PIEZO mechanoreceptors play critical roles in fundamental physiological processes such as proprioception or musculoskeletal biomechanics. However, their complex gating mechanisms and downstream signaling are still not completely understood, mainly due to the lack of effective probing tools. Here, we combine molecular imprinting and magnetic concepts to develop tailor-made nanoswitches enabling wireless targeted actuation of PIEZO1. Two epitopes selected in silico from distinct domains of PIEZO1 were used as templates for synthesizing magnetically responsive molecularly imprinted nanoparticles. These nanoswitches showed sub-nanomolar affinity for their respective epitope and recognized PIEZO1 in endothelial cells, similarly to antibodies. Applying magnetic fields to actuate PIEZO1 through nanoswitches led to increased calcium signaling, Yes-associated protein (YAP) nuclear translocation, and significant changes in the expression of genes related to PIEZO1 activity, implying that they can transduce the stimulus into intracellular signaling effects. Finally, this wireless actuation system proved to be effective in differentially modulating the behavior of mesenchymal stem cells. Remarkably, the selective targeting of each epitope led to contrasting downstream signaling cascades, implying distinct roles for each superstructure domain in the sophisticated function of these channels. Overall, this technology constitutes a promising tool for studying PIEZO-mediated mechanobiology, opening perspectives for harnessing its potential toward therapeutic approaches.

Breast cancer remains the most prevalent malignancy affecting women worldwide. Nanomedicine offers promising strategies to drastically enhance the efficacy of current treatments by enabling spatiotemporal control over cancer diagnostics and therapy. Here, we developed a triple-modal nanocarrier integrating chemo-, photothermal and photodynamic therapies within a single nanoplatform to address the limitations of conventional monotherapies. The nanoparticles were synthesized via polyelectrolyte complexation of sodium alginate and oligochitosan, yielding a biocompatible, non-toxic nanosystem suitable for localized therapies. The designed nanocarriers displayed an average hydrodynamic diameter of 206.1 ± 6.8 nm, with a ζ-potential of -23.8 ± 2.5 mV, features that favor their tumor retention. Additionally, they demonstrated effective encapsulation of doxorubicin and indocyanine green, high photostability, and protection of the loaded drugs from degradation, while remaining responsive to near-infrared stimulation. In vitro, the nanoparticles demonstrate low drug release coupled with substantial cellular uptake and pronounced cytotoxicity against triple-negative breast cancer cells. These results highlight the potential of the designed alginate-oligochitosan-based nanoplatform as a multimodal therapeutic system, capable of additively combining multiple treatment modalities for localized breast cancer therapy, with predicted improvements in therapeutic efficacy and reduced off-target effects, pending validation in further studies.

Tendons heal poorly after injury, and the molecular mechanisms governing tendon healing remain incompletely understood. Among the regulatory molecules involved in tissue repair, microRNAs have emerged as important modulators of gene expression and represent attractive candidates for directing tendon regeneration. In this study, we investigated the in situ delivery of miRNA-16-5p to modulate tendon-associated gene expression in encapsulated human adipose-derived stem cells (hASCs) using anisotropic gelatin methacryloyl (GelMA) hydrogels as tendon-mimetic culture platform. For this, hASCs were encapsulated in the GelMA hydrogels containing miRNA-16-5p mimics or inhibitors formulated as transfection complexes. Hydrogel retention of the miRNA complexes, transfection efficiency, SMAD3 modulation, and the expression of tendon-associated genes were evaluated over 21 days of culture. The hydrogels retained the miRNA complexes during the initial culture period and supported in situ transfection of the encapsulated hASCs. Delivery of miRNA-16-5p mimics resulted in sustained downregulation of SMAD3 expression for up to 14 days and was associated with increased expression of TNMD, TNC, and DCN, together with reduced COL3A1 expression, compared with the miRNA-16-5p inhibitor group. Together, these findings show that, modulation of miRNA-16-5p influences tendon-associated gene expression of the hASCs cultured within a tendon-mimetic microenvironment, and provide a foundation for further studies investigating the role of this miRNA in tendon biology.

