonic emission of the inorganic HNP core at 395 nm. This protocol induced a significant growth inhibition in DU145 cells, which was only observed upon specific irradiation at 790 nm, highlighting the promising capabilities of LNO-CM-ELA NPs for theranostic applications.

Islet Encapsulation: New Developments for the Treatment of Type 1 Diabetes.

Islet transplantation is a promising approach for the treatment of type 1 diabetes (T1D). Currently, clinical islet transplantation is limited by allo - and autoimmunity that may cause partial or complete loss of islet function within a short period of time, and long-term immunosuppression is required to prevent rejection. Encapsulation into semipermeable biomaterials provides a strategy that allows nutrients, oxygen and secreted hormones to diffuse through the membrane while blocking immune cells and the like out of the capsule, allowing long-term graft survival and avoiding long-term use of immunosuppression. In recent years, a variety of engineering strategies have been developed to improve the composition and properties of encapsulation materials and to explore the clinical practicality of islet cell transplantation from different sources. In particular, the encapsulation of porcine islet and the co-encapsulation of islet cells with other by-standing cells or active ingredients for promoting long-term functionality, attracted significant research efforts. Hydrogels have been widely used for cell encapsulation as well as other therapeutic applications including tissue engineering, cell carriers or drug delivery. Here, we review the current status of various hydrogel biomaterials, natural and synthetic, with particular focus on islet transplantation applications. Natural hydrophilic polymers include polysaccharides (starch, cellulose, alginic acid, hyaluronic acid, chitosan) and peptides (collagen, poly-L-lysine, poly-L-glutamic acid). Synthetic hydrophilic polymers include alcohol, acrylic acid and their derivatives [poly (acrylic acid), poly (methacrylic acid), poly(acrylamide)]. By understanding the advantages and disadvantages of materials from different sources and types, appropriate materials and encapsuling methods can be designed and selected as needed to improve the efficacy and duration of islet. Islet capsule transplantation is emerging as a promising future treatment for T1D.

Recent Developments on the Use of Nanomaterials for the Treatment of Epilepsy.

Epilepsy affects more than 40 million people worldwide, constituting one of the most debilitating disorders of the Central Nervous System (CNS). It results from an imbalance in the electrical activity of neurons, which is primarily mediated by calcium ions. In many cases, treatment with Antiepileptic Drugs (AEDs) that regulate calcium channel activity results in successful seizure control. However, AEDs frequently cause adverse effects that range in severity from minimal impairment of the CNS to death from aplastic anemia or hepatic failure. Moreover, 30% of epileptic patients show drug-resistant epilepsy and do not respond to any form of medical treatment. In this context, nanotechnology has emerged as an excellent tool to overcome AEDs limitations. Numerous nano-strategies have been proposed as therapeutics and diagnostics for epilepsy through inhibition of different calcium channel types in the brain. In addition, limited brain access of classical AEDs in patients showing refractory epilepsy could be improved through the design of targeted drug delivery nanosystems. This report presents a review of the nanocarriers developed so far that could facilitate the interaction with calcium channels in the brain and the transport of AEDs through the blood-brain-barrier, mapping out a potential future direction in the research of epilepsy treatment.

Design and Preparation of Sensing Surfaces for Capacitive Biodetection.

Despite their high sensitivity and their suitability for miniaturization, biosensors are still limited for clinical applications due to the lack of reproducibility and specificity of their detection performance. The design and preparation of sensing surfaces are suspected to be a cause of these limitations. Here, we first present an updated overview of the current state of use of capacitive biosensors in a medical context. Then, we summarize the encountered strategies for the fabrication of capacitive biosensing surfaces. Finally, we describe the characteristics which govern the performance of the sensing surfaces, along with recent developments that were suggested to overcome their main current limitations.

Chitosan Functionalization: Covalent and Non-Covalent Interactions and Their Characterization.

Chitosan (CS) is a natural biopolymer that has gained great interest in many research fields due to its promising biocompatibility, biodegradability, and favorable mechanical properties. The versatility of this low-cost polymer allows for a variety of chemical modifications via covalent conjugation and non-covalent interactions, which are designed to further improve the properties of interest. This review aims at presenting the broad range of functionalization strategies reported over the last five years to reflect the state-of-the art of CS derivatization. We start by describing covalent modifications performed on the CS backbone, followed by non-covalent CS modifications involving small molecules, proteins, and metal adjuvants. An overview of CS-based systems involving both covalent and electrostatic modification patterns is then presented. Finally, a special focus will be given on the characterization techniques commonly used to qualify the composition and physical properties of CS derivatives.

In Vitro Models for the Study of the Intracellular Activity of Antibiotics.

