A novel, microfluidic high-throughput single-cell encapsulation of human bone marrow mesenchymal stromal cells.

The efficacy of stem-cell therapy depends on the ability of the transplanted cells to escape early immunological reactions and to be retained at the site of transplantation. The use of tissue engineering scaffolds or injectable biomaterials as carriers has been proposed, but they still present limitations linked to a reliable manufacturing process, surgical practice and clinical outcomes. Alginate microbeads are potential candidates for the encapsulation of mesenchymal stromal cells with the aim of providing a delivery carrier suitable for minimally-invasive and scaffold-free transplantation, tissue-adhesive properties and protection from the immune response. However, the formation of stable microbeads relies on the cross-linking of alginate with divalent calcium ions at concentrations that are toxic for the cells, making control over the beads' size and a single-cell encapsulation unreliable. The present work demonstrates the efficiency of an innovative, high throughput, and reproducible microfluidic system to produce single-cell, calcium-free alginate coatings of human mesenchymal stromal cells. Among the various conditions tested, visible light and confocal microscopy following staining of the cell nuclei by DAPI showed that the microfluidic system yielded an optimal single-cell encapsulation of 2000 cells/min in 2% w/v alginate microcapsules of reproducible morphology and an average size of 28.2 ± 3.7 µm. The adhesive properties of the alginate microcapsules, the viability of the encapsulated cells and their ability to escape the alginate microcapsule were demonstrated by the relatively rapid adherence of the beads onto tissue culture plastic and the cells' ability to gradually disrupt the microcapsule shell after 24 h and proliferate. To mimic the early inflammatory response upon transplantation, the encapsulated cells were exposed to proliferating macrophages at different cell seeding densities for up to 2 days and the protection effect of the microcapsule on the cells assessed by time-lapse microscopy showing a shielding effect for up to 48 h. This work underscores the potential of microfluidic systems to precisely encapsulate cells by good manufacturing practice standards while favouring cell retention on substrates, viability and proliferation upon transplantation.

Synthetic peptides of IL-1Ra and HSP70 have anti-inflammatory activity on human primary monocytes and macrophages: Potential treatments for inflammatory diseases.

Chronic inflammatory diseases are increasing in developed societies, thus new anti-inflammatory approaches are needed in the clinic. Synthetic peptides complexes can be designed to mimic the activity of anti-inflammatory mediators, in order to alleviate inflammation. Here, we evaluated the anti-inflammatory efficacy of tethered peptides mimicking the interleukin-1 receptor antagonist (IL-1Ra) and the heat-shock protein 70 (HSP70). We tested their biocompatibility and anti-inflammatory activity in vitro in primary human monocytes and differentiated macrophages activated with two different stimuli: the TLR agonists (LPS + IFN-γ) or Pam3CSK4. Our results demonstrate that IL-1Ra and HSP70 synthetic peptides present a satisfactory biocompatible profile and significantly inhibit the secretion of several pro-inflammatory cytokines (IL-6, IL-8, IL-1ß and TNFα). We further confirmed their anti-inflammatory activity when peptides were coated on a biocompatible material commonly employed in surgical implants. Overall, our findings support the potential use of IL-1Ra and HSP70 synthetic peptides for the treatment of inflammatory conditions.

Exploiting the Features of Short Peptides to Recognize Specific Cell Surface Markers.

Antibodies are the macromolecules of choice to ensure specific recognition of biomarkers in biological assays. However, they present a range of shortfalls including a relatively high production cost and limited tissue penetration. Peptides are relatively small molecules able to reproduce sequences of highly specific paratopes and, although they have less biospecificity than antibodies, they offer advantages like ease of synthesis, modifications of their amino acid sequences and tagging with fluorophores and other molecules required for detection. This work presents a strategy to design peptide sequences able to recognize the CD44 hyaluronic acid receptor present in the plasmalemma of a range of cells including human bone marrow stromal mesenchymal cells. The protocol of identification of the optimal amino acid sequence was based on the combination of rational design and in silico methodologies. This protocol led to the identification of two peptide sequences which were synthesized and tested on human bone marrow mesenchymal stromal cells (hBM-MSCs) for their ability to ensure specific binding to the CD44 receptor. Of the two peptides, one binds CD44 with sensitivity and selectivity, thus proving its potential to be used as a suitable alternative to this antibody in conventional immunostaining. In the context of regenerative medicine, the availability of this peptide could be harnessed to functionalize tissue engineering scaffolds to anchor stem cells as well as to be integrated into systems such as cell sorters to efficiently isolate MSCs from biological samples including various cell subpopulations. The data here reported can represent a model for developing peptide sequences able to recognize hBM-MSCs and other types of cells and for their integration in a range of biomedical applications.