Nowadays, cancer remains a global leading cause of death, with therapeutic advances often hindered by drug resistance and adverse side effects. The integration of nanotechnology with immunotherapy has emerged as a promising approach to enhance specificity and efficacy of oncological treatments. A key immunotherapeutic target is the so-called programmed death-ligand 1 (PD-L1), a protein that enables tumors to evade immune surveillance and increase their chemotherapy resistance. Interestingly, RNA interference using small interfering RNA (siRNA) targeting PD-L1, has shown potential in reactivating anti-cancer immune responses. However, efficient delivery of siRNA still faces challenges in terms of stability, cellular uptake, and/or targeted release. In this study, we developed a multifunctional theranostic nanoplatform based on gold nanorods (GNRs) surface-engineered through a layer-by-layer assembly with poly(styrene sulfonate), poly(L-lysine), and hyaluronic acid, to provide enhanced stability and active targeting towards CD44 receptors overexpressed in cancer cells. Within the polymeric multilayers PD-L1 siRNA, doxorubicin and indocyanine green were loaded for multimodal therapeutic activity. The anti-tumor effect, siRNA transfection efficiency and cell death mechanism of the nanoplatform was evaluated on HeLa cells expressing PD-L1 and CD44 and Balb/3T3 fibroblasts. The surface-engineered GNRs-based nanosystem efficiently transfected PD-L1 siRNA and allowed subsequent application of multimodal chemo-, photodynamic and photothermal therapy with enhanced cytotoxicity.

The development of novel approaches to bone regeneration remains a challenge in medicine. For such, the control release of biochemical factors appears key to successfully regulate the regeneration process. In this work, the characterization of methacrylated gelatin (GelMA)-hyaluronic acid (HAMA) hydrogels that incorporated dexamethasone-loaded poly(lactic-co-glycolic acid) nanoparticles (DEX-PLGA NPs) was explored as potential scaffolds for bone tissue regeneration. The DEX-PLGA NPs were synthesized and incorporated into the polymeric hydrogels to achieve a controlled and sustained release of the drug in order to ensure the progressive osteogenic differentiation of adipose-derived mesenchymal stem cells (adMSC) within the scaffold. The physicochemical properties of the NP-loaded hydrogels were not affected by the incorporation of DEX-PLGA NPs. In vitro studies demonstrated that these hybrid hydrogels are biocompatible and presented excellent cell adhesion, proliferation, and differentiation properties promoted by the sustained release of dexamethasone as observed, for example, by the alkaline phosphatase (ALP) assay, which confirmed large concentrations of phosphate ions after the first 14 days of incubation. Furthermore, Alizarin Red S staining corroborated a good mineralization, indicative of effective bone matrix formation.

This work explores the role of CTAB as a surfactant, stabilizer, and modulator in determining the structural characteristics as morphology and size of ZIF-67 nanoMOFs. Furthermore, the influence of these structural changes on the physico-chemical and catalytic properties of the derived nanostructures is analyzed. By modifying the CTAB concentration in the synthesis process, ZIF-67 experienced a morphological transition from perfectly cubic, monodisperse nanoparticles with edge sizes of ca. 80 nm at the highest CTAB concentration used (4 mM) to cross-spike or flower-like structures within the micro size range in its absence, without evident changes in the inherent MOF sodalite-type structure with high thermal stability and crystalline structure. The activation of peroxymonosulphate (PMS) as oxidizing agent by these nanoMOFs to degrade RhB proved to be highly effective achieving very large efficiencies (>95 %). Several factors affecting PMS activation were also evaluated, such as catalyst concentration, pH, temperature, UV irradiation and ultrasonication. Optimal conditions for RhB degradation included slightly acidic pH, high temperature (65 degrees C) and UV light (365 nm). It was also observed that the ZIF-67 nanocube morphology showed lower catalytic activity in comparison with the flower-like structures. ZIF-67 nanoMOFs also showed potential as photocatalysts under visible light irradiation. As the concentration of CTAB increased during the synthesis, the range of allowed bandgap energies progressively decreases. This change correlates with a change in the electronic properties of the nanomaterial, which can affect its ability to absorb and utilize visible light for photocatalytic reactions.

This study delves into the development and characterization of dipalmitoyl phosphatidylcholine (DPPC) liposomes incorporated with gemini surfactant (tetradecamethylene-1,14 bis(dimethyl tetradecyl ammonium bromide); 14-14-14) and atorvastatin, aimed at enhancing drug delivery efficiency for cardiovascular diseases. The integration of gemini surfactants into liposomes is investigated for its potential to improve atorvastatin encapsulation and retention, addressing the drug's poor water solubility and the limitations of conventional liposomal systems. Through a combination of dynamic light scattering (DLS), differential scanning calorimetry (DSC), and molecular dynamics (MD) simulations, the study reveals that the presence of gemini surfactants significantly reduces liposome size and polydispersity, indicative of a more uniform and potentially unilamellar structure. DSC analysis highlights a decrease in transition temperatures and an alteration in transition symmetry, suggesting enhanced stability and a favourable drug release profile at physiological temperatures. MD simulations provide insight into the internalization mechanism of gemini surfactants and atorvastatin within the liposomal bilayer, demonstrating their mutual incorporation facilitated by polar interactions. Spectrophotometry-based retention studies further confirmed that liposomes containing gemini surfactants exhibit superior atorvastatin retention capabilities, nearly doubling the encapsulation efficiency compared to conventional liposomes. This research highlights the promising role of gemini surfactant-incorporated liposomes as an efficient drug delivery platform for cardiovascular therapeutics, offering insights into the molecular interactions and structural dynamics underlying their enhanced performance.

