Comparative analysis of lipid-peptide nanoparticles prepared via microfluidics, reverse phase evaporation, and ouzo techniques for efficient plasmid DNA delivery.

In the current "era of lipid carriers," numerous strategies have been developed to manufacture lipid nanoparticles (LNPs). Nevertheless, the potential impact of various preparation methods on the characteristics, use, and/or stability of these LNPs remains unclear. In this work, we attempted to compare the effects of three different preparation methods: microfluidics (MF), reverse phase evaporation (RV), and ouzo (OZ) on lipid-peptide NPs (LPNPs) as plasmid DNA delivery carriers. These LPNPs had the same components, namely DOTMA cationic lipid, DSPC, cholesterol, and protamine. Subsequently, we compared the LPNPs in terms of their physicochemical features, functionality as gene delivery vehicles in two distinct cell lines (NT2 and D1-MSCs), and finally, their storage stability over a six-month period. It was clear that all three LPNP formulations worked to deliver EGFP-pDNA while keeping cells alive, and their physicochemical stability was high for 6 months. However, the preparation technique had a significant impact on their physicochemical characteristics. The MF produced LPNPs with a lesser size, polydispersity index, and zeta potential than the other synthesis methods. Additionally, their DNA entrapment efficiency, cell viability, and functional stability profiles were generally superior. These findings provide new insights for comparing different manufacturing methods to create LPNPs with the desired characteristics for effective and safe gene delivery.

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.

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.

Signet ring cell component predicts the response to neoadjuvant chemoradiotherapy in rectal cancer. Long interim results of a single institution experience.

There are limited studies evaluating the correlation between the presence of signet ring carcinoma and tumor response to neoadjuvant therapy in the rectum. Hereby, we aimed to report for the first time our experience from Upper Egypt through assessing the predictive role of signet ring cell component (SRCC) in the response to preoperative chemoradiotherapy (PCRT) and the impact of histological types (SRCC versus other types) on survival. This retrospective study analysed the medical records of 195 patients with locally advanced rectal cancer treated from 2011, to 2018. Patients were divided into two groups according to histological types: SRCC group and non SRCC group. All patients received PCRT followed by surgery. SRCC group was associated with significant higher rate of complete clinical response (cCR) and pathologic complete response (pCR) (83.3% and 88.9% respectively) as compared to non SRCC group (9.0% and 10.2% respectively); P<0.0001. Fifteen cases (93.8%) who were diagnosed by magnetic resonance tumor regression grade (mrTRG) and diffusion weighted imaging (DWI) as cCR after PCRT, also achieved pCR, in contrast to 88.9% of cases without SRCC. Signet ring histology was the only predictor of pCR in multivariate analysis (P=0.027). There was no statistically significant difference between both histological groups as regard to survival. SRCC is an important predictor of pCR and assessing their response to PCRT using mrTRG and DWI showed high sensitivity for the detection of cCR, making them good candidates for watch-and-wait approach. Histological types did not significantly affect the survival outcome.

The efficacy and safety of extended adjuvant temozolomide following concurrent radio-chemotherapy among Egyptian patients with newly diagnosed glioblastoma multiforme.

Although concurrent radio-chemotherapy and adjuvant temozolomide (TMZ) treatment for 6 cycles has been established as a standard of care for newly diagnosed glioblastoma multiforme (GBM) patients, the recommended duration of adjuvant TMZ remains a matter of debate. Hereby, we aimed to report for the first time our experience from Upper Egypt through comparing survival and toxicity profile between two treatment modalities of adjuvant TMZ (> six cycles versus six cycles) and delineating factors of prognostic significance in Egyptian patients with newly diagnosed GBM treated by radiation therapy with concomitant and adjuvant TMZ. Between June 2016 and February 2018, the medical records of 121 patients were eligible to be retrospectively reviewed to extract the study relevant data. All patients received concurrent radio-chemotherapy, followed by TMZ for 6 cycles in 29 patients (Group 1) and for >6 cycles in 26 patients (Group 2). Patients in Group 1 had a median PFS of 15 months (95% CI: 10.215-19.785), while those in Group 2 had a median PFS of 18 months (95% CI: 16.611-19.389). After a median follow up duration of 20 months (range: 12-41), the median OS was 18 months (95% CI: 13.420-22.580) in Group 1 and 22 months (95% CI: 18.777-25.223) in Group 2. There was no statistically significant correlation between the number of chemotherapy cycles and PFS (P=0.513) or OS (P=0.867). The extent of surgical resection was the only independent prognostic factor for both PFS (P=0.015) and OS (P=0.028) by multivariate analysis. Three grade ≥3 hematologic toxicity were encountered in 3 patients. One in the six-cycle group (neutropenia), and two in the extended cycles group (one had neutropenia and the other one developed thrombocytopenia). No statistically significant difference in the toxicity profile between both groups. The results of our study suggest that extended TMZ therapy is safe and tolerable, however it did not significantly improve PFS or OS as compared to the standard six-cycle course. Larger randomized studies are required to shed more light on this issue.

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.

Cell-Based Therapy for the Treatment of Glioblastoma: An Update from Preclinical to Clinical Studies.

Glioblastoma (GB), an aggressive primary tumor of the central nervous system, represents about 60% of all adult primary brain tumors. It is notorious for its extremely low (~5%) 5-year survival rate which signals the unsatisfactory results of the standard protocol for GB therapy. This issue has become, over time, the impetus for the discipline of bringing novel therapeutics to the surface and challenging them so they can be improved. The cell-based approach in treating GB found its way to clinical trials thanks to a marvelous number of preclinical studies that probed various types of cells aiming to combat GB and increase the survival rate. In this review, we aimed to summarize and discuss the up-to-date preclinical studies that utilized stem cells or immune cells to treat GB. Likewise, we tried to summarize the most recent clinical trials using both cell categories to treat or prevent recurrence of GB in patients. As with any other therapeutics, cell-based therapy in GB is still hampered by many drawbacks. Therefore, we highlighted several novel techniques, such as the use of biomaterials, scaffolds, nanoparticles, or cells in the 3D context that may depict a promising future when combined with the cell-based approach.

Mesenchymal Stem Cells: The Past Present and Future.

The biomedical applications of mesenchymal stem cells (MSCs) have gained expanding attention over the past three decades. MSCs are easily obtained from various tissue types (e.g. bone marrow, fat, cord blood, etc.), are capable of self-renewal, and could be induced to differentiate into several cell lineages for countless biomedical applications. In addition, when transplanted, MSCs are not detected by immune surveillance, thus do not lead to graft rejection. Moreover, they can home towards affected tissues and induce their therapeutic effect in a cell-base and/or a cell-free manner. These properties, and many others, have made MSCs appealing therapeutic cell candidates (for cell and/or gene therapy) in myriad clinical conditions. However, similar to any other therapeutic tool, MSCs still have their own limitations and grey areas that entail more research for better understanding and optimization. Herein, we present a brief overview of various pre-clinical/clinical applications of MSCs in regenerative medicine and discuss limitations and future challenges.

The Therapeutic Potential of Extracellular Vesicles Versus Mesenchymal Stem Cells in Liver Damage.

BACKGROUND: The extracellular vesicles (EVs) secreted by bone marrow-derived mesenchymal stem cells (MSCs) hold significant potential as a novel alternative to whole-cell therapy. We herein compare the therapeutic potential of BM-MSCs versus their EVs (MSC-EVs) in an experimental Carbon tetrachloride (CCl4)-induced liver damage rat model. METHODS: Rats with liver damage received a single IV injection of MSC-EVs, 1 million MSCs, or 3 million MSCs. The therapeutic efficacy of each treatment was assessed using liver histopathology, liver function tests and immunohistochemistry for liver fibrosis and hepatocellular injury. RESULTS: Animals that received an injection of either MSCs-EVs or 3 million MSCs depicted significant regression of collagen deposition in the liver tissue and marked attenuation of hepatocellular damage, both structurally and functionally. CONCLUSION: Similar to high doses of MSC-based therapy (3 million MSCs), MSC-EVs mitigated the fibrogenesis and hepatocellular injury in a rat model of CCl4-induced liver fibrosis. The anti-fibrinogenic effect was induced by attenuating hepatic stellate cell activation. Therefore, the administration of MSC-EVs could be considered as a candidate cell-free therapeutic strategy for liver fibrosis and hepatocellular damage.

Non-viral vectors based on cationic niosomes as efficient gene delivery vehicles to central nervous system cells into the brain.

Development of safe and efficient non-viral vectors to deliver DNA into the CNS represents a huge challenge to face many neurological disorders. We elaborated niosomes based on DOTMA cationic lipid, lycopene "helper" lipid and polysorbate 60 as non-ionic surfactants for gene delivery to the CNS. Niosomes, and their corresponding nioplexes obtained after the addition of the pCMS-EGFP plasmid, were characterized in terms of size, charge, morphology and capacity to condense, release and protect DNA. In vitro experiments were performed in NT2 cells to evaluate transfection efficiency, viability, cellular uptake and intracellular distribution. Additionally, transfection in primary cortex cells were performed prior to brain administration into rat cerebral cortex. Data obtained showed that nioplexes exhibited not only adequate physicochemical properties for gene delivery applications, but also relevant transfection efficiencies (17%), without hampering viability (90%). Interestingly, In vivo experiments depicted promising protein expression in both cortical glial cells and blood vessels.

Cerium oxide nanoparticles: In pursuit of liver protection against doxorubicin-induced injury in rats.

Doxorubicin (DOX) is considered as a backbone in several chemotherapeutic regimens. Nevertheless, the reported systemic toxicity usually hampers its broad application. Interestingly, Cerium oxide nanoparticles (CeONPs) depicted promising regenerative antioxidant and hepatoprotective potentials against multiple oxidative stress-induced pathologies. Thus, the aim of the present study was to determine either CeONPs would display hepatoprotective properties once concomitantly administered with DOX or not. Male Sprague Dawley rats were divided into four groups (n = 10) in a two weeks study: Control (received saline, IP injection thrice a week), CeO (0.5 mg/kg, IP injection once a week), DOX (2.5 mg/kg, IP injections thrice a week) and DOX + CeO (received both treatments). Hepatic toxicity was assessed by histological and ultrastructural studies. In addition, serum transaminases (ALT, AST) and malondialdehyde (MDA), an oxidative stress marker, were evaluated. CeONPs were not only proved to be safe at the proposed dose, but also their concomitant administration with DOX managed to mitigate DOX-induced hepatic insult on both histological and biochemical aspects. Such hepatoprotective behavior was referred to the noticed antioxidant action CeONPs as highlighted by the significant difference in MDA levels.

Stem cell-based gene delivery mediated by cationic niosomes for bone regeneration.

Bone morphogenetic protein-7(BMP-7) plays a pivotal role in the transformation of mesenchymal stem cells (MSCs) into bone. However, its impact is hampered due to its short half-life. Therefore, gene therapy may be an interesting approach to deliver BMP-7 gene to D1-MSCs. In this manuscript we prepared and characterized niosomes based on cationic lipid 2,3-di(tetradecyloxy)propan-1-amine, combined with polysorbate 80 for gene delivery purposes. Niosomes were characterized and combined initially with pCMS-EGFP reporter plasmid, and later with pUNO1-hBMP-7 plasmid to evaluate osteogenesis differentiation. Additionally, specific blockers of most relevant endocytic pathways were used to evaluate the intracellular disposition of complexes. MSCs transfected with niosomes showed increased growth rate, enhanced alkaline phosphatase activity (ALP) and extracellular matrix deposition which suggested the formation of osteoblast-like cells. We concluded that hBMP-7-transfected MSCs could be considered not only as an effective delivery tool of hBMP-7, but also as proliferating and bone forming cells for bone regeneration.

Transcatheter arterial chemoembolization of hepatocellular carcinoma in patients with celiac axis occlusion using pancreaticoduodenal arcade as a challenging alternative route.

INTRODUCTION: Celiac axis occlusion is a challenging condition when catheterization of the hepatic artery is required for chemoembolization of hepatocellular carcinoma (HCC). As a result, the hepatic artery has to be catheterized through the pancreaticoduodenal arcades (PDA) and the gastroduodenal artery (GDA) from the superior mesenteric artery (SMA) which is a tortuous course with acute angles and small caliber branches. OBJECTIVE: To assess new techniques for facilitating catheterization of the tortuous PDA and the GDA to reach the proper hepatic artery (PHA) and tumor-feeding branches in patients with celiac axis occlusion undergoing chemoembolization of HCC. METHODS AND MATERIALS: The study included eleven patients all admitted to do transcatheter arterial chemoembolization (TACE) for treatment of unresectable HCC. During angiography occlusion of the celiac axis was diagnosed and hypertrophied PDA and GDA was noted in SMA angiography. Catheterization of the PDA was performed by preshