Mesenchymal Stem Cells as a Gene Delivery Tool: Promise, Problems, and Prospects

The cell-based approach in gene therapy arises as a promising strategy to provide safe, targeted, and efficient gene delivery. Owing to their unique features, as homing and tumor-tropism, mesenchymal stem cells (MSCs) have recently been introduced as an encouraging vehicle in gene therapy. Nevertheless, non-viral transfer of nucleic acids into MSCs remains limited due to various factors related to the main stakeholders of the process (e.g., nucleic acids, carriers, or cells). In this review, we have summarized the main types of nucleic acids used to transfect MSCs, the pros and cons, and applications of each. Then, we have emphasized on the most efficient lipid-based carriers for nucleic acids to MSCs, their main features, and some of their applications. While a myriad of studies have demonstrated the therapeutic potential for engineered MSCs therapy in various illnesses, optimization for clinical use is an ongoing challenge. On the way of improvement, genetically modified MSCs have been combined with various novel techniques and tools (e.g., exosomes, spheroids, 3D-Bioprinting, etc.,) aiming for more efficient and safe applications in biomedicine.Authors wish to thank: ICTS “NANBIOSIS”, specifically the Drug Formulation Unit (U10) of the CIBER in Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN) for the intellectual and technical assistance. The Department of Education, University and Research of the Basque Country Government (Consolidated Groups, IT907-16); the Spanish Ministry of Science and Innovation (GrantsPID2019-106199RB-C21)

Malaria Vaccine Adjuvants: Latest Update and Challenges in Preclinical and Clinical Research

There is no malaria vaccine currently available, and the most advanced candidate has recently reported a modest 30% efficacy against clinical malaria. Although many efforts have been dedicated to achieve this goal, the research was mainly directed to identify antigenic targets. Nevertheless, the latest progresses on understanding how immune system works and the data recovered from vaccination studies have conferred to the vaccine formulation its deserved relevance. Additionally to the antigen nature, the manner in which it is presented (delivery adjuvants) as well as the immunostimulatory effect of the formulation components (immunostimulants) modulates the immune response elicited. Protective immunity against malaria requires the induction of humoral, antibody-dependent cellular inhibition (ADCI) and effector and memory cell responses. This review summarizes the status of adjuvants that have been or are being employed in the malaria vaccine development, focusing on the pharmaceutical and immunological aspects, as well as on their immunization outcomings at clinical and preclinical stages.This project was partially supported by the "Ministerio de Ciencia e Innovacion" (SAF2007-66115), the University of the Basque Country (UPV/EHU) (UFI 11/32), and FEDER funds. E. Mata thanks the Basque Government for a fellowship grant

Encapsulation of Oleuropein in Nanostructured Lipid Carriers: Biocompatibility and Antioxidant Efficacy in Lung Epithelial Cells

Oxidative damage has been linked to a number of diseases. Oleuropein (OLE), a natural occurring polyphenol from olive leaves (Olea europaea L.), is known to be a potent antioxidant compound with inherent instability and compromised bioavailability. Therefore, in this work, nanostructured lipid carriers (NLCs) were proposed for OLE encapsulation to protect and improve its antioxidant efficacy. The lipid matrix, composed of olive oil and Precirol, was optimized prior to OLE encapsulation. The characterization of the optimized oleuropein-loaded NLCs (NLC-OLE) showed a mean size of 150 nm, a zeta potential of −21 mV, an encapsulation efficiency of 99.12%, sustained release profile, and improved radical scavenging activity. The cellular in vitro assays demonstrated the biocompatibility of the NLCs, which were found to improve and maintain OLE antioxidant efficacy in the A549 and CuFi-1 lung epithelial cell lines, respectively. Overall, these findings suggest a promising potential of NLC-OLE to further design a pulmonary formulation for OLE delivery in lung epithelia.A.H.-C. thanks the Spanish Ministry of Economy and Competitiveness for the Industrial Doctorate fellowship grant (DI-15-07513). This work was done under the R&D projects of BIOSASUN and supported by the University of the Basque Country (UPV/EHU) and the Basque Country Government (Grupos Consolidados, No. IT907-16 to J.L.P.)

Cell Microencapsulation Technologies for Sustained Drug Delivery: Latest Advances in Efficacy and Biosafety

The development of cell microencapsulation systems began several decades ago. However, today few systems have been tested in clinical trials. For this reason, in the last years, researchers have directed efforts towards trying to solve some of the key aspects that still limit efficacy and biosafety, the two major criteria that must be satisfied to reach the clinical practice. Regarding the efficacy, which is closely related to biocompatibility, substantial improvements have been made, such as the purification or chemical modification of the alginates that normally form the microspheres. Each of the components that make up the microcapsules has been carefully selected to avoid toxicities that can damage the encapsulated cells or generate an immune response leading to pericapsular fibrosis. As for the biosafety, researchers have developed biological circuits capable of actively responding to the needs of the patients to precisely and accurately release the demanded drug dose. Furthermore, the structure of the devices has been subject of study to adequately protect the encapsulated cells and prevent their spread in the body. The objective of this review is to describe the latest advances made by scientist to improve the efficacy and biosafety of cell microencapsulation systems for sustained drug delivery, also highlighting those points that still need to be optimized

Design of double functionalized carbon nanotube for amphotericin B and genetic material delivery.

In the present work, single wall carbon nanotubes (SWCNT) were successively functionalized with phospholipid DSPE-PEG carboxylic acid, and then, with ethylenediamine (EDA), to obtain double functionalized single wall carbon nanotube (DFSWCNT). Then, DFSWCNT was applied as a carrier for delivering amphotericin B (Amb) and EGFP plasmid. FSWCNT’s concentration obtained via UV–visible analysis was 0.99 mg/mL. The TGA analysis results provided the lost weights of DSPE-PEG-COOH, EDA, Amb and SWCNT impurities. XPS results showed that carbon atoms’ percentage decreased during the functionalization processes from 97.2% (SWCNT) to 76.4% (FSWCNT) and 69.9% (DFSWNCT). Additionally, the oxygen atoms’ percentage increased from 2.3% (SWCNT) to 21% and 22.5% for FSWCNT and DFSWCNT, respectively. New bonds such as C–N and N–C=O appeared in the synthesized nanocarrier. The IG/ID ratio in Raman analysis decreased from 7.15 (SWCNT) to 4.08 (FSWCNT). The amount of Amb released to phosphate buffer saline medium was about 33% at pH = 5.5 and 75% at pH = 7.4 after 48 h. CCK8 results confirmed that the toxicity of functionalized SWCNT had decreased. In a 2:1 ratio of DFSWCNT/EGFP plasmid, the cell viability (87%) and live transfected cells (56%) were at their maximum values. The results indicate that carbon nanotubes have the potential to be applied as drug/gene delivery systems with outstanding properties such as high loading capacity and easy penetration to cell membrane.This work was supported by the Basque Country Government (IT907-16). Additional funding was provided by the CIBER of Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), an initiative of the Carlos III Health Institute (ISCIII)

Decellularized Extracellular Matrix-Based Bioinks for Tendon Regeneration in Three-Dimensional Bioprinting

In the last few years, attempts to improve the regeneration of damaged tendons have been rising due to the growing demand. However, current treatments to restore the original performance of the tissue focus on the usage of grafts; although, actual grafts are deficient because they often cannot provide enough support for tissue regeneration, leading to additional complications. The beneficial effect of combining 3D bioprinting and dECM as a novel bioink biomaterial has recently been described. Tendon dECMs have been obtained by using either chemical, biological, or/and physical treatments. Although decellularization protocols are not yet standardized, recently, different protocols have been published. New therapeutic approaches embrace the use of dECM in bioinks for 3D bioprinting, as it has shown promising results in mimicking the composition and the structure of the tissue. However, major obstacles include the poor structural integrity and slow gelation properties of dECM bioinks. Moreover, printing parameters such as speed and temperature have to be optimized for each dECM bioink. Here, we show that dECM bioink for 3D bioprinting provides a promising approach for tendon regeneration for future clinical applications.This work was funded by the University of the Basque Country UPV/EHU and the Basque Country Government (IT1448-22). Supported by the fellowships granted to Fouad Al-Hakim Khalak (PRE_2021_2_0181) and Sandra Ruiz-Alonso (PRE_2021_2_0153). Likewise, the authors thank ICTS “NANBIOSIS”, in particular the Drug Formulation Unit (U10) of the CIBER in Bioengineering, Biomaterials, and Nanomedicine (CIBER-BBN), at the University of the Basque Country (UPV/EHU) in Vitoria-Gasteiz

Nanomedicine for autophagy modulation in cancer therapy: a clinical perspective

In recent years, progress in nanotechnology provided new tools to treat cancer more effectively. Advances in biomaterials tailored for drug delivery have the potential to overcome the limited selectivity and side effects frequently associated with traditional therapeutic agents. While autophagy is pivotal in determining cell fate and adaptation to different challenges, and despite the fact that it is frequently dysregulated in cancer, antitumor therapeutic strategies leveraging on or targeting this process are scarce. This is due to many reasons, including the very contextual effects of autophagy in cancer, low bioavailability and non-targeted delivery of existing autophagy modulatory compounds. Conjugating the versatile characteristics of nanoparticles with autophagy modulators may render these drugs safer and more effective for cancer treatment. Here, we review current standing questions on the biology of autophagy in tumor progression, and precursory studies and the state-of-the-art in harnessing nanomaterials science to enhance the specificity and therapeutic potential of autophagy modulators.Work in the laboratories of the authors is funded by grants from the Italian Ministries for Health (Ricerca Corrente) to M.T., from Education, University and Research (MIUR; 000003_17_MAP_STRIP and FISR 2020-Covid FISR2020IP_03366) to R.S; and from the Spanish Ministerio de Ciencia e Innovación (PID2021-128106NA-I00). M.C. is currently a recipient of a Ramón y Cajal tenure track contract from the Spanish Ministry of Science and Innovation (RYC2021-031003-I) and was funded by “Maria Zambrano” contract from Spanish Ministry of Universities and Complutense University of Madrid. M.S-A is recipient of a Ramón y Cajal tenure track contract from the Spanish Ministry of Science and Innovation (RYC2020-029690-I)

Therapeutic Opportunities and Delivery Strategies for Brain Revascularization in Stroke, Neurodegeneration, and Aging

[EN] Central nervous system (CNS) diseases, especially acute ischemic events and neurodegenerative disorders, constitute a public health problem with no effective treatments to allow a persistent solution. Failed therapies targeting neuronal recovery have revealed the multifactorial and intricate pathophysiology underlying such CNS disorders as ischemic stroke, Alzheimers disease, amyotrophic lateral sclerosis, vascular Parkisonism, vascular dementia, and aging, in which cerebral microvasculature impairment seems to play a key role. In fact, a reduction in vessel density and cerebral blood flow occurs in these scenarios, contributing to neuronal dysfunction and leading to loss of cognitive function. In this review, we provide an overview of healthy brain microvasculature structure and function in health and the effect of the aforementioned cerebral CNS diseases. We discuss the emerging new therapeutic opportunities, and their delivery approaches, aimed at recovering brain vascularization in this context. SIGNIFICANCE STATEMENT: The lack of effective treatments, mainly focused on neuron recovery, has prompted the search of other therapies to treat cerebral central nervous system diseases. The disruption and degeneration of cerebral microvasculature has been evidenced in neurodegenerative diseases, stroke, and aging, constituting a potential target for restoring vascularization, neuronal functioning, and cognitive capacities by the development of therapeutic pro-angiogenic strategies.This work was supported by the University of the Basque Country (UPV/EHU) [Grant ESPDOC19/47] (postdoctoral fellowship to I.V.B.); and the Basque Country Government (Consolidated Groups) [Grant IT907-16]

Benefits of cryopreservation as long-term storage method of encapsulated cardiosphere-derived cells for cardiac therapy: A biomechanical analysis

[EN]Cardiosphere-derived cells (CDCs) encapsulated within alginate-poly-L-lysine-alginate (APA) microcapsules present a promising treatment alternative for myocardial infarction. However, clinical translatability of encapsulated CDCs requires robust long-term preservation of microcapsule and cell stability, since cell culture at 37 degrees C for long periods prior to patient implantation involve high resource, space and manpower costs, sometimes unaffordable for clinical facilities. Cryopreservation in liquid nitrogen is a well-established procedure to easily store cells with good recovery rate, but its effects on encapsulated cells are understudied. In this work, we assess both the biological response of CDCs and the mechanical stability of microcapsules after long-term (i.e., 60 days) cryopreservation and compare them to encapsulated CDCs cultured at 37 degrees C. We investigate for the first time the effects of cryopreservation on stiffness and topographical features of microcapsules for cell therapy. Our results show that functionality of encapsulated CDCs is optimum during 7 days at 37 degrees C, while cryopreservation seems to better guarantee the stability of both CDCs and APA microcapsules properties during longer storage than 15 days. These results point out cryopreservation as a suitable technique for long-term storage of encapsulated cells to be translated from the bench to the clinic.This work has been supported by the European Union's H2020 Framework Program (H2020/2014-2020) and National Authorities through the Electronic Components and Systems for European Leadership Joint Undertaking (ECSEL JU) program under grant agreement Ecsel-78132-Position-II-2017-IA. The regional Government of Aragon provided L.P. studentship. This research was partially funded by Instituto de Salud Carlos III (PI20/00247) and Agencia Estatal de Investigacion (PID2019-107329RA-C22), cofunded by European Regional Development Fund "A way to make Europe.

Characterization and assessment of new fibrillar collagen inks and bioinks for 3D printing and bioprinting

Collagen is a cornerstone protein for tissue engineering and 3D bioprinting due to its outstanding biocompatibility, low immunogenicity, and natural abundance in human tissues. Nonetheless, it still poses some important challenges, such as complicated and limited extraction processes, usually accompanied by batchto-batch reproducibility and influence of factors, such as temperature, pH, and ionic strength. In this work, we evaluated the suitability and performance of new, fibrillar type I collagen as standardized and reproducible collagen source for 3D printing and bioprinting. The acidic, native fibrous collagen formulation (5% w/w) performed remarkably during 3D printing, which was possible to print constructs of up to 27 layers without collapsing. On the other hand, the fibrous collagen mass has been modified to provide a fast, reliable, and easily neutralizable process. The neutralization with TRIS-HCl enabled the inclusion of cells without hindering printability. The cell-laden constructs were printed under mild conditions (50–80 kPa, pneumatic 3D printing), providing remarkable cellular viability (>90%) as well as a stable platform for cell growth and proliferation in vitro. Therefore, the native, type I collagen masses characterized in this work offer a reproducible and reliable source of collagen for 3D printing and bioprinting purposes.This research was funded by Viscofan (S.A.) Centro para el Desarrollo Tecnológico Industrial (CDTI) IDI-20210050 and the Basque Country Government/Eusko Jaurlaritza (Department of Education, University and Research, Consolidated Groups IT907-16). Author Sandra Ruiz-Alonso thanks the Basque Country Government for the granted fellowship PRE_2021_2_0153. José M. Rey thanks the funding from the European’s Union Horizon 2020 research and Innovation framework program (Triankle Project; Grant Agreement #952981). BioRender.com has been used as support for some figures assembly