Expanding the horizon of transient CAR T therapeutics using virus-free technology.

The extraordinary success that chimeric antigen receptor (CAR) T cell therapies have shown over the years on fighting hematological malignancies is evidenced by the six FDA-approved products present on the market. CAR T treatments have forever changed the way we understand cellular immunotherapies, as current research in the topic is expanding even outside the field of cancer with very promising results. Until now, virus-based strategies have been used for CAR T cell manufacturing. However, this methodology presents relevant limitations that need to be addressed prior to wide spreading this technology to other pathologies and in order to optimize current cancer treatments. Several approaches are being explored to overcome these challenges such as virus-free alternatives that additionally offer the possibility of developing transient CAR expression or in vivo T cell modification. In this review, we aim to spotlight a pivotal juncture in the history of medicine where a significant change in perspective is occurring. We review the current progress made on viral-based CAR T therapies as well as their limitations and we discuss the future outlook of virus-free CAR T strategies to overcome current challenges and achieve affordable immunotherapies for a wide variety of pathologies, including cancer.

Carbon-Based Nanostructures as Emerging Materials for Gene Delivery Applications.

Gene therapeutics are promising for treating diseases at the genetic level, with some already validated for clinical use. Recently, nanostructures have emerged for the targeted delivery of genetic material. Nanomaterials, exhibiting advantageous properties such as a high surface-to-volume ratio, biocompatibility, facile functionalization, substantial loading capacity, and tunable physicochemical characteristics, are recognized as non-viral vectors in gene therapy applications. Despite progress, current non-viral vectors exhibit notably low gene delivery efficiency. Progress in nanotechnology is essential to overcome extracellular and intracellular barriers in gene delivery. Specific nanostructures such as carbon nanotubes (CNTs), carbon quantum dots (CQDs), nanodiamonds (NDs), and similar carbon-based structures can accommodate diverse genetic materials such as plasmid DNA (pDNA), messenger RNA (mRNA), small interference RNA (siRNA), micro RNA (miRNA), and antisense oligonucleotides (AONs). To address challenges such as high toxicity and low transfection efficiency, advancements in the features of carbon-based nanostructures (CBNs) are imperative. This overview delves into three types of CBNs employed as vectors in drug/gene delivery systems, encompassing their synthesis methods, properties, and biomedical applications. Ultimately, we present insights into the opportunities and challenges within the captivating realm of gene delivery using CBNs.

Calcium Carbonate/Polydopamine Composite Nanoplatform Based on TGF-ß Blockade for Comfortable Cancer Immunotherapy.

Cancer pain seriously reduces the quality of life of cancer patients. However, most research about cancer focuses solely on inhibiting tumor growth, neglecting the issue of cancer pain. Therefore, the development of therapeutic agents with both tumor suppression and cancer pain relief is crucial to achieve human-centered treatment. Here, the work reports curcumin (CUR) and ropivacaine (Ropi) coincorporating CaCO3/PDA nanoparticles (CaPNMCUR+Ropi) that realized efficient tumor immunotherapy and cancer pain suppression. The therapeutic efficiency and mechanism are revealed in vitro and in vivo. The results indicate that CaPNMCUR+Ropi underwent tumor microenvironment-responsive degradation and realized rapid release of calcium ions, Ropi, and CUR. The excessive intracellular calcium triggered the apoptosis of tumor cells, and the transient pain caused by the tumor injection was relieved by Ropi. Simultaneously, CUR reduced the levels of immunosuppressive factor (TGF-ß) and inflammatory factor (IL-6, IL-1ß, and TNF-α) in the tumor microenvironment, thereby continuously augmenting the immune response and alleviating inflammatory pain of cancer animals. Meanwhile, the decrease of TGF-ß leads to the reduction of transient receptor potential vanilloid 1 (TRPV1) expression, thereby alleviating hyperalgesia and achieving long-lasting analgesic effects. The design of the nanosystem provides a novel idea for human-centered tumor treatment in the future.

Liposome-based vaccines for minimally or noninvasive administration: an update on current advancements.

INTRODUCTION: Vaccination requires innovation to provide effective protection. Traditional vaccines have several drawbacks, which can be overcome with advanced technologies and different administration routes. Over the past 10 years, a significant amount of research has focussed on the delivery of antigens into liposomes due to their dual role as antigen-carrying systems and vaccine adjuvants able to increase the immunogenicity of the carried antigen. AREAS COVERED: This review encompasses the progress made over the last 10 years with liposome-based vaccines designed for minimally or noninvasive administration, filling the gaps in previous reviews and providing insights on composition, administration routes, results achieved, and Technology Readiness Level of the most recent formulations. EXPERT OPINION: Liposome-based vaccines administered through minimally or noninvasive routes are expected to improve efficacy and complacency of vaccination programs. However, the translation from lab-scale production to large-scale production and collaborations with hospitals, research centers, and companies are needed to allow new products to enter the market and improve the vaccination programs in the future.

Lenvatinib-Loaded Poly(lactic-co-glycolic acid) Nanoparticles with Epidermal Growth Factor Receptor Antibody Conjugation as a Preclinical Approach to Therapeutically Improve Thyroid Cancer with Aggressive Behavior.

BACKGROUND: Lenvatinib, a tyrosine kinase inhibitor (TKI) approved for the treatment of progressive and radioactive iodine (RAI)-refractory differentiated thyroid cancer (DTC), is associated with significant adverse effects that can be partially mitigated through the development of novel drug formulations. The utilization of nanoparticles presents a viable option, as it allows for targeted drug delivery, reducing certain side effects and enhancing the overall quality of life for patients. This study aimed to produce and assess, both in vitro and in vivo, the cytotoxicity, biodistribution, and therapeutic efficacy of lenvatinib-loaded PLGA nanoparticles (NPs), both with and without decoration using antibody conjugation (cetuximab), as a novel therapeutic approach for managing aggressive thyroid tumors. METHODS: Poly(lactic-co-glycolic acid) nanoparticles (NPs), decorated with or without anti-EGFR, were employed as a lenvatinib delivery system. These NPs were characterized for size distribution, surface morphology, surface charge, and drug encapsulation efficiency. Cytotoxicity was evaluated through MTT assays using two cellular models, one representing normal thyroid cells (Nthy-ori 3-1) and the other representing anaplastic thyroid cells (CAL-62). Additionally, an in vivo xenograft mouse model was established to investigate biodistribution and therapeutic efficacy following intragastric administration. RESULTS: The NPs demonstrated success in terms of particle size, polydispersity index (PDI), zeta potential, morphology, encapsulation efficiency, and cetuximab distribution across the surface. In vitro analysis revealed cytotoxicity in both cellular models with both formulations, but only the decorated NPs achieved an ID50 value in CAL-62 cells. Biodistribution analysis following intragastric administration in xenografted thyroid mice demonstrated good stability in terms of intestinal barrier function and tumor accumulation. Both formulations were generally well tolerated without inducing pathological effects in the examined organs. Importantly, both formulations increased tumor necrosis; however, decorated NPs exhibited enhanced parameters related to apoptotic/karyolytic forms, mitotic index, and vascularization compared with NPs without decoration. CONCLUSIONS: These proof-of-concept findings suggest a promising strategy for administering TKIs in a more targeted and effective manner.

Stability of Monoclonal Antibodies as Solid Formulation for Auto-Injectors: A Pilot Study.

Drug adherence is a significant medical issue, often responsible for sub-optimal outcomes during the treatment of chronic diseases such as rheumatoid or psoriatic arthritis. Monoclonal antibodies (which are exclusively given parenterally) have been proven to be an effective treatment in these cases. The use of auto-injectors is an effective strategy to improve drug adherence in parenteral treatments since these pen-like devices offer less discomfort and increased user-friendliness over conventional syringe-based delivery. This study aims to investigate the feasibility of including a monoclonal antibody as a solid formulation inside an auto-injector pen. Specifically, the objective was to evaluate the drug stability after a concentration (to reduce the amount of solvent and space needed) and freeze-drying procedure. A preliminary screening of excipients to improve stability was also performed. The nano-DSC results showed that mannitol improved the stability of the concentrated, freeze-dried antibody in comparison to its counterpart without it. However, a small instability of the CH2 domain was still found for mannitol samples, which will warrant further investigation. The present results serve as a stepping stone towards advancing future drug delivery systems that will ultimately improve the patient experience and associated drug adherence.

3D Bioprinting tissue analogs: Current development and translational implications.

Three-dimensional (3D) bioprinting is a promising and rapidly evolving technology in the field of additive manufacturing. It enables the fabrication of living cellular constructs with complex architectures that are suitable for various biomedical applications, such as tissue engineering, disease modeling, drug screening, and precision regenerative medicine. The ultimate goal of bioprinting is to produce stable, anatomically-shaped, human-scale functional organs or tissue substitutes that can be implanted. Although various bioprinting techniques have emerged to develop customized tissue-engineering substitutes over the past decade, several challenges remain in fabricating volumetric tissue constructs with complex shapes and sizes and translating the printed products into clinical practice. Thus, it is crucial to develop a successful strategy for translating research outputs into clinical practice to address the current organ and tissue crises and improve patients' quality of life. This review article discusses the challenges of the existing bioprinting processes in preparing clinically relevant tissue substitutes. It further reviews various strategies and technical feasibility to overcome the challenges that limit the fabrication of volumetric biological constructs and their translational implications. Additionally, the article highlights exciting technological advances in the 3D bioprinting of anatomically shaped tissue substitutes and suggests future research and development directions. This review aims to provide readers with insight into the state-of-the-art 3D bioprinting techniques as powerful tools in engineering functional tissues and organs.

Assessment of Different Niosome Formulations for Optogenetic Applications: Morphological and Electrophysiological Effects.

Gene therapy and optogenetics are becoming promising tools for treating several nervous system pathologies. Currently, most of these approaches use viral vectors to transport the genetic material inside the cells, but viruses present some potential risks, such as marked immunogenicity, insertional mutagenesis, and limited insert gene size. In this framework, non-viral nanoparticles, such as niosomes, are emerging as possible alternative tools to deliver genetic material, avoiding the aforementioned problems. To determine their suitability as vectors for optogenetic therapies in this work, we tested three different niosome formulations combined with three optogenetic plasmids in rat cortical neurons in vitro. All niosomes tested successfully expressed optogenetic channels, which were dependent on the ratio of niosome to plasmid, with higher concentrations yielding higher expression rates. However, we found changes in the dendritic morphology and electrophysiological properties of transfected cells, especially when we used higher concentrations of niosomes. Our results highlight the potential use of niosomes for optogenetic applications and suggest that special care must be taken to achieve an optimal balance of niosomes and nucleic acids to achieve the therapeutic effects envisioned by these technologies.

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 batch- to-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.

The role of microfluidics and 3D-bioprinting in the future of exosome therapy.

Exosome-based strategies constitute a promising tool for therapeutics, avoiding potential immunogenic and tumorigenic side-effects of cell therapies. However, the collection of a suitable exosome pool, and the need for high doses with conventional administration approaches, hamper their clinical translation. To overcome these challenges, versatile exosome collection strategies together with advanced delivery platforms may represent major progress in this field. Microfluidics enables large-scale gathering of both natural and synthetic exosomes for their implementation into bioinks, while 3D-bioprinting holds great promise in regenerative medicine with the use of exosome-loaded scaffolds that mimic the target tissue with controlled pharmacokinetics and pharmacodynamics. Hence, the combination of both strategies might become the key for the translation of exosome therapies to clinical practice.

Stability of polymeric cationic niosomes and their plasmid DNA-based complexes as gene delivery carriers.

This study aims to explore the stability of lipo-polymeric niosomes/niosome-based pCMS-EGFP complexes under different storage temperatures (25 °C, 4 °C, and -20 °C). To date, the question of nucleic acid-complex stability is one of the most vital issues in gene delivery applications. The need for stable vaccines during the COVID-19 pandemic has merely highlighted it. In the case of niosomes as gene carriers, the scientific literature still lacks comprehensive stability studies. In this study, the physicochemical features of niosomes/nioplexes in terms of size, surface charge, and polydispersity index (PDI), along with transfection efficiency, and cytotoxicity in NT2 cells were evaluated for 8 weeks. Compared to day 0, the physicochemical features of the niosomes stored at 25 °C and -20 °C changed dramatically in terms of size, zeta potential, and PDI, while remaining in reasonable values when stored at 4 °C. However, niosomes and nioplexes stored at 4 °C and -20 °C showed nearly stable transfection efficiency values, yet an obvious decrease at 25 °C. This article provides a proof of concept into the stability of polymeric cationic niosomes and their nioplexes as promising gene delivery vehicles. Moreover, it highlights the practical possibility of storing nioplexes at 4 °C for up to 2 months, as an alternative to niosomes, for gene delivery purposes.