The application of gold nanoparticles in the biomedical field has been widely explored in the last decades owing to their high biocompatibility, easy functionalization, and their structurally highly dependent optical properties. Among the different types of gold-based nanostructures, gold nanoshells (GNSs) are a particularly interesting option due to their complex dual structure, which allows the integration of several functionalities and a wide tunability of their optical response. Despite the remarkable progress made for the design of GNSs with the optimal characteristics for the intended application, the development of nanosystems that display the desired functionalities and preserve them in biological environments while showing effective targeting capability is still a challenge. In this review, the different synthetic routes proposed for the design of GNSs, their most remarkable optical properties, and the most relevant concerns associated with their interaction with biological systems are discussed. The application of these nanostructures for different diagnostic and/or therapeutic purposes is also reviewed. Finally, major remaining barriers to the clinical translation of GNSs and potential future directions of research in this field are critically commented.

In the biological milieu, nanoparticles (NPs) interact with different biomolecules, particularly proteins, leading to the formation of an interfacial corona, which gives rise to a new biological identity affecting NP biodistribution, cytotoxicity and biological fate. The surface coating of NPs plays a key role in regulating such biocorona formation and composition. We here investigated the interactions between bovine serum albumin (BSA) and bovine fibrinogen (FIB) with gold nanorods (Au NRs) bearing different surface coatings (cetyltrimethylammonium bromide, CTAB, and carboxylic acid and amine-terminated polyethylene glycols (PEGs). It was revealed that CTAB-coated NPs interact with both proteins with high affinity (ca. 108-109 M−1) whereas for PEG ones the extent of protein binding decreases thanks to the stealth properties of PEG, but a protein corona is still formed, with binding affinities between 104-106 M−1. In addition, present results indicated that thicknesses of protein coronas and the aggregation behavior of AuNPs were closely related to their surface properties and protein structure. We also found that BSA and FIB underwent different conformational changes upon adsorption depending on the surface-modified Au NRs. Hence, these findings offered important insights into the essence of the interactions between NPs and proteins toward the development of safe and effective nanomaterials.

Most tissues of the human body present hierarchical fibrillar extracellular matrices (ECMs) that have a strong influence over their physicochemical properties and biological behavior. Of great interest is the introduction of this fibrillar structure to hydrogels, particularly due to the water-rich composition, cytocompatibility, and tunable properties of this class of biomaterials. Here, the main bottom-up fabrication strategies for the design and production of hierarchical biomimetic fibrillar hydrogels and their most representative applications in the fields of tissue engineering and regenerative medicine are reviewed. For example, the controlled assembly/arrangement of peptides, polymeric micelles, cellulose nanoparticles (NPs), and magnetically responsive nanostructures, among others, into fibrillar hydrogels is discussed, as well as their potential use as fibrillar-like hydrogels (e.g., those from cellulose NPs) with key biofunctionalities such as electrical conductivity or remote stimulation. Finally, the major remaining barriers to the clinical translation of fibrillar hydrogels and potential future directions of research in this field are discussed.

A major obstacle in biofabrication is replicating the organization of the extracellular matrix and cellular patterns found in anisotropic tissues within bioengineered constructs. While magnetically-assisted 3D bioprinting techniques have the potential to create scaffolds that mimic natural biological structures, they currently lack the ability to accurately control the dispersion of magnetic substances within the bioinks without compromising the fidelity of the intended composite. To overcome this dichotomy, the concepts of magnetically- and matrix-assisted 3D bioprinting are combined here. This method preserves the resolution of printed structures by keeping low viscosity bioinks uncrosslinked during printing, which allows for the arrangement of magnetically-responsive microfibers without compromising the structural integrity of the design. Solidification is induced after the microfibers are arranged in the desired pattern. Furthermore, the precise design of these magnetic microfillers permits the utilization of low levels of inorganic materials and weak magnetic field strengths, which reduces the potential risks that may be associated with their use. The effectiveness of this approach is evaluated in the context of tendon tissue engineering, and the results demonstrate that combining the tendons like anisotropic fibrous microstructure with remote magneto-mechanical stimulation during in vitro maturation provides both biochemical and biophysical cues that effectively guide human adipose-derived stem cells towards a tenogenic phenotype In summary, the developed strategy allows the fabrication of anisotropic high-resolution magnetic composites with remote stimulation functionalities, opening new horizons for tissue engineering applications.Acknowledgments: ERC Grant CoG MagTendon nr 772817, BioChips PoC project nr 10106930, (PD/BD/129403/2017), (CEECIND/01375/2017), (2020.03410.CEECIND), (2022.05526.PTDC), (ED481B2019/025).