Magnetic separation and concentration of Aß 1-42 molecules dispersed at the threshold concentration for Alzheimer's disease diagnosis in clinically-relevant volumes of sample.

BACKGROUND: Alzheimer's disease (AD) is the leading cause of dementia and loss of autonomy in the elderly, implying a progressive cognitive decline and limitation of social activities. The progressive aging of the population is expected to exacerbate this problem in the next decades. Therefore, there is an urgent need to develop quantitative diagnostic methodologies to assess the onset the disease and its progression especially in the initial phases. RESULTS: Here we describe a novel technology to extract one of the most important molecular biomarkers of AD (Aß1-42) from a clinically-relevant volume - 100 µl - therein dispersed in a range of concentrations critical for AD early diagnosis. We demonstrate that it is possible to immunocapture Aß1-42 on 20 nm wide magnetic nanoparticles functionalized with hyperbranced KVLFF aptamers. Then, it is possible to transport them through microfluidic environments to a detection system where virtually all (~ 90%) the Aß1-42 molecules are concentrated in a dense plug of ca.50 nl. The technology is based on magnetic actuation by permanent magnets, specifically designed to generate high gradient magnetic fields. These fields, applied through submillimeter-wide channels, can concentrate, and confine magnetic nanoparticles (MNPs) into a droplet with an optimized shape that maximizes the probability of capturing highly diluted molecular biomarkers. These advancements are expected to provide efficient protocols for the concentration and manipulation of molecular biomarkers from clinical samples, enhancing the accuracy and the sensitivity of diagnostic technologies. CONCLUSIONS: This easy to automate technology allows an efficient separation of AD molecular biomarkers from volumes of biological solutions complying with the current clinical protocols and, ultimately, leads to accurate measurements of biomarkers. The technology paves a new way for a quantitative AD diagnosis at the earliest stage and it is also adaptable for the biomarker analysis of other pathologies.

Bone Regeneration Guided by a Magnetized Scaffold in an Ovine Defect Model.

The reconstruction of large segmental defects still represents a critical issue in the orthopedic field. The use of functionalized scaffolds able to create a magnetic environment is a fascinating option to guide the onset of regenerative processes. In the present study, a porous hydroxyapatite scaffold, incorporating superparamagnetic Fe3O4 nanoparticles (MNPs), was implanted in a critical bone defect realized in sheep metatarsus. Superparamagnetic nanoparticles functionalized with hyperbranched poly(epsilon-Lysine) peptides and physically complexed with vascular endothelial growth factor (VEGF) where injected in situ to penetrate the magnetic scaffold. The scaffold was fixed with cylindrical permanent NdFeB magnets implanted proximally, and the magnetic forces generated by the magnets enabled the capture of the injected nanoparticles forming a VEGF gradient in its porosity. After 16 weeks, histomorphometric measurements were performed to quantify bone growth and bone-to-implant contact, while the mechanical properties of regenerated bone via an atomic force microscopy (AFM) analysis were investigated. The results showed increased bone regeneration at the magnetized interface; this regeneration was higher in the VEGF-MNP-treated group, while the nanomechanical behavior of the tissue was similar to the pattern of the magnetic field distribution. This new approach provides insights into the ability of magnetic technologies to stimulate bone formation, improving bone/scaffold interaction.

The Real-Time Validation of the Effectiveness of Third-Generation Hyperbranched Poly(ɛ-lysine) Dendrons-Modified KLVFF Sequences to Bind Amyloid-ß1-42 Peptides Using an Optical Waveguide Light-Mode Spectroscopy System.