219Three-dimensional printing as a cutting-edge, versatile and personalizable vascular stent manufacturing procedure: Toward tailor-made medical devices.

Vascular stents (VS) have revolutionized the treatment of cardiovascular diseases, as evidenced by the fact that the implantation of VS in coronary artery disease (CAD) patients has become a routine, easily approachable surgical intervention for the treatment of stenosed blood vessels. Despite the evolution of VS throughout the years, more efficient approaches are still required to address the medical and scientific challenges, especially when it comes to peripheral artery disease (PAD). In this regard, three-dimensional (3D) printing is envisaged as a promising alternative to upgrade VS by optimizing the shape, dimensions and stent backbone (crucial for optimal mechanical properties), making them customizable for each patient and each stenosed lesion. Moreover, the combination of 3D printing with other methods could also upgrade the final device. This review focuses on the most recent studies using 3D printing techniques to produce VS, both by itself and in combination with other techniques. The final aim is to provide an overview of the possibilities and limitations of 3D printing in the manufacturing of VS. Furthermore, the current situation of CAD and PAD pathologies is also addressed, thus highlighting the main weaknesses of the already existing VS and identifying research gaps, possible market niches and future directions.

Recent advances in nanomedicine development for traumatic brain injury.

Traumatic brain injury (TBI) is one of the major causes of morbidity and mortality worldwide, and it is also a risk factor for neurodegeneration. However, there has not been perceptible progress in treating acute TBI over the last few years, mainly due to the inability of therapeutic drugs to cross the blood-brain barrier (BBB), failing to exert significant pharmacological effects on the brain parenchyma. Recently, nanomedicines are emerging as a powerful tool for the treatment of TBI where nanoscale materials (also called nanomaterials) are employed to deliver therapeutic agents. The advantages of using nanomaterials as a drug carrier include their high solubility and stability, high carrier capacity, site-specific, improved pharmacokinetics, and biodistribution. Keeping these points in consideration, this article reviews the pathophysiology, current treatment options, and emerging nanomedicine strategies for the treatment of TBI. The review will help readers to gain insight into the state-of-the-art of nanomedicine as a new tool for the treatment of TBI.

Long-term biophysical stability of nanodiamonds combined with lipid nanocarriers for non-viral gene delivery to the retina.

Nanodiamonds were combined with niosome, and resulting formulations were named as nanodiasomes, which were evaluated in terms of physicochemical features, cellular internalization, cell viability and transfection efficiency both in in vitro and in in vivo conditions. Such parameters were analyzed at 4 and 25 °C, and at 15 and 30 days after their elaboration. Nanodiasomes showed a particle size of 128 nm that was maintained over time inside the ± 10% of deviation, unless after 30 days of storage at 25 °C. Something similar occurred with the initial zeta potential value, 35.2 mV, being both formulations more stable at 4 °C. The incorporation of nanodiamonds into niosomes resulted in a 4-fold increase of transfection efficiency that was maintained over time at 4 and 25 °C. In vivo studies reported high transgene expression of nanodiasomes after subretinal and intravitreal administration in mice, when injected freshly prepared and after 30 days of storage at 4 °C.

Impact of nanotechnology on conventional and artificial intelligence-based biosensing strategies for the detection of viruses.

Recent years have witnessed the emergence of several viruses and other pathogens. Some of these infectious diseases have spread globally, resulting in pandemics. Although biosensors of various types have been utilized for virus detection, their limited sensitivity remains an issue. Therefore, the development of better diagnostic tools that facilitate the more efficient detection of viruses and other pathogens has become important. Nanotechnology has been recognized as a powerful tool for the detection of viruses, and it is expected to change the landscape of virus detection and analysis. Recently, nanomaterials have gained enormous attention for their value in improving biosensor performance owing to their high surface-to-volume ratio and quantum size effects. This article reviews the impact of nanotechnology on the design, development, and performance of sensors for the detection of viruses. Special attention has been paid to nanoscale materials, various types of nanobiosensors, the internet of medical things, and artificial intelligence-based viral diagnostic techniques.