Tendons and tendon-to-bone entheses don't usually regenerate after injury, and the hierarchical organization of such tissues makes them challenging sites of study for tissue engineers. In this study, we have tried a novel approach using miRNA and a bioactive bioink to stimulate the regeneration of the enthesis. microRNAs (miRNAs) are short, non-coding sequences of RNA that act as post-transcriptional regulators of gene and protein expression [1]. Mimics or inhibitors of specific miRNAs can be used to restore lost functions at the cell level or improve healing at the tissue level [2,3]. We characterized the healing of a rat patellar enthesis and found that miRNA-16-5p was upregulated in the fibrotic portion of the injured tissue 10 days after the injury. Based on the reported interactions of miRNA-16-5p with the TGF-β pathway via targeting of SMAD3, we aimed to explore the effects of miRNA-16-5p mimics on the tenogenic differentiation of adipose-derived stem cells (ASCs) encapsulated in a bioactive bioink [4,5]. Bioinks with different properties are used for the 3D printing of biomimetic constructs. By integrating cells, materials, and bioactive molecules it is possible to tailor the regenerative capacity of the ink to meet the particular requirements of the tissue to engineer [5]. Here we have encapsulated ASCs in a gelatin-methacryloyl (GelMa) bioink that incorporates miR-16-5p mimics and magnetically responsive microfibers (MRFs). When the bioink is crosslinked in the presence of a magnetic field, the MRFs align unidirectionally to create an anisotropic construct with the ability to promote the tenogenic differentiation of the encapsulated ASCs. Additionally, the obtained GelMA hydrogels retained the encapsulated miRNA probes, which permitted the effective 3D transfection of the ASC and therefore, the regulation of gene expression, allowing to investigate the effects of the miR-16-5p mimics on the tenogenic differentiation of the ASCs in a biomimetic scenario.

Lanthanide-doped up-converting nanoparticles (UCNPs) have emerged as promising biomedical tools in recent years. Most research efforts were devoted to the synthesis of inorganic cores with the optimal physicochemical properties. However, the careful design of UCNPs with the adequate surface coating to optimize their biological performance still remains a significant challenge. Here, we propose the functionalization of UCNPs with four distinct types of surface coatings, which were compared in terms of the provided colloidal stability and resistance to degradation in different biological-relevant media, including commonly avoided analysis in acidic lysosomal-mimicking fluids. Moreover, the influence of the type of particle surface coating on cell cytotoxicity and endocytosis/exocytosis was also evaluated. The obtained results demonstrated that the functionalization of UCNPs with poly(isobutylene-alt-maleic anhydride) grafted with dodecylamine (PMA-g-dodecyl) constitutes an outstanding strategy for their subsequent biomedical application, whereas poly(ethylene glycol) (PEG) coating, although suitable for colloidal stability purposes, hinders extensive cell internalization. Conversely, surface coating with small ligand were found not to be suitable, leading to large degradation degrees of UCNPs. The analysis of particle’ behavior in different biological media and in vitro conditions here performed pretends to help researchers to improve the design and implementation of UCNPs as theranostic nanotools.

