Advances in Microfluidics for the Implementation of Liquid Biopsy in Clinical Routine.

In recent years, we have seen major advances in the field of liquid biopsy and its implementation in the clinic, mainly driven by breakthrough developments in the area of molecular biology. New developments have seen an integration of microfluidics and also biosensors in liquid biopsy systems, bringing advantages in terms of cost, sensitivity and automation. Without a doubt, the next decade will bring the clinical validation and approval of these combined solutions, which is expected to be crucial for the wide implementation of liquid biopsy systems in clinical routine.

Ibuprofen-loaded fibrous patches-taming inhibition at the spinal cord injury site.

It is now widely accepted that a therapeutic strategy for spinal cord injury (SCI) demands a multi-target approach. Here we propose the use of an easily implantable bilayer polymeric patch based on poly(trimethylene carbonate-co-ε-caprolactone) (P(TMC-CL)) that combines physical guidance cues provided by electrospun aligned fibres and the delivery of ibuprofen, as a mean to reduce the inhibitory environment at the lesion site by taming RhoA activation. Bilayer patches comprised a solvent cast film onto which electrospun aligned fibres have been deposited. Both layers were loaded with ibuprofen. In vitro release (37°C, in phosphate buffered saline) of the drug from the loaded scaffolds under sink condition was found to occur in the first 24 h. The released ibuprofen was shown to retain its bioactivity, as indicated by the reduction of RhoA activation when the neuronal-like cell line ND7/23 was challenged with lysophosphatidic acid. Ibuprofen-loaded P(TMC-CL) bilayer scaffolds were successfully implanted in vivo in a dorsal hemisection rat SCI model mediating the reduction of RhoA activation after 5 days of implantation in comparison to plain P(TMC-CL) scaffolds. Immunohistochemical analysis of the tissue shows ßIII tubulin positive cells close to the ibuprofen-loaded patches further supporting the use of this strategy in the context of regeneration after a lesion in the spinal cord.

Quality of Life of Colorectal Cancer Survivors: Mapping the Key Indicators by Expert Consensus and Measures for Their Assessment.

Quality of life (QoL) assessments are integral to cancer care, yet their effectiveness in providing essential information for supporting survivors varies. This study aimed to elucidate key indicators of QoL among colorectal cancer survivors from the perspective of healthcare professionals, and to evaluate existing QoL questionnaires in relation to these indicators. Two studies were conducted: a Delphi study to identify key QoL indicators and a scoping review of questionnaires suitable for colorectal cancer survivors. Fifty-four healthcare professionals participated in the Delphi study's first round, with 25 in the second. The study identified two primary QoL domains (physical and psychological) and 17 subdomains deemed most critical. Additionally, a review of 12 questionnaires revealed two instruments assessing the most important general domains. The findings underscored a misalignment between existing assessment tools and healthcare professionals' clinical priorities in working with colorectal cancer survivors. To enhance support for survivors' QoL, efforts are needed to develop instruments that better align with the demands of routine QoL assessment in clinical practice.

It takes two to remyelinate: A bioengineered platform to study astrocyte-oligodendrocyte crosstalk and potential therapeutic targets in remyelination.

The loss of the myelin sheath insulating axons is the hallmark of demyelinating diseases. These pathologies often lead to irreversible neurological impairment and patient disability. No effective therapies are currently available to promote remyelination. Several elements contribute to the inadequacy of remyelination, thus understanding the intricacies of the cellular and signaling microenvironment of the remyelination niche might help us to devise better strategies to enhance remyelination. Here, using a new in vitro rapid myelinating artificial axon system based on engineered microfibres, we investigated how reactive astrocytes influence oligodendrocyte (OL) differentiation and myelination ability. This artificial axon culture system enables the effective uncoupling of molecular cues from the biophysical properties of the axons, allowing the detailed study of the astrocyte-OL crosstalk. Oligodendrocyte precursor cells (OPCs) were cultured on poly(trimethylene carbonate-co-ε-caprolactone) copolymer electrospun microfibres that served as surrogate axons. This platform was then combined with a previously established tissue engineered glial scar model of astrocytes embedded in 1 % (w/v) alginate matrices, in which astrocyte reactive phenotype was acquired using meningeal fibroblast conditioned medium. OPCs were shown to adhere to uncoated engineered microfibres and differentiate into myelinating OL. Reactive astrocytes were found to significantly impair OL differentiation ability, after six and eight days in a co-culture system. Differentiation impairment was seen to be correlated with astrocytic miRNA release through exosomes. We found significantly reduction on the expression of pro-myelinating miRNAs (miR-219 and miR-338) and an increase in anti-myelinating miRNA (miR-125a-3p) content between reactive and quiescent astrocytes. Additionally, we show that OPC differentiation inhibition could be reverted by rescuing the activated astrocytic phenotype with ibuprofen, a chemical inhibitor of the small rhoGTPase RhoA. Overall, these findings show that modulating astrocytic function might be an interesting therapeutic avenue for demyelinating diseases. The use of these engineered microfibres as an artificial axon culture system will enable the screening for potential therapeutic agents that promote OL differentiation and myelination while providing valuable insight on the myelination/remyelination processes.

Establishing an Expert Consensus on Key Indicators of the Quality of Life among Breast Cancer Survivors: A Modified Delphi Study.

(1) Background: The needs of cancer survivors are often not reflected in practice. One of the main barriers of the use of patient-reported outcomes is associated with data collection and the interpretation of patient-reported outcomes (PROs) due to a multitude of instruments and measuring approaches. The aim of the study was to establish an expert consensus on the relevance and key indicators of quality of life in the clinical practice of breast cancer survivors. (2) Methods: Potential indicators of the quality of life of breast cancer survivors were extracted from the established quality of life models, depicting survivors' perspectives. The specific domains and subdomains of quality of life were evaluated in a two-stage online Delphi process, including an international and multidisciplinary panel of experts. (3) Results: The first round of the Delphi process was completed by 57 and the second by 37 participants. A consensus was reached for the Physical and Psychological domains, and on eleven subdomains of quality of life. The results were further supported by the additional ranking of importance of the subdomains in the second round. (4) Conclusions: The current findings can serve to optimize the use of instruments and address the challenges related to data collection and interpretation as the facilitators of the adaption in routine practice.

HER2 Expression in Circulating Tumour Cells Isolated from Metastatic Breast Cancer Patients Using a Size-Based Microfluidic Device.

HER2 is a prognostic and predictive biomarker in breast cancer, normally assessed in tumour biopsy and used to guide treatment choices. Circulating tumour cells (CTCs) escape the primary tumour and enter the bloodstream, exhibiting great metastatic potential and representing a real-time snapshot of the tumour burden. Liquid biopsy offers the unique opportunity for low invasive sampling in cancer patients and holds the potential to provide valuable information for the clinical management of cancer patients. This study assesses the performance of the RUBYchip™, a microfluidic system for CTC capture based on cell size and deformability, and compares it with the only FDA-approved technology for CTC enumeration, CellSearch®. After optimising device performance, 30 whole blood samples from metastatic breast cancer patients were processed with both technologies. The expression of HER2 was assessed in isolated CTCs and compared to tissue biopsy. Results show that the RUBYchipTM was able to isolate CTCs with higher efficiency than CellSearch®, up to 10 times more, averaging all samples. An accurate evaluation of different CTC subpopulations, including HER2+ CTCs, was provided. Liquid biopsy through the use of the RUBYchipTM in the clinic can overcome the limitations of histological testing and evaluate HER2 status in patients in real-time, helping to tailor treatment during disease evolution.

Dissolving microneedles for the delivery of peptides - Towards tolerance-inducing vaccines.

Tolerance inducing vaccines have re-appeared in recent years as a mean to re-establish immunological tolerance in the context of autoimmune disease. In the case of multiple sclerosis, several myelin-related peptides have been identified. The use of microneedles (MNs) allows the painless administration of molecules to the epidermal and intradermal space. This approach has been considered particularly promising in the scope of vaccination as the skin represents an immunologically super-active organ. This work explores the preparation of a MN patch that can deliver immunologically active peptides foreseeing the establishment of tolerance in the context of multiple sclerosis. A new MN design was achieved by microfabrication. The patches are composed of a dense MN array containing 33 × 33 needles with 200 or 125 µm diameter and height around 600 µm. Polymeric MNs composed by poly(vinyl alcohol), poly(vinyl pyrrolidone) and chitosan were successfully obtained, replicating the silicon masters morphology. The polymer MN patches showed to perforate pig skin, reaching more than 400 µm depth of penetration when assessed using agarose as a model for the skin viscoelastic properties. The MNs with 200 µm diameter showed improved mechanical properties in comparison to 125 µm diameter MNs. The presence of chitosan in the MN structure was explored and found not to affect mechanical properties or significantly alter the drug loading or release profile. The immunomodulatory peptide associated with the proteolipid protein PLP139-151 was loaded in 200 µm diameter MN patch and it is released in physiological conditions at therapeutic doses of the peptide, putting forward this strategy to integrate a new tolerance-inducing therapy for multiple sclerosis successfully.