The aggregation of cytotoxic amyloid peptides (Aß1-42) is widely recognised as the cause of brain tissue degeneration in Alzheimer's disease (AD). Indeed, evidence indicates that the deposition of cytotoxic Aß1-42 plaques formed through the gradual aggregation of Aß1-42 monomers into fibrils determines the onset of AD. Thus, distinct Aß1-42 inhibitors have been developed, and only recently, the use of short linear peptides has shown promising results by either preventing or reversing the process of Aß1-42 aggregation. Among them, the KLVFF peptide sequence, which interacts with the hydrophobic region of Aß16-20, has received widespread attention due to its ability to inhibit fibril formation of full-length Aß1-42. In this study, hyperbranched poly-L-lysine dendrons presenting sixteen KLVFF at their uppermost molecular branches were designed with the aim of providing the KLVFF sequence with a molecular scaffold able to increase its stability and of improving Aß1-42 fibril formation inhibitory effect. These high-purity branched KLVFF were used to functionalise the surface of the metal oxide chip of the optical waveguide lightmode spectroscopy sensor showing the more specific, accurate and rapid measurement of Aß1-42 than that detected by linear KLVFF peptides.

Structural analysis of a simplified model reproducing SARS-CoV-2 S RBD/ACE2 binding site.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an RNA virus identified as the cause of the coronavirus outbreak in December 2019 (COVID-19). Like all the RNA viruses, SARS-CoV-2 constantly evolves through mutations in its genome, accumulating 1-2 nucleotide changes every month, giving the virus a selective advantage through enhanced transmissibility, greater pathogenicity, and the possibility of circumventing immunity previously acquired by an individual either by natural infection or by vaccination. Several SARS-CoV-2 variants of concern (VoC) have been identified, among which we find Alpha (Lineage B.1.1.7), Beta (Lineage B.1.351), and Gamma (Lineage P.1) variants. Most of the mutations occur in the spike (S) protein, a surface glycoprotein that plays a crucial role in viral infection; the S protein binds the host cell receptor, the angiotensin-converting enzyme of type 2 (ACE2) via the receptor binding domain (RBD) and catalyzes the fusion of the viral membrane with the host cell. In this work, we present the development of a simplified system that would afford to study the change in the SARS-CoV-2 S RBD/ACE2 binding related to the frequent mutations. In particular, we synthesized and studied the structure of short amino acid sequences, mimicking the two proteins' critical portions. Variations in the residues were easily managed through the one-point alteration of the sequences. Nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopies provide insights into ACE2 and SARS-CoV-2 S RBD structure with its related three variants (Alpha, Beta, and Gamma). Spectroscopy data supported by molecular dynamics lead to the description of an ACE2/RBD binding model in which the effect of a single amino acid mutation in changing the binding of S protein to the ACE2 receptor is predictable.

The composition of cell-based therapies obtained from point-of-care devices/systems which mechanically dissociate lipoaspirate: a scoping review of the literature.

PURPOSE: Cell-based therapies using lipoaspirate are gaining popularity in orthopaedics due to their hypothesised regenerative potential. Several 'point-of-care' lipoaspirate-processing devices/systems have become available to isolate cells for therapeutic use, with published evidence reporting their clinical relevance. However, few studies have analysed the composition of their 'minimally-manipulated' cellular products in parallel, information that is vital to understand the mechanisms by which these therapies may be efficacious. This scoping review aimed to identify devices/systems using mechanical-only processing of lipoaspirate, the constituents of their cell-based therapies and where available, clinical outcomes. METHODS: PRISMA extension for scoping reviews guidelines were followed. MEDLINE, Embase and PubMed databases were systematically searched to identify relevant articles until 21st April 2022. Information relating to cellular composition and clinical outcomes for devices/systems was extracted. Further information was also obtained by individually searching the devices/systems in the PubMed database, Google search engine and contacting manufacturers. RESULTS: 2895 studies were screened and a total of 15 articles (11 = Level 5 evidence) fulfilled the inclusion criteria. 13 unique devices/systems were identified from included studies. All the studies reported cell concentration (cell number regardless of phenotype per millilitre of lipoaspirate) for their devices/systems (range 0.005-21 × 106). Ten reported cell viability (the measure of live cells- range 60-98%), 11 performed immuno-phenotypic analysis of the cell-subtypes and four investigated clinical outcomes of their cellular products. Only two studies reported all four of these parameters. CONCLUSION: When focussing on cell concentration, cell viability and MSC immuno-phenotypic analysis alone, the most effective manual devices/systems were ones using filtration and cutting/mincing. However, it was unclear whether high performance in these categories would translate to improved clinical outcomes. Due to the lack of standardisation and heterogeneity of the data, it was also not possible to draw any reliable conclusions and determine the role of these devices/systems in clinical practice at present. LEVEL OF EVIDENCE: Level V Therapeutic.

