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Negar Hassani Besheli,1 Juul Verbakel,1 Maryam Hosseini,1 Lea Andrée,1 Ben Joosten,2 X Frank Walboomers,1 Alessandra Cambi,2 Fang Yang,1 Sander CG Leeuwenburgh1 1Department of Dentistry – Regenerative Biomaterials, Radboud Institute for Molecular Life Sciences, Radboudumc, Nijmegen, The Netherlands; 2Department of Cell Biology, Radboud Institute for Molecular Life Sciences, Radboudumc, Nijmegen, The NetherlandsCorrespondence: Sander CG Leeuwenburgh, Tel +31 6 15 40 9006, Fax +31 2 43 61 4657, Email sander.leeuwenburgh@radboudumc.nlIntroduction: There has recently been a surge of interest in mesoporous bioactive glass nanoparticles (MBGNs) as multi-functional nanocarriers for application in bone-reconstructive and -regenerative surgery. Their excellent control over their structural and physicochemical properties renders these nanoparticles suitable for the intracellular delivery of therapeutic agents to combat degenerative bone diseases, such as bone infection, or bone cancer. Generally, the therapeutic efficacy of nanocarriers strongly depends on the efficacy of their cellular uptake, which is determined by numerous factors including cellular features and the physicochemical characteristics of nanocarriers, particularly surface charge. In this study, we have systematically investigated the effect of the surface charge of MBGNs doped with copper as a model therapeutic agent on cellular uptake by both macrophages and pre-osteoblast cells involved in bone healing and bone infections to guide the future design of MBGN-based nanocarriers.Methods: Cu-MBGNs with negative, neutral, and positive surface charges were synthesized and their cellular uptake efficiency was assessed. Additionally, the intracellular fate of internalized nanoparticles along with their ability to deliver therapeutic cargo was studied in detail.Results: The results showed that both cell types internalized Cu-MBGNs regardless of their surface charge, indicating that cellular uptake of nanoparticles is a complex process influenced by multiple factors. This similarity in cellular uptake was attributed to the formation of a protein corona surrounding the nanoparticles when exposed to protein-rich biological media, which masks the original nanoparticle surface. Once internalized, the nanoparticles were found to mainly colocalize with lysosomes, exposing them to a more compartmentalized and acidic environment. Furthermore, we verified that Cu-MBGNs released their ionic components (Si, Ca, and Cu ions) in both acidic and neutral environments, leading to the delivery of these therapeutic cargos intracellularly.Conclusion: The effective internalization of Cu-MBGNs and their ability to deliver cargos intracellularly highlight their potential as intracellular delivery nanocarriers for bone-regenerative and -healing applications.Graphical Abstract: Keywords: intracellular delivery, nanocarrier, bioactive glass, copper, surface charge

Colloidal gels assembled from gelatin nanoparticles (GNPs) as particulate building blocks show strong promise to solve challenges in cell delivery and biofabrication, such as low cell survival and limited spatial retention. These gels offer evident advantages to facilitate cell encapsulation, but research on this topic is still limited, which hampers our understanding of the relationship between the physicochemical and biological properties of cell-laden colloidal gels. Human adipose-derived mesenchymal stem cells were successfully encapsulated in gelatin colloidal gels and evaluated their mechanical and biological performance over 7 days. The cells dispersed well within the gels without compromising gel cohesiveness, remained viable, and spread throughout the gels. Cells partially coated with silica were introduced into these gels, which increased their storage moduli and decreased their self-healing capacity after 7 days. This finding demonstrates the ability to modulate gel stiffness by incorporating cells partially coated with silica, without altering the solid content or introducing additional particles. Our work presents an efficient method for cell encapsulation while preserving gel integrity, expanding the applicability of colloidal hydrogels for tissue engineering and bioprinting. Overall, our study contributes to the design of improved cell delivery systems and biofabrication techniques., (© 2024 The Authors. Biotechnology Journal published by Wiley-VCH GmbH.)

Gelatin nanoparticles (GNPs) have been widely studied for a plethora of biomedical applications, but their formation mechanism remains poorly understood, which precludes precise control over their physicochemical properties. This leads to time-consuming parameter adjustments without a fundamental grasp of the underlying mechanism. Here, we analyze and visualize in a time-resolved manner the mechanism by which GNPs are formed during desolvation of gelatin as a function of gelatin molecular weight and type of desolvating agent. Through various analytical and imaging techniques, we unveil a multistage process that is initiated by the formation of primary particles that are ∼18 nm in diameter (wet state). These primary particles subsequently assemble into colloidally stable GNPs with a raspberry-like structure and a hydrodynamic diameter of ∼300 nm. Our results create a basic understanding of the formation mechanism of gelatin nanoparticles, which opens new opportunities for precisely tuning their physicochemical and biofunctional properties.

Messenger RNA (mRNA) is increasingly gaining interest as a modality in vaccination and protein replacement therapy. In regenerative medicine, the mRNA-mediated expression of growth factors has shown promising results. In contrast to protein delivery, successful mRNA delivery requires a vector to induce cellular uptake and subsequent endosomal escape to reach its end destination, the ribosome. Current non-viral vectors such as lipid- or polymer-based nanoparticles have been successfully used to express mRNA-encoded proteins. However, to advance the use of mRNA in regenerative medicine, it is required to assess the compatibility of mRNA with biomaterials that are typically applied in this field. Herein, we investigated the complexation, cellular uptake and maintenance of the integrity of mRNA complexed with gelatin nanoparticles (GNPs). To this end, GNPs with positive, neutral or negative surface charge were synthesized to assess their ability to bind and transport mRNA into cells. Positively charged GNPs exhibited the highest binding affinity and transported substantial amounts of mRNA into pre-osteoblastic cells, as assessed by confocal microscopy using fluorescently labeled mRNA. Furthermore, the GNP-bound mRNA remained stable. However, no expression of mRNA-encoded protein was detected, which is likely related to insufficient endosomal escape and/or mRNA release from the GNPs. Our results indicate that gelatin-based nanomaterials interact with mRNA in a charge-dependent manner and also mediate cellular uptake. These results create the basis for the incorporation of further functionality to yield endosomal release., Competing Interests: R.B. is a co-founder of Mercurna and RiboPro, companies directed at mRNA therapeutics and services.

Implant coatings are frequently applied to modulate tissue response and delivery of drugs. Copper (Cu)-containing coatings on dental implant abutments have been proposed to improve soft tissue integration and reduce the risk for peri-implant infections. However, precise control over Cu loading and release kinetics remains a major challenge. In this study, we introduced a bottom-up coating deposition method based on nanoparticle assembly to allow for local release of Cu ions from implant surfaces. We first doped mesoporous bioactive glass (MBG) nanoparticles with various amounts of Cu. Subsequently, we suspended these Cu-doped MBG (Cu-MBG), Cu-free MBG nanoparticles, or mixtures thereof in chitosan solution and prepared a series of composite coatings on commercially pure titanium disks as model surfaces for transmucosal components of bone implants through electrophoretic deposition (EPD). By changing the Cu-MBG:MBG ratio of the composite coatings, we controlled the Cu release kinetics without changing other coating properties. Human gingival fibroblasts proliferated on the composite coatings except for coatings with the highest amount of Cu, which inhibited their proliferation. The migration rate of human umbilical vein endothelial cells cultured on the composite coatings was highest on coatings containing equal amounts of Cu-MBG and Cu-free MBG. Antibacterial tests confirmed that Cu-containing coatings reduced the growth of Porphyromonas gingivalis up to fivefold compared with uncoated implants. In conclusion, our data indicate that the EPD method is suitable to deposit nanoparticle-based coatings onto dental implants, which enhance endothelial cell migration and reduce bacterial growth. Impact statement Precise control over the release of therapeutic agents remains a major challenge for implant coatings. In this study, we introduce a simple and cost-effective way to tune the release of angiogenic and antibacterial copper ions using the electrophoretic deposition technique. Due to the flexibility and mild processing conditions of this technique, our method can also be used to incorporate other therapeutic agents onto implant surfaces.

To investigate the delivery of next-generation macromolecular drugs, such as engineered proteins and mRNA-containing nanoparticles, there is an increasing push towards the use of physiologically relevant disease models that incorporate human cells and do not face ethical dilemmas associated with animal use. Here, we illustrate the versatility and ease of use of a microfluidic platform for studying drug delivery using high-resolution microscopy in 3D. Using this microfluidic platform, we successfully demonstrate the specific targeting of carbonic anhydrase IX (CAIX) on cells overexpressing the protein in a tumor-mimicking chip system using affibodies, with CAIX-negative cells and non-binding affibodies as controls. Furthermore, we demonstrate this system's feasibility for testing mRNA-containing biomaterials designed to regenerate bone defects. To this end, peptide- and lipid-based mRNA formulations were successfully mixed with colloidal gelatin in microfluidic devices, while translational activity was studied by the expression of a green fluorescent protein. This microfluidic platform enables the testing of mRNA delivery from colloidal biomaterials of relatively high densities, which represents a first important step towards a bone-on-a-chip platform. Collectively, by illustrating the ease of adaptation of our microfluidic platform towards use in distinct applications, we show that our microfluidic chip represents a powerful and flexible way to investigate drug delivery in 3D disease-mimicking culture systems that recapitulate key parameters associated with in vivo drug application.

One of the main challenges of using biomaterials for inducing bone regeneration is the bacterial resistance before complete bone repair. Biomaterials with both antibacterial and bone regeneration properties are more promising for bone repair. In the present study, vascular endothelial growth factor (VEGF) was loaded on silk fibroin nanoparticles (SFNPs) and then embedded in silk scaffold containing vancomycin to form a dual drug release system. The chemical and physical properties of the fabricated structure were confirmed by Fourier transform infrared, scanning electron microscopy, and ζ-potential analysis. The size of spherical SFNPs was ∼92 nm. The release kinetics of vancomycin and VEGF showed that ∼99.56% of vancomycin and ∼14% of VEGF were released during 21 and 28 days, respectively. The bioactivity of VEGF was ∼75%. Disk diffusion test confirmed the ability of this drug delivery system against methicillin-resistant Staphylococcus aureus (MRSA). Moreover, expression of the endothelial markers (FLK-1, vWF, and VE-cadherin), alkaline phosphatase, and matrix mineral production were higher in VEGF loaded groups. Taken together, the results indicated that the fabricated codelivery system was able to simultaneously deliver antibiotic and angiogenic factor, which can be considered as a potential candidate for the treatment of contaminated bone injuries.

The successful treatment of bone infections is a major challenge in the field of orthopedics. There are some common methods for treating bone infections, including systemic antibiotic administration, local nondegradable drug vehicles, and surgical debridement, and each of these approaches has advantages and disadvantages. In the present study, the antibiotic vancomycin (VANCO) was loaded in silk fibroin nanoparticles (SFNPs) and the complexes were then entrapped in silk scaffolds to form sustained drug delivery systems. The release kinetics of VANCO from SFNPs alone and when the SFNPs were entrapped in silk scaffolds were assessed at two different pH values, 4.5 and 7.4, that affected the release profiles of VANCO. Disk diffusion tests performed with pathogens causing osteomyelitis methicillin-resistant Staphylococcus aureus (MRSA) showed antibacterial activity of the released drug at two different pH values. Additionally, injection of 8 × 10 6 CFU MRSA in rat's tibia induced severe osteomyelitis disease. Radiographic and histopathological analyses were performed to evaluate the effectiveness of treatment after 6 weeks. The VANCO-loaded silk fibroin nanoparticles entrapped in scaffolds reduced bone infections at the defected site with better outcomes than the other treatment groups. In conclusion, the delivery system with good biocompatibility and sustained release properties would be appropriate for further study in the context of osteomyelitis disease.