Tendon diseases are prevalent health concerns for which current therapies present limited success, in part due to the intrinsically low regenerative ability of tendons. Therefore, tissue engineering presents a potential to improve this outcome. Here, we hypothesize that a concurrent control over both biophysical and biochemical stimuli will boost the tenogenic commitment of stem cells, thus promoting regeneration. To achieve this, we combine molecularly imprinted nanoparticles (MINPs), which act as artificial amplifiers for endogenous growth factor (GF) activity, with bioinspired anisotropic hydrogels2 to manufacture 3D tenogenic constructs. MINPs were solid phase-imprinted using a TGF-β3 epitope as template and their affinity for the target was assessed by SPR and dot blot. Magnetically-responsive microfibers were produced by cryosectioning electrospun meshes containing iron oxide nanoparticles. The constructs were prepared by encapsulating adipose tissue-derived stem cells (ASCs), microfibers, and MINPs within gelatin hydrogels, while aligning the microfibers with an external magnetostatic field during gelation. This allows an effective modulation of hydrogel fibrillar topography, mimicking the native tissue's anisotropic architecture. Cell responses were analyzed by multiplex immunoassay, quantitative polymerase chain reaction, and immunocytochemistry. MINPs showed an affinity for the template comparable to monoclonal antibodies. Encapsulated ASCs acquired an elongated shape and predominant orientation along the alignment direction. Cellular studies revealed that combining MINPs with aligned microfibers increased TGF-β signaling via non-canonical Akt/ERK pathways and upregulated tendon-associated gene expression, contrasting with randomly oriented gels. Immunostaining of tendon-related proteins presented analogous outcomes, corroborating our hypothesis.Our results thus demonstrate that microstructural cues and biological signals synergistically direct stem cell fate commitment, suggesting that this strategy holds potential for improving tendon healing and might be adaptable for other biological tissues. The proposed concept highlights the GF-sequestering ability of MINPs which allows a cost-effective alternative to recombinant GF supplementation, potentially decreasing the translational costs of tissue engineering strategies.Acknowledgements: The authors acknowledge the funding from the European Union's Horizon 2020 under grant No. 772817; from FCT/MCTES for scholarships PD/BD/143039/2018 & COVID/BD/153025/2022 (S.P.B.T.), and PD/BD/129403/2017 (S.M.B.), co-financed by POCH and NORTE 2020, under the Portugal 2020 partnership agreement through the European Social Fund, for contract 2020.03410.CEECIND (R.M.A.D.) and project 2022.05526.PTDC; and from Xunta de Galicia for grant ED481B2019/025 (A.P.).

Tendon injuries and tendinopathies are increasingly prevalent health problems currently lacking effective treatments. Tissue engineering offers promising strategies to boost the low innate regenerative ability of tendons. Within this context, the simultaneous leveraging of both physical and biochemical cues by engineered scaffolding systems can be explored to promote a stronger tenogenic response from stem cells. Here, molecularly imprinted polymeric nanoparticles (MINPs) against transforming growth factor (TGF)-beta 3 are combined with bioinspired anisotropic hydrogels to produce tenogenesis-inductive constructs. MINPs are first solid phase-imprinted against a TGF-beta 3 epitope, achieving an affinity comparable to monoclonal antibodies. MINPs and magnetically-responsive microfibers are then encapsulated together with adipose-derived stem cells within gelatin-based hydrogels, applying a magnetostatic field during gelation to align the microfibers. The created anisotropic microstructure guides cell growth and elongation unidirectionally, while MINPs act as artificial receptors for TGF-beta 3, potentiating its paracrine action in the cellular microenvironment. The combination of both stimuli proves effective at increasing TGF-beta signaling, which promotes the expression of tendon-associated genes and corresponding protein synthesis, suggesting that microstructural cues and biomolecule sequestration act in tandem to direct cell fate commitment. Overall, this system recapitulates several elements of tendon development, constituting a promising strategy for the regeneration of this tissue. Endogenous growth factor sequestration by molecularly imprinted nanoparticles is combined with microcontact guidance by magnetic microfibers inside bioinspired hydrogel systems. Simultaneous control over biochemical and biophysical cues synergistically promotes a more robust tenogenic commitment of adipose tissue-derived stem cells. The principles of scalability and cost-effectiveness here demonstrated can be repurposed toward engineering other tissues in the future. image

The lack of representative in vitro models recapitulating human tendon (patho)physiology is among the major factors hindering consistent progress in the knowledge-based development of adequate therapies for tendinopathy.Here, an organotypic 3D tendon-on-chip model is designed that allows studying the spatiotemporal dynamics of its cellular and molecular mechanisms.Combining the synergistic effects of a bioactive hydrogel matrix with the biophysical cues of magnetic microfibers directly aligned on the microfluidic chip, it is possible to recreate the anisotropic architecture, cell patterns, and phenotype of tendon intrinsic (core) compartment. When incorporated with vascular-like vessels emulating the interface between its intrinsic-extrinsic compartments, crosstalk with endothelial cells are found to drive stromal tenocytes toward a reparative profile. This platform is further used to study adaptive immune cell responses at the onset of tissue inflammation, focusing on interactions between tendon compartment tenocytes and circulating T cells.The proinflammatory signature resulting from this intra/inter-cellular communication induces the recruitment of T cells into the inflamed core compartment and confirms the involvement of this cellular crosstalk in positive feedback loops leading to the amplification of tendon inflammation.Overall, the developed 3D tendon-on-chip provides a powerful new tool enabling mechanistic studies on the pathogenesis of tendinopathy as well as for assessing new therapies.