New Aß(1-42) ligands from anti-amyloid antibodies: Design, synthesis, and structural interaction.

Alzheimer's disease (AD), is the most common neurodegenerative disorder of the aging population resulting in progressive cognitive and functional decline. Accumulation of amyloid plaques around neuronal cells is considered a critical pathogenetic event and, in most cases, a hallmark of the pathology. In the attempt to identify anti-AD drug candidates, hundreds of molecules targeting Aß peptides have been screened. Peptide molecules have been widely explored, appreciating chemical stability, biocompatibility, and low production cost. More recently, many anti-Aß(1-42) monoclonal antibodies have been developed, given the excellent potential of immunotherapy for treating or preventing AD. Antibodies are versatile ligands that bind a large variety of molecules with high affinity and specificity; however, their extensive therapeutic application is complex and requires huge economic investments. Novel approaches to identify alternative antibody formats are considered with great interest. In this context, taking advantage of the favorable peptide properties and the availability of Aß-antibodies structural data, we followed an innovative research approach to identify short peptide sequences on the model of the binding sites of Aß(1-42)/antibodies. WAibH and SYSTPGK were designed as mimics of solanezumab and aducanumab, respectively. Circular dichroism and nuclear magnetic resonance analysis reveal that the antibody-derived peptides interact with Aß(1-42) in the soluble monomeric form. Moreover, AFM microscopy imaging shows that WAibH and SYSTPGK are capable of controlling the Aß(1-42) aggregation. The strategy to identify WAibH and SYSTPGK is innovative and can be widely applied for new anti-Aß antibody mimicking peptides.

Development of scaffold-free vascularized pancreatic beta-islets in vitro models by the anchoring of cell lines to a bioligand-functionalized gelatine substrate.

Bioengineered pancreatic ß-islets have been widely advocated for the research and treatment of diabetes by offering both suitable cell culture models for the study of the pathology and the testing of new drugs and a therapy in those patients no longer responding to insulin administration and as an alternative to the shortage of donors for organ and islet transplantation. Unlike most of the studies published so far where pancreatic islets of pancreatic ß-cells are encapsulated in hydrogels, this study demonstrate the formation of bioengineered pancreatic islets through cell anchoring to a gelatine-based biomaterial, PhenoDrive-Y, able to mimic the basement membrane of tissues. Through simple culture conditions, PhenoDrive-Y led human pancreatic ß-cell lines and human umbilical endothelial cell lines to form organized structures closely resembling the natural vascularized pancreatic islets. When compared to gelatine, the cultures in presence of PhenoDrive-Y show higher degree of organization in tissue-like structures, a more pronounced endothelial sprouting and higher expression of typical cell markers. Noticeably, when challenged by hyperglycaemic conditions, the cells embedded in the PhenoDrive-Y assembled spheroids responded with higher levels of insulin production. In conclusion, the present work demonstrates the potential of PhenoDrive-Y as substrate for the development of bioengineered vascularized pancreatic islets and to be particularly suitable as a model for in vitro studies and testing of new therapeutics. Graphical abstract.

A multistep in vitro hemocompatibility testing protocol recapitulating the foreign body reaction to nanocarriers.

The development of drug nanocarriers based on polymeric, lipid and ceramic biomaterials has been paving the way to precision medicine, where the delivery of poorly soluble active compounds and personalized doses are made possible. However, the nano-size character of these carriers has been demonstrated to have the potential to elicit pathways of the host response different from those of the same biomaterials when engineered as larger size implants and of the drugs when administered without a carrier. Therefore, a specific regulatory framework needs to be made available that can offer robust scientific insights and provide safety data by reliable tests of these novel nano-devices. In this context, the present work presents a multistep protocol for the in vitro assessment of the hemocompatibility of nanocarriers of different physicochemical properties. Poly (ethyl butyl cyanoacrylate) nanoparticles and lipid-based (LipImage™ 815) nanoparticles of comparable hydrodynamic diameter were tested through a battery of assays using human peripheral blood samples and recapitulating the main pathways of the host response upon systemic administration; i.e., protein interactions, fibrinogen-platelet binding, cytotoxicity, and inflammatory response. The data showed the sensitivity and reproducibility of the methods adopted that were also demonstrated to determine individual variability as well as to discriminate between activation of pathways of inflammation and unintended release of inflammatory signaling caused by loss of cell integrity. Therefore, this multistep testing is proposed as a reliable protocol for nanoparticle development and emerging regulatory frameworks.