Immunomodulatory and Anti-inflammatory effect of Neural Stem/Progenitor Cells in the Central Nervous System.

Neuroinflammation is a critical event that responds to disturbed homeostasis and governs various neurological diseases in the central nervous system (CNS). The excessive inflammatory microenvironment in the CNS can adversely affect endogenous neural stem cells, thereby impeding neural self-repair. Therapies with neural stem/progenitor cells (NSPCs) have shown significant inhibitory effects on inflammation, which is mainly achieved through intercellular contact and paracrine signalings. The intercellular contact between NSPCs and immune cells, the activated CNS- resident microglia, and astrocyte plays a critical role in the therapeutic NSPCs homing and immunomodulatory effects. Moreover, the paracrine effect mainly regulates infiltrating innate and adaptive immune cells, activated microglia, and astrocyte through the secretion of bioactive molecules and extracellular vesicles. However, the molecular mechanism involved in the immunomodulatory effect of NSPCs is not well discussed. This article provides a systematic analysis of the immunomodulatory mechanism of NSPCs, discusses efficient ways to enhance its immunomodulatory ability, and gives suggestions on clinical therapy.

Bioengineered Bacterial Membrane Vesicles with Multifunctional Nanoparticles as a Versatile Platform for Cancer Immunotherapy.

Inducing immunogenic cell death (ICD) is a critical strategy for enhancing cancer immunotherapy. However, inefficient and risky ICD inducers along with a tumor hypoxia microenvironment seriously limit the immunotherapy efficacy. Non-specific delivery is also responsible for this inefficiency. In this work, we report a drug-free bacteria-derived outer membrane vesicle (OMV)-functionalized Fe3O4-MnO2 (FMO) nanoplatform that realized neutrophil-mediated targeted delivery and photothermally enhanced cancer immunotherapy. In this system, modification of OMVs derived from Escherichia coli enhanced the accumulation of FMO NPs at the tumor tissue through neutrophil-mediated targeted delivery. The FMO NPs underwent reactive decomposition in the tumor site, generating manganese and iron ions that induced ICD and O2 that regulated the tumor hypoxia environment. Moreover, OMVs are rich in pathogen-associated pattern molecules that can overcome the tumor immunosuppressive microenvironment and effectively activate immune cells, thereby enhancing specific immune responses. Photothermal therapy (PTT) caused by MnO2 and Fe3O4 can not only indirectly stimulate systemic immunity by directly destroying tumor cells but also promote the enrichment of neutrophil-equipped nanoparticles by enhancing the inflammatory response at the tumor site. Finally, the proposed multi-modal treatment system with targeted delivery capability realized effective tumor immunotherapy to prevent tumor growth and recurrence.

Stem Cell-Derived Extracellular Vesicles as a Potential Therapeutic Tool for Eye Diseases: From Benchtop to Bedside.

Stem cell-derived extracellular vesicles (SC-EVs) have remarkably drawn clinicians' attention in treating ocular diseases. As a paracrine factor of stem cells and an appealing alternative for off-the-shelf cell-free therapeutics, SC-EVs can be conveniently applied topically on the ocular surface or introduced to the retina via intravitreal injection, without increasing the risks of immunogenesis or oncogenesis. This chapter aims to assess the potential applications for EV, obtained from various types of stem cells, in myriad eye diseases (traumatic, inflammatory, degenerative, immunological, etc.). To the best of our knowledge, all relevant pre-clinical studies are summarized here. Furthermore, we highlight the up-to-date status of clinical trials in the same realm and emphasize where future research efforts should be directed. For a successful clinical translation, various drawbacks of EVs therapy should be overcome (e.g., contamination, infection, insufficient yield, etc.). Moreover, standardized, and scalable extraction, purification, and characterization protocols are highly suggested to determine the exosome quality before they are offered to patients with ocular disorders.

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.