Intracellular bacteria are poorly responsive to antibiotic treatment. Pharmacological studies are thus needed to determine the antibiotics which are the most potent or effective against intracellular bacteria as well as to explore the reasons for poor bacterial responsiveness. An in vitro pharmacodynamic model is described, consisting of (1) phagocytosis of preopsonized bacteria by eukaryotic cells, (2) elimination of noninternalized bacteria with gentamicin, (3) incubation of infected cells with antibiotics, and (4) determination of surviving bacteria by viable cell counting and normalization of the counts based on sample protein content. The use of strains expressing fluorescent proteins under the control of an inducible promoter allows to follow intracellular bacterial division at the individual level and therefore to monitor bacterial persisters that do not multiply anymore.

Development of Covalent Chitosan-Polyethylenimine Derivatives as Gene Delivery Vehicle: Synthesis, Characterization, and Evaluation.

There is an increasing interest in cationic polymers as important constituents of non-viral gene delivery vectors. In the present study, we developed a versatile synthetic route for the production of covalent polymeric conjugates consisting of water-soluble depolymerized chitosan (dCS; MW 6-9 kDa) and low molecular weight polyethylenimine (PEI; 2.5 kDa linear, 1.8 kDa branched). dCS-PEI derivatives were evaluated based on their physicochemical properties, including purity, covalent bonding, solubility in aqueous media, ability for DNA condensation, and colloidal stability of the resulting polyplexes. They were complexed with non-integrating DNA vectors coding for reporter genes by simple admixing and assessed in vitro using liver-derived HuH-7 cells for their transfection efficiency and cytotoxicity. Using a rational screening cascade, a lead compound was selected (dCS-Suc-LPEI-14) displaying the best balance of biocompatibility, cytotoxicity, and transfection efficiency. Scale-up and in vivo evaluation in wild-type mice allowed for a direct comparison with a commercially available non-viral delivery vector (in vivo-jetPEI). Hepatic expression of the reporter gene luciferase resulted in liver-specific bioluminescence, upon intrabiliary infusion of the chitosan-based polyplexes, which exceeded the signal of the in vivo jetPEI reference formulation by a factor of 10. We conclude that the novel chitosan-derivative dCS-Suc-LPEI-14 shows promise and potential as an efficient polymeric conjugate for non-viral in vivo gene therapy.

Multipotent mesenchymal stromal cells derived from porcine exocrine pancreas improve insulin secretion from juvenile porcine islet cell clusters.

Neonatal and juvenile porcine islet cell clusters (ICC) present an unlimited source for islet xenotransplantation to treat type 1 diabetes patients. We isolated ICC from pancreata of 14 days old juvenile piglets and characterized their maturation by immunofluorescence and insulin secretion assays. Multipotent mesenchymal stromal cells derived from exocrine tissue of same pancreata (pMSC) were characterized for their differentiation potential and ability to sustain ICC insulin secretion in vitro and in vivo. Isolation of ICC resulted in 142 ± 50 × 103 IEQ per pancreas. Immunofluorescence staining revealed increasing presence of insulin-positive beta cells between day 9 and 21 in culture and insulin content per 500IEC of ICC increased progressively over time from 1178.4 ± 450 µg/L to 4479.7 ± 1954.2 µg/L from day 7 to 14, P < .001. Highest glucose-induced insulin secretion by ICC was obtained at day 7 of culture and reached a fold increase of 2.9 ± 0.4 compared to basal. Expansion of adherent cells from the pig exocrine tissue resulted in a homogenous CD90+ , CD34- , and CD45- fibroblast-like cell population and differentiation into adipocytes and chondrocytes demonstrated their multipotency. Insulin release from ICC was increased in the presence of pMSC and dependent on cell-cell contact (glucose-induced fold increase: ICC alone: 1.6 ± 0.2; ICC + pMSC + contact: 3.2 ± 0.5, P = .0057; ICC + pMSC no-contact: 1.9 ± 0.3; theophylline stimulation: alone: 5.4 ± 0.7; pMSC + contact: 8.4 ± 0.9, P = .013; pMSC no-contact: 5.2 ± 0.7). After transplantation of encapsulated ICC using Ca2+ -alginate (alg) microcapsules into streptozotocin-induced diabetic and immunocompetent mice, transient normalization of glycemia was obtained up to day 7 post-transplant, whereas ICC co-encapsulated with pMSC did not improve glycemia and showed increased pericapsular fibrosis. We conclude that pMSC derived from juvenile porcine exocrine pancreas improves insulin secretion of ICC by direct cell-cell contact. For transplantation purposes, the use of pMSC to support beta-cell function will depend on the development of new anti-fibrotic polymers and/or on genetically modified pigs with lower immunogenicity.

Selection of Water-Soluble Chitosan by Microwave-Assisted Degradation and pH-Controlled Precipitation.

In the field of gene therapy, chitosan (CS) gained interest for its promise as a non-viral DNA vector. However, commercial sources of CS lack precise characterization and do not generally reach sufficient solubility in aqueous media for in vitro and in vivo evaluation. As low molecular weight CS showed improved solubility, we investigated the process of CS depolymerization by acidic hydrolysis, using either long time heating at 80 °C or short time microwave-enhanced heating. The resulting depolymerized chitosan (dCS) were analyzed by gel permeation chromatography (GPC) and 1H nuclear magnetic resonance (NMR) to determine their average molecular weight (Mn, Mp and Mw), polydispersity index (PD) and degree of deacetylation (DD). We emphasized the production of water-soluble CS (solubility > 5 mg/mL), obtained in reproducible yield and characteristics, and suitable for downstream functionalization. Optimal microwave-assisted conditions provided dCS with a molecular weight (MW) = 12.6 ± 0.6 kDa, PD = 1.41 ± 0.05 and DD = 85%. While almost never discussed in the literature, we observed the partial post-production aggregation of dCS when exposed to phase changes (from liquid to solid). Repeated cycles of freezing/thawing allowed the selection of dCS fractions which were exempt of crystalline particles formation upon solubilization from frozen samples.

Strategies to Functionalize the Anionic Biopolymer Na-Alginate without Restricting Its Polyelectrolyte Properties.

The natural anionic polyelectrolyte alginate and its derivatives are of particular interest for pharmaceutical and biomedical applications. Most interesting for such applications are alginate hydrogels, which can be processed into various shapes, self-standing or at surfaces. Increasing efforts are underway to functionalize the alginate macromolecules prior to hydrogel formation in order to overcome the shortcomings of purely ionically cross-linked alginate hydrogels that are hindering the progress of several sophisticated biomedical applications. Particularly promising are derivatives of alginate, which allow simultaneous ionic and covalent cross-linking to improve the physical properties and add biological activity to the hydrogel. This review will report recent progress in alginate modification and functionalization with special focus on synthesis procedures, which completely conserve the ionic functionality of the carboxyl groups along the backbone. Recent advances in analytical techniques and instrumentation supported the goal-directed modification and functionalization.

Two-Photon-Triggered Photorelease of Caged Compounds from Multifunctional Harmonic Nanoparticles.

The design of stimuli-responsive nanocarriers has raised much attention to achieve higher local concentration of therapeutics and mitigate the appearance of drug resistance. The combination of imaging properties and controlled photorelease of active molecules within the same nanoconjugate has a great potential for theranostic applications. In this study, a system for NIR light-triggered release of molecular cargos induced by the second harmonic emission from bismuth ferrite harmonic nanoparticles (BFO HNPs) is presented. Silica-coated BFO HNPs were covalently conjugated to a photocaging tether based on coumarin (CM) and l-tryptophan (Trp) as a model molecular cargo. Upon femtosecond pulsed irradiation at 790 nm, Trp was efficiently released from the NP surface in response to the harmonic emission of the nanomaterial at 395 nm. The emitted signal induced the photocleavage of the CM-Trp carbamate linkage resulting in the release of Trp, which was monitored and quantified by ultrahigh performance liquid chromatography-mass spectrometry (UHPLC-MS). While a small fraction of the uncaging process could be attributed to the nonlinear absorption of CM derivatives, the main trigger responsible for Trp release was established as the second harmonic signal from BFO HNPs. This strategy may provide a new way for the application of functionalized HNPs in dual imaging delivery theranostic protocols.

Inhibitor-conjugated harmonic nanoparticles targeting fibroblast activation protein.

The recent progress in the engineering of nanosized inorganic materials presenting tailored physical properties and reactive surface for post-functionalization has opened promising avenues for the use of nanoparticles (NPs) in diagnosis and therapeutic intervention. Surface decoration of metal oxide NPs with ligands modulating circulation time, cellular uptake, affinity and extravasation through active targeting led to efficient cancer specific bioimaging probes. The most relevant cancer biomarkers studied so far include surface and transmembrane cancer cell receptors. More recently, tumor microenvironments and more specifically the fibroblastic element of the tumor stroma have emerged as a valuable target for diagnosis and treatment of several types of cancers. In this study, a low molecular weight ligand targeting fibroblast activation protein α (FAP), which is specifically expressed by activated fibroblasts of the tumor stroma, was synthesized. This ligand demonstrated nanomolar inhibition of FAP with high selectivity with respect to prolyl oligopeptidase (PREP) and dipeptidyl peptidase (DPP) IV, as well as good biocompatibility toward a human lung tissue model. Bismuth ferrite (BFO) harmonic nanoparticles (HNPs) conjugated to this ligand showed target-specific association to FAP as demonstrated by reverse ELISA-type assay using Human Fibroblast Activation Protein alpha/FAP Alexa Fluor® 594-conjugated Antibody and multiphoton multispectral microscopy experiments. These functionalized HNPs may provide new nanocarriers to explore the role of FAP in tumorigenesis and to target the fibroblastic component of the tumor microenvironment.