A Substrate-Mimicking Basement Membrane Drives the Organization of Human Mesenchymal Stromal Cells and Endothelial Cells Into Perivascular Niche-Like Structures.

Extracellular matrix-derived products (e.g. Matrigel) are widely used for in vitro cell cultures both as two-dimensional (2D) substrates and as three-dimensional (3D) encapsulation gels because of their ability to control cell phenotypes through biospecific cues. However, batch-to-batch variations, poor stability, cumbersome handling, and the relatively high costs strictly limit their use. Recently, a new substrate known as PhenoDrive-Y has been used as 2D coating of tissue culture plastic showing to direct the bone marrow mesenchymal stromal cells (MSCs) toward the formation of 3D spheroids. When organized into 3D spheroids, the MSCs expressed levels of pluripotency markers and of paracrine angiogenic activity higher than those of the MSCs adhering as fibroblast-like colonies on tissue culture plastic. The formation of the spheroids was attributed to the properties of this biomaterial that resemble the main features of the basement membrane by mimicking the mesh structure of collagen IV and by presenting the cells with orderly spaced laminin bioligands. In this study, PhenoDrive-Y was compared to Matrigel for its ability to drive the formation of perivascular stem cell niche-like structures in 2D co-culture conditions of human endothelial cells and adult bone marrow MSCs. Morphological analyses demonstrated that, when compared to Matrigel, PhenoDrive-Y led endothelial cells to sprout into a more consolidated tubular network and that the MSCs nestled as compact spheroids above the anastomotic areas of this network resemble more closely the histological features of the perivascular stem cell niche. A study of the expressions of relevant markers led to the identification of the pathways linking the PhenoDrive-Y biomimicking properties to the acquired histological features, demonstrating the enhanced levels of stemness, renewal potential, predisposition to migration, and paracrine activities of the MSCs.

A Peptide-Based Nanocarrier for an Enhanced Delivery and Targeting of Flurbiprofen into the Brain for the Treatment of Alzheimer's Disease: An In Vitro Study.

Alzheimer's disease (AD) is an age-related disease caused by abnormal accumulation of amyloid-ß in the brain leading to progressive tissue degeneration. Flurbiprofen (FP), a drug used to mitigate the disease progression, has low efficacy due to its limited permeability across the blood-brain barrier (BBB). In a previous work, FP was coupled at the uppermost branching of an ε-lysine-based branched carrier, its root presenting a phenylalanine moiety able to increase the hydrophobicity of the complex and enhance the transport across the BBB by adsorptive-mediated transcytosis (AMT). The present study explores a different molecular design of the FP-peptide delivery system, whereby its root presents an ApoE-mimicking peptide, a targeting ligand that could enhance transport across the BBB by receptor-mediated transcytosis (RMT). The functionalised complex was synthesised using a solid-phase peptide synthesis and characterised by mass spectrometry and FTIR. Cytotoxicity and permeability of this complex across an in vitro BBB model were analysed. Moreover, its activity and degradation to release the drug were investigated. The results revealed successful synthesis and grafting of FP molecules at the uppermost molecular branches of the lysine terminal without observed cytotoxicity. When covalently linked to the nanocarrier, FP was still active on target cells, albeit with a reduced activity, and was released as a free drug upon hydrolysis in a lysosome-mimicking medium. Noticeably, this work shows the high efficiency of RMT-driven FP delivery over delivery systems relying on AMT.

Complexation of Injectable Biphasic Calcium Phosphate with Phosphoserine-Presenting Dendrons with Enhanced Osteoregenerative Properties.

Injectable biphasic calcium phosphates have been proposed as a solution in the treatment of a range of clinical applications including as fillers in the augmentation of osteoporotic bone. To date, various biodegradable natural or synthetic organics have been used as a polymer component of bone materials to increase their cohesiveness. Herein, a novel bone material was developed combining osteoconductive biphasic calcium phosphate (BCP) nanoparticles with phosphoserine-tethered generation 3 poly(epsilon-lysine) dendron (G3-K PS), a class of hyperbranched peptides previously shown to induce biomineralization and stem cell osteogenic differentiation. Strontium was also incorporated into the BCP nanocrystals (SrBCP) to prevent bone resorption. Within 24 h, an antiwashout behavior was observed in G3-K PS-integrated pure BCP group (BCPG3). Moreover, both in vitro tests by relevant cell phenotypes and an in vivo tissue regeneration study by an osteoporotic animal bone implantation showed that the integration of G3-K PS would downregulate Cxcl9 gene and protein expressions, thus enhancing bone regeneration measured as bone mineral density, new bone volume ratio, and trabecular microarchitectural parameters. However, no synergistic effect was found when Sr was incorporated into the BCPG3 bone pastes. Notably, results indicated a concomitant reduction of bone regeneration potential assessed as reduced Runx2 and PINP expression when bone resorptive RANKL and CTX-I levels were reduced by Sr supplementation. Altogether, the results suggest the potential of injectable BCPG3 bone materials in the treatment of osteoporotic bone defects.

Assessment of Cytocompatibility and Anti-Inflammatory (Inter)Actions of Genipin-Crosslinked Chitosan Powders.

Chitosan is a polysaccharide commonly used, together with its derivatives, in the preparation of hydrogel formulations, scaffolds and films for tissue engineering applications. Chitosan can be used as such, but it is commonly stabilized by means of chemical crosslinkers. Genipin is one of the crosslinkers that has been considered that is a crystalline powder extracted from the fruit of Gardenia jasminoides and processed to obtain an aglycon compound. Genipin is gaining interest in biological applications because of its natural origin and anti-inflammatory actions. In this paper, the ability of chitosan-based materials crosslinked with genipin to exert anti-inflammation properties in applications such as bone regeneration was studied. Powders obtained from chitosan-genipin scaffolds have been tested in order to mimic the natural degradation processes occurring during biomaterials implantation in vivo. The results from osteoblast-like cells showed that specific combinations of chitosan and genipin stimulate high permissiveness towards cells, with higher performance than the pure chitosan. In parallel, evidences from monocyte-like cells showed that the crosslinker, genipin, seems to promote slowing of the monocyte-macrophage transition at morphological level. This suggests a sort of modularity of pro-inflammatory versus anti-inflammatory behavior of our chitosan-based biomaterials. Being both the cell types exposed to microscale powders, as an added value our results bring information on the cell-material interactions in the degradative dynamics of chitosan scaffold structures during the physiological resorption processes.

A comparative in vitro study of the effect of biospecific integrin recognition processes and substrate nanostructure on stem cell 3D spheroid formation.

The in vitro study of the properties of the human mesenchymal stem cells as well as their manipulation in culture for clinical purposes depends on the elimination of artefacts caused by the lack of their natural environment. It is now widely accepted that mesenchymal stem cells should be studied when they are organised as 3D spheroids rather than fibroblast-like colonies. Although this can be achieved with the use of some extracellular matrix proteins or by non-adherent conditions these suffer of significant limitations. The recent development of synthetic substrates resembling the physicochemical and biochemical properties of the adult stem cell niche has prompted questions about the role played by nanotopography and receptor-mediated adhesion. In the present paper, the influence of two types of substrates bearing the same nanostructure, but exposing either a non-specific or an integrin-specific binding motif was studied. Carboxybetaine-tethered hyperbranched poly(ɛ-lysine) dendrons showed that the hyperbranched structure was fundamental to induce spheroid formation, but these were forming more slowly, were of reduced size and less stable than those growing on substrates based on the same hyperbranched structures that had been functionalised at their uppermost branching generation by a laminin amino acid sequence, i.e. YIGSR. The study shows that both nanostructure and biorecognition need to be combined to achieve a substrate for stem cell spheroid formation as that observed in vivo in the adult stem cell niche.

Designing and Characterization of a Novel Delivery System for Improved Cellular Uptake by Brain Using Dendronised Apo-E-Derived Peptide.

Neurodegenerative diseases (ND) are characterized by the progressive loss of neuronal structure or function mostly associated with neuronal death. The presence of the blood-brain barrier (BBB) is considered the main obstacle that prevents the penetration of almost all drugs rendering the diseases untreatable. Currently, one of the most promising approaches for drug delivery to the brain is by employing endogenous transcytosis to improve endothelial cell uptake. This study aimed to exploit this potential route of enhanced drug uptake through the design and characterization of low generations lysine dendrons with further functionalization of dendron with ApoE-derived peptide (AEP) ligand to improve cellular uptake and targeting of delivery to the brain. Dendrons and peptide were synthesized using solid phase peptide chemistry and the products were characterized by mass spectrometry and high performance liquid chromatography which confirmed the successful synthesis of dendrons and functionalization with the AEP. Cell viability and lactate dehydrogenase release were conducted to study the cytotoxicity of the materials against an immortalized brain endothelial cell line (bEnd.3) which demonstrated that no toxicity was seen at the concentration range used (up to 400 µM) for up to 48 h incubation. Cellular uptake of the synthesized molecules was examined using confocal microscopy and flow cytometer which clearly showed the cellular uptake of the dendronised carrier systems and that the highest percentage of cellular uptake was achieved with the AEP-functionalized dendron. This study has therefore demonstrated the successful synthesis of dendronised carrier systems with the potential to act as carriers for improved delivery and targeting the brain.

Dendrimeric Poly(Epsilon-Lysine) Delivery Systems for the Enhanced Permeability of Flurbiprofen across the Blood-Brain Barrier in Alzheimer's Disease.

Alzheimer's disease (AD) is a progressive brain disorder and age-related disease characterised by abnormal accumulation of ß-amyloid (Aß). The development of drugs to combat AD is hampered by the lack of therapeutically-active molecules able to cross the blood-brain barrier (BBB). It is agreed that specifically-designed carriers, such as dendrimers, could support the drug penetration across the BBB. The aim of this study was to design biocompatible and biodegradable dendrimeric delivery systems able to carry Flurbiprofen (FP), as drug for AD treatment, across the BBB and liberate it at the target tissue. These dendrons were synthesised using solid-phase peptide synthesis method and characterised by mass spectrometry and fourier-transform infrared spectroscopy (FTIR). The results revealed successful synthesis of dendrons having FP been integrated during the synthesis at their branching ends. Cytotoxicity assays demonstrated the biocompatibility of the delivery systems, whereas HPLC analysis showed high percentages of permeability across an in vitro BBB model for FP-integrated dendrons. Results also revealed the efficiency of drug conjugates on the γ-secretase enzyme in target cells with evidence of eventual drug release by hydrolysis of the carrier. This study demonstrates that the coupling of FP to dendrimeric delivery systems can successfully be achieved during the synthesis of the poly(epsilon-lysine) macromolecules to improve the transport of the active drug across the BBB.

Gadolinium Tagged Osteoprotegerin-Mimicking Peptide: A Novel Magnetic Resonance Imaging Biospecific Contrast Agent for the Inhibition of Osteoclastogenesis and Osteoclast Activity.

The control of osteoblast/osteoclast cross-talk is crucial in the bone remodelling process and provides a target mechanism in the development of drugs for bone metabolic diseases. Osteoprotegerin is a key molecule in this biosignalling pathway as it inhibits osteoclastogenesis and osteoclast activation to prevent run-away bone resorption. This work reports the synthesis of a known osteoprotegerin peptide analogue, YCEIEFCYLIR (OP3-4), and its tagging with a gadolinium chelate, a standard contrast agent for magnetic resonance imaging. The resulting contrast agent allows the simultaneous imaging and treatment of metabolic bone diseases. The gadolinium-tagged peptide was successfully synthesised, showing unaltered magnetic resonance imaging contrast agent properties, a lack of cytotoxicity, and dose-dependent inhibition of osteoclastogenesis in vitro. These findings pave the way toward the development of biospecific and bioactive contrast agents for the early diagnosis, treatment, and follow up of metabolic bone diseases such as osteoporosis and osteosarcoma.