Poly(d,l-lactide-co-glycolide) (PLGA) Nanoparticles Loaded with Proteolipid Protein (PLP)-Exploring a New Administration Route.

The administration of specific antigens is being explored as a mean to re-establish immunological tolerance, namely in the context of multiple sclerosis (MS). PLP139-151 is a peptide of the myelin's most abundant protein, proteolipid protein (PLP), which has been identified as a potent tolerogenic molecule in MS. This work explored the encapsulation of the peptide into poly(lactide-co-glycolide) nanoparticles and its subsequent incorporation into polymeric microneedle patches to achieve efficient delivery of the nanoparticles and the peptide into the skin, a highly immune-active organ. Different poly(d,l-lactide-co-glycolide) (PLGA) formulations were tested and found to be stable and to sustain a freeze-drying process. The presence of trehalose in the nanoparticle suspension limited the increase in nanoparticle size after freeze-drying. It was shown that rhodamine can be loaded in PLGA nanoparticles and these into poly(vinyl alcohol)-poly(vinyl pyrrolidone) microneedles, yielding fluorescently labelled structures. The incorporation of PLP into the PLGA nanoparticles resulted in nanoparticles in a size range of 200 µm and an encapsulation efficiency above 20%. The release of PLP from the nanoparticles occurred in the first hours after incubation in physiological media. When loading the nanoparticles into microneedle patches, structures were obtained with 550 µm height and 180 µm diameter. The release of PLP was detected in PLP-PLGA.H20 nanoparticles when in physiological media. Overall, the results show that this strategy can be explored to integrate a new antigen-specific therapy in the context of multiple sclerosis, providing minimally invasive administration of PLP-loaded nanoparticles into the skin.

Rapidly dissolving microneedles for the delivery of cubosome-like liquid crystalline nanoparticles with sustained release of rapamycin.

In this study, we developed a system for the transdermal delivery and controlled release of the hydrophobic immunosuppressive drug rapamycin, foreseeing an application in psoriasis treatment. To do so, rapamycin was encapsulated in phytantriol-based cubosome-like liquid crystalline nanoparticles stabilized with pluronic F127. The final mass percent composition of the lipid nanoparticles was 0.25% phytantriol, 0.1% pluronic F127, 4.75% ethanol and 94.9% water. These particles showed a rapamycin encapsulation efficiency above 95% and a sustained in vitrodrug release profile throughout 14 days. Subsequently the rapamycin-carrying particles were incorporated into rapidly dissolving microneedle patches composed of a polymeric matrix of poly(vinylpyrrolidone) and poly(vinyl alcohol). Confocal microscopy allowed to infer the preferential distribution of the cubosome-like particles at the tip and baseplate of the microneedles. The fabricated microneedles showed successful piercing and deposition of the loaded cubosome-like particles on a skin-mimicking agarose gel. Finally, the rapamycin-loaded cubosome-like particles showed antiproliferative activity in natural killer cells in vitro. The results here presented show the potential of the developed system to deliver cubosome-like particles into the skin and promote the sustained release of rapamycin in the context of immunomodulation.

A Perspective on Microneedle-Based Drug Delivery and Diagnostics in Paediatrics.

Microneedles (MNs) have been extensively explored in the literature as a means to deliver drugs in the skin, surpassing the stratum corneum permeability barrier. MNs are potentially easy to produce and may allow the self-administration of drugs without causing pain or bleeding. More recently, MNs have been investigated to collect/assess the interstitial fluid in order to monitor or detect specific biomarkers. The integration of these two concepts in closed-loop devices holds the promise of automated and minimally invasive disease detection/monitoring and therapy. These assure low invasiveness and, importantly, open a window of opportunity for the application of population-specific and personalised therapies.

Nano- and micro-based systems for immunotolerance induction in multiple sclerosis.

It is estimated that more than 2.5 million individuals worldwide have multiple sclerosis (MS). MS is an autoimmune neurodegenerative disease resulting from the destruction of the myelin sheath that enwraps axons driven by an immune cell attack to the central nervous system. Current therapeutic programs for MS focus in immunosuppression and more recently in the use of immunomodulatory molecules. These therapeutic approaches provide significant improvements in the management of the disease, but are frequently associated with an increased susceptibility of opportunistic infection. In this commentary, we highlight the application of nano and micro-technologies as emerging and innovative solutions for MS therapy with the potential to restore immune homeostasis via antigen-specific interactions. Furthermore, we propose and discuss the usage of a minimally invasive approach, namely microneedle patches, as a new therapeutic route. Microneedle patches for the delivery of specific antigens to restore immunotolerance in the context of multiple sclerosis.

Ibuprofen-loaded poly(trimethylene carbonate-co-ε-caprolactone) electrospun fibres for nerve regeneration.

The development of scaffolds that combine the delivery of drugs with the physical support provided by electrospun fibres holds great potential in the field of nerve regeneration. Here it is proposed the incorporation of ibuprofen, a well-known non-steroidal anti-inflammatory drug, in electrospun fibres of the statistical copolymer poly(trimethylene carbonate-co-ε-caprolactone) [P(TMC-CL)] to serve as a drug delivery system to enhance axonal regeneration in the context of a spinal cord lesion, by limiting the inflammatory response. P(TMC-CL) fibres were electrospun from mixtures of dichloromethane (DCM) and dimethylformamide (DMF). The solvent mixture applied influenced fibre morphology, as well as mean fibre diameter, which decreased as the DMF content in solution increased. Ibuprofen-loaded fibres were prepared from P(TMC-CL) solutions containing 5% ibuprofen (w/w of polymer). Increasing drug content to 10% led to jet instability, resulting in the formation of a less homogeneous fibrous mesh. Under the optimized conditions, drug-loading efficiency was above 80%. Confocal Raman mapping showed no preferential distribution of ibuprofen in P(TMC-CL) fibres. Under physiological conditions ibuprofen was released in 24 h. The release process being diffusion-dependent for fibres prepared from DCM solutions, in contrast to fibres prepared from DCM-DMF mixtures where burst release occurred. The biological activity of the drug released was demonstrated using human-derived macrophages. The release of prostaglandin E2 to the cell culture medium was reduced when cells were incubated with ibuprofen-loaded P(TMC-CL) fibres, confirming the biological significance of the drug delivery strategy presented. Overall, this study constitutes an important contribution to the design of a P(TMC-CL)-based nerve conduit with anti-inflammatory properties.

Bridging the lesion-engineering a permissive substrate for nerve regeneration.

Biomaterial-based strategies to restore connectivity after lesion at the spinal cord are focused on bridging the lesion and providing an favourable substrate and a path for axonal re-growth. Following spinal cord injury (SCI) a hostile environment for neuronal cell growth is established by the activation of multiple inhibitory mechanisms that hamper regeneration to occur. Implantable scaffolds can provide mechanical support and physical guidance for axon re-growth and, at the same time, contribute to alleviate the hostile environment by the in situ delivery of therapeutic molecules and/or relevant cells. Basic research on SCI has been contributing with the description of inhibitory mechanisms for regeneration as well as identifying drugs/molecules that can target inhibition. This knowledge is the background for the development of combined strategies with biomaterials. Additionally, scaffold design is significantly evolving. From the early simple hollow conduits, scaffolds with complex architectures that can modulate cell fate are currently being tested. A number of promising pre-clinical studies combining scaffolds, cells, drugs and/or nucleic acids are reported in the open literature. Overall, it is considered that to address the multi-factorial inhibitory environment of a SCI, a multifaceted therapeutic approach is imperative. The progress in the identification of molecules that target inhibition after SCI and its combination with scaffolds and/or cells are described and discussed in this review.

The role of the surface on microglia function: implications for central nervous system tissue engineering.

In tissue engineering, it is well accepted that a scaffold surface has a decisive impact on cell behaviour. Here we focused on microglia-the resident immune cells of the central nervous system (CNS)-and on their response to poly(trimethylene carbonate-co-ε-caprolactone) (P(TMC-CL)) fibrous and flat surfaces obtained by electrospinning and solvent cast, respectively. This study aims to provide cues for the design of instructive surfaces that can contribute to the challenging process of CNS regeneration. Cell morphology was evidently affected by the substrate, mirroring the surface main features. Cells cultured on flat substrates presented a round shape, while cells with elongated processes were observed on the electrospun fibres. A higher concentration of the pro-inflammatory cytokine tumour necrosis factor-α was detected in culture media from microglia on fibres. Still, astrogliosis is not exacerbated when astrocytes are cultured in the presence of microglia-conditioned media obtained from cultures in contact with either substrate. Furthermore, a significant percentage of microglia was found to participate in the process of myelin phagocytosis, with the formation of multinucleated giant cells being observed only on films. Altogether, the results presented suggest that microglia in contact with the tested substrates may contribute to the regeneration process, putting forward P(TMC-CL) substrates as supporting matrices for nerve regeneration.

Imidazole-grafted chitosan-mediated gene delivery: in vitro study on transfection, intracellular trafficking and degradation.

AIM: To study the mechanism of transfection mediated by imidazole-grafted chitosan (CHimi) nanoparticles, to propose new strategies to control and improve the expression of a delivered gene in the context of regenerative medicine. METHODS: Biochemical and microscopy methods were used to establish transfection efficiency and nanoparticle intracellular trafficking. The role of CHimi and degree of N-acetylation (DA) on transfection was explored. RESULTS: CHimi was found to promote the expression of a delivered gene during a minimum 7-day period. Additionally, the production of a protein of interest could be upheld by consecutive transfections, without compromising cell viability. Transfection was found to be a time-dependent process, requiring CHimi-DNA complex disassembling. The DA was found to have an impact on transfection kinetics in line with the observation that the rate of lysozyme-mediated nanoparticle degradation increases with the polymer DA. CONCLUSION: The adjustment of the CH degradation rate can be used as a tool for tuning the expression of a gene delivered by CH-based nanoparticle systems.

Chitosan-based gene delivery vectors targeted to the peripheral nervous system.

A non-toxic, targeted, simple and efficient system that can specifically transfect peripheral sensorial neurons can pave the way towards the development of new therapeutics for the treatment of peripheral neuropathies. In this study chitosan (CH), a biodegradable polymer, was used as the starting material in the design of a multicomponent vector targeted to the peripheral nervous system (PNS). Polycation-DNA complexes were optimized using imidazole- and thiol-grafted CH (CHimiSH), in order to increase transfection efficiency and allow the formation of ligand conjugated nanocomplexes, respectively. The 50 kDa non-toxic fragment from the tetanus toxin (HC), shown to interact specifically with peripheral neurons and undergo retrograde transport, was grafted to the binary complex via a bi-functional poly(ethylene glycol) (HC-PEG) reactive for the thiol moieties present in the complex surface. The targeting of the developed nanocomplexes was assessed by means of internalization and transfection studies in the ND7/23 (neuronal) vs. NIH 3T3 (fibroblast) cell lines. Targeted transfection was further confirmed in dorsal root ganglion dissociated primary cultures. A versatile, multi-component nanoparticle system that successfully targets and transfects neuronal cell lines, as well as dorsal root ganglia (DRG) primary neuron cultures was obtained for the 1.0 (w/w) HC-PEG/DNA formulation.

Targeted gene delivery into peripheral sensorial neurons mediated by self-assembled vectors composed of poly(ethylene imine) and tetanus toxin fragment c.

A simple, safe and efficient system that can specifically transfect peripheral sensorial neurons can bring new answers to address peripheral neuropathies. A multi-component non-viral gene delivery vector targeted to peripheral nervous system cells was developed, using poly(ethylene imine) (PEI) as starting material. A binary DNA/polymer complex based on thiolated PEI (PEISH) was optimized, considering complex size and zeta potential and the ability to transfect a sensorial neuron cell line (ND7/23). The 50 kDa non-toxic fragment from tetanus toxin (HC), which has been previously shown to interact specifically with peripheral neurons and to undergo retrograde transport, was grafted to the complex core via a bifunctional PEG (HC-PEG) reactive for the thiol moieties present in the complex surface. Several formulations of HC-PEG ternary complexes were tested for targeting, by assessing the extent of cellular internalization and levels of transfection, in both the ND7/23 and NIH 3 T3 (fibroblast) cell lines. Targeted gene transfer to the neuronal cell line was observed for the complex formulations containing 5 and 7.5 microg of HC-PEG. Finally, our results demonstrate that the developed ternary vectors are able to transfect primary cultures of dorsal root ganglion dissociated neurons in a targeted manner and elicit the expression of a relevant neurotrophic factor.