Intracellular protein delivery: New insights into the therapeutic applications and emerging technologies.

The inability to cross the plasma membranes traditionally limited the therapeutic use of recombinant proteins. However, in the last two decades, novel technologies made delivering proteins inside the cells possible. This allowed researchers to unlock intracellular targets, once considered 'undruggable', bringing a new research area to emerge. Protein transfection systems display a large potential in a plethora of applications. However, their modality of action is often unclear, and cytotoxic effects are elevated, whereas experimental conditions to increase transfection efficacy and cell viability still need to be identified. Furthermore, technical complexity often limits in vivo experimentation, while challenging industrial and clinical translation. This review highlights the applications of protein transfection technologies, and then critically discuss the current methodologies and their limitations. Physical membrane perforation systems are compared to systems exploiting cellular endocytosis. Research evidence of the existence of either extracellular vesicles (EVs) or cell-penetrating peptides (CPPs)- based systems, that circumvent the endosomal systems is critically analysed. Commercial systems, novel solid-phase reverse protein transfection systems, and engineered living intracellular bacteria-based mechanisms are finally described. This review ultimately aims at finding new methodologies and possible applications of protein transfection systems, while helping the development of an evidence-based research approach.

Cold stress combined with salt or abscisic acid supplementation enhances lipogenesis and carotenogenesis in Phaeodactylum tricornutum (Bacillariophyceae).

Compounds from microalgae such as ω3-fatty acids or carotenoid are commercially exploited within the pharmacology, nutraceutical, or cosmetic sectors. The co-stimulation of several compounds of interest may improve the cost-effectiveness of microalgal biorefinery pipelines. This study focussed on Phaeodactylum tricornutum to investigate the effects on lipogenesis and carotenogenesis of combined stressors, here cold temperature and addition of NaCl salt or the phytohormone abscisic acid, using a two-stage cultivation strategy. Cold stress with NaCl or phytohormone addition increased the neutral lipid content of the biomass (20 to 35%). These treatments also enhanced the proportions of EPA (22% greater than control) in the fatty acid profile. Also, these treatments had a stimulatory effect on carotenogenesis, especially the combination of cold stress with NaCl addition, which returned the highest production of fucoxanthin (33% increase). The gene expression of diacylglycerol acyltransferase (DGAT) and the ω-3 desaturase precursor (PTD15) were enhanced 4- and 16-fold relative to the control, respectively. In addition, zeaxanthin epoxidase 3 (ZEP3), was downregulated at low temperature when combined with abscisic acid. These results highlight the benefits of applying a combination of low temperature and salinity stress, to simultaneously enhance the yields of the valuable metabolites EPA and fucoxanthin in Phaeodactylum tricornutum.

Development of a fibrin-mediated gene delivery system for the treatment of cystinosis via design of experiment.

Cystinosis is a rare disease, caused by a mutation in the gene cystinosin and characterised by the accumulation of cystine crystals. Advantages of biomaterial-mediated gene delivery include reduced safety concerns and the possibility to cure organs that are difficult to treat using systemic gene transfer methods. This study developed novel fibrin hydrogels for controlled, localised gene delivery, for the treatment of cystinosis. In the first part, fabrication parameters (i.e., DNA, thrombin, and aprotinin concentrations) were optimised, using a Design of Experiment (DOE) methodology. DOE is a statistical engineering approach to process optimisation, which increases experimental efficiency, reduces the number of experiments, takes into consideration interactions between different parameters, and allows the creation of predictive models. This study demonstrated the utility of DOE to the development of gene delivery constructs. In the second part of the study, primary fibroblasts from a patient with cystinosis were seeded on the biomaterials. Seeded cells expressed the recombinant CTNS and showed a decrease in cystine content. Furthermore, conditioned media contained functional copies of the recombinant CTNS. These were taken up by monolayer cultures of non-transfected cells. This study described a methodology to develop gene delivery constructs by using a DOE approach and ultimately provided new insights into the treatment of cystinosis.

Clinical Development of Cell Therapies to Halt Lysosomal Storage Diseases: Results and Lessons Learned.

Although cross-correction was discovered more than 50 years ago, and held the promise of drastically improving disease management, still no cure exists for lysosomal storage diseases (LSDs). Cell therapies have the potential to halt disease progression: either a subset of autologous cells can be ex vivo/ in vivo transfected with the functional gene or allogenic wild type stem cells can be transplanted. However, the majority of cell-based attempts have been ineffective, due to the difficulties in reversing neuronal symptomatology, in finding appropriate gene transfection approaches, in inducing immune tolerance, reducing the risk of graft versus host disease (GVHD) when allogenic cells are used and that of immune response when engineered viruses are administered, coupled with a limited secretion and uptake of some enzymes. In the last decade, due to advances in our understanding of lysosomal biology and mechanisms of cross-correction, coupled with progresses in gene therapy, ongoing pre-clinical and clinical investigations have remarkably increased. Even gene editing approaches are currently under clinical experimentation. This review proposes to critically discuss and compare trends and advances in cell-based and gene therapy for LSDs. Systemic gene delivery and transplantation of allogenic stem cells will be initially discussed, whereas proposed brain targeting methods will be then critically outlined.

Physical and mechanical cues affecting biomaterial-mediated plasmid DNA delivery: insights into non-viral delivery systems.

BACKGROUND: Whilst traditional strategies to increase transfection efficiency of non-viral systems aimed at modifying the vector or the polyplexes/lipoplexes, biomaterial-mediated gene delivery has recently sparked increased interest. This review aims at discussing biomaterial properties and unravelling underlying mechanisms of action, for biomaterial-mediated gene delivery. DNA internalisation and cytoplasmic transport are initially discussed. DNA immobilisation, encapsulation and surface-mediated gene delivery (SMD), the role of extracellular matrix (ECM) and topographical cues, biomaterial stiffness and mechanical stimulation are finally outlined. MAIN TEXT: Endocytic pathways and mechanisms to escape the lysosomal network are highly variable. They depend on cell and DNA complex types but can be diverted using appropriate biomaterials. 3D scaffolds are generally fabricated via DNA immobilisation or encapsulation. Degradation rate and interaction with the vector affect temporal patterns of DNA release and transgene expression. In SMD, DNA is instead coated on 2D surfaces. SMD allows the incorporation of topographical cues, which, by inducing cytoskeletal re-arrangements, modulate DNA endocytosis. Incorporation of ECM mimetics allows cell type-specific transfection, whereas in spite of discordances in terms of optimal loading regimens, it is recognised that mechanical loading facilitates gene transfection. Finally, stiffer 2D substrates enhance DNA internalisation, whereas in 3D scaffolds, the role of stiffness is still dubious. CONCLUSION: Although it is recognised that biomaterials allow the creation of tailored non-viral gene delivery systems, there still are many outstanding questions. A better characterisation of endocytic pathways would allow the diversion of cell adhesion processes and cytoskeletal dynamics, in order to increase cellular transfection. Further research on optimal biomaterial mechanical properties, cell ligand density and loading regimens is limited by the fact that such parameters influence a plethora of other different processes (e.g. cellular adhesion, spreading, migration, infiltration, and proliferation, DNA diffusion and release) which may in turn modulate gene delivery. Only a better understanding of these processes may allow the creation of novel robust engineered systems, potentially opening up a whole new area of biomaterial-guided gene delivery for non-viral systems.

Therapeutic Potential of Reactive Oxygen Species: State of the Art and Recent Advances.

In the last decade, several studies have proven that when at low concentration reactive oxygen species (ROS) show an adaptive beneficial effect and posited the idea that they can be utilized as inexpensive and convenient inducers of tissue regeneration. On the other hand, the recent discovery that cancer cells are more sensitive to oxidative damage paved the way for their use in the selective killing of tumor cells, and sensors to monitor ROS production during cancer treatment are under extensive investigation. Nevertheless, although ROS-activated signaling pathways are well established, less is known about the mechanisms underlying the switch from an anabolic to a cytotoxic response. Furthermore, a high variability in biological response is observed between different modalities of administration, cell types, donor ages, eventual concomitant diseases, and external microenvironment. On the other hand, available preclinical studies are scarce, whereas the quest for the most suitable systems for in vivo delivery is still elusive. Furthermore, new strategies to control the temporal pattern of ROS release need to be developed, if considering their tumorigenic potential. This review initially discusses ROS mechanisms of action and their potential application in stem cell biology, tissue engineering, and cancer therapy. It then outlines the state of art of ROS-based drugs and identifies challenges faced in translating ROS research into clinical practice.

Chasing Chimeras - The elusive stable chondrogenic phenotype.

The choice of the best-suited cell population for the regeneration of damaged or diseased cartilage depends on the effectiveness of culture conditions (e.g. media supplements, three-dimensional scaffolds, mechanical stimulation, oxygen tension, co-culture systems) to induce stable chondrogenic phenotype. Herein, advances and shortfalls in in vitro, preclinical and clinical setting of various in vitro microenvironment modulators on maintaining chondrocyte phenotype or directing stem cells towards chondrogenic lineage are critically discussed. Chondrocytes possess low isolation efficiency, limited proliferative potential and rapid phenotypic drift in culture. Mesenchymal stem cells are relatively readily available, possess high proliferation potential, exhibit great chondrogenic differentiation capacity, but they tend to acquire a hypertrophic phenotype when exposed to chondrogenic stimuli. Embryonic and induced pluripotent stem cells, despite their promising in vitro and preclinical data, are still under-investigated. Although a stable chondrogenic phenotype remains elusive, recent advances in in vitro microenvironment modulators are likely to develop clinically- and commercially-relevant therapies in the years to come.

Macromolecular crowding as a means to assess the effectiveness of chondrogenic media.

Chondrocyte-based tissue engineering requires in vitro cell expansion, which is associated with phenotypic losses, decrease in Collagen Type II synthesis and increase in Collagen Type I synthesis. Another major obstacle in clinical translation of chondrocyte-based therapies is the lack of extracellular matrix (ECM) in the engineered cartilage substitutes. Various research and commercially available media claim that they can maintain chondrogenic phenotype, whereas macromolecular crowding (MMC) has been shown to increase tissue-specific ECM deposition and maintain cell phenotype in vitro. Herein, we hypothesised that the combination of chondrogenic media with MMC will enable chondrogenic phenotype maintenance during in vitro expansion and increase cartilage-specific ECM deposition, enabling that way the development of a tissue-engineered cartilage substitute. Immunocytochemistry analysis of Passage 3 human chondrocytes in normal media in monolayer revealed that MMC significantly increased Collagen Type I deposition, whereas no statistical difference was observed in Collagen Type II deposition. When Passage 3 human chondrocytes were cultured in normal media and alginate beads, immunocytochemistry analysis revealed that MMC increased, albeit not significantly, both Collagen Type I and Collagen Type II deposition. Subsequently, human chondrocytes were expanded up to Passage 6 in either fetal bovine serum or human serum and redifferentiated using commercially available chondrogenic media in either monolayer or alginate beads. Immunocytochemistry analysis revealed that MMC, independently of the serum used, significantly increased Collagen Type I deposition in human-redifferentiated monolayer and alginate bead chondrocyte cultures, whereas almost no Collagen Type II was detected. These data clearly illustrate that an optimal chondrogenic medium is still elusive.

State of art and limitations in genetic engineering to induce stable chondrogenic phenotype.

Current protocols for chondrocyte expansion and chondrogenic differentiation of stem cells fail to reduce phenotypic loss and to mitigate hypertrophic tendency. To this end, cell genetic manipulation is gaining pace as a means of generating cells with stable chondrocyte phenotype. Herein, we provide an overview of candidate genes that either induce cartilage regeneration or inhibit cartilage degeneration. We further discuss in vitro, ex vivo and in vivo viral transduction and non-viral transfection strategies for targeted cells (chondrocytes, mesenchymal stem cells, induced pluripotent stem cells and synovial cells), along with the most representative results obtained in pre-clinical models and in clinical trials. We highlight current challenges and associated risks that slowdown clinical acceptance and commercialisation of gene transfer technologies.

An experimental toolbox for characterization of mammalian collagen type I in biological specimens.

Collagen type I is the most abundant extracellular matrix protein, and collagen type I supramolecular assemblies (e.g., tissue grafts, biomaterials and cell-assembled systems) are used extensively in tissue engineering and regenerative medicine. Many studies, for convenience or economic reasons, do not accurately determine collagen type I purity, concentration, solubility and extent of cross-linking in biological specimens, frequently resulting in erroneous conclusions. In this protocol, we describe solubility; normal, reduced and delayed (interrupted) SDS-PAGE; hydroxyproline; Sircol collagen and Pierce BCA protein; denaturation temperature; ninhydrin/trinitrobenzene sulfonic acid; and collagenase assays and assess them in a diverse range of biological samples (e.g., tissue samples; purified solutions or lyophilized materials; 3D scaffolds, such as sponges and hydrogels; and cell media and layers). Collectively, the described protocols provide a comprehensive, yet fast and readily implemented, toolbox for collagen type I characterization in any biological specimen.

Collagen Quantification in Tissue Specimens.

Collagen is the major extracellular protein in mammals. Accurate quantification of collagen is essential in the biomaterials (e.g., reproducible collagen scaffold fabrication), drug discovery (e.g., assessment of collagen in pathophysiologies, such as fibrosis), and tissue engineering (e.g., quantification of cell-synthesized collagen) fields. Although measuring hydroxyproline content is the most widely used method to quantify collagen in biological specimens, the process is very laborious. To this end, the Sircol™ Collagen Assay is widely used due to its inherent simplicity and convenience. However, this method leads to overestimation of collagen content due to the interaction of Sirius red with basic amino acids of non-collagenous proteins. Herein, we describe the addition of an ultrafiltration purification step in the process to accurately determine collagen content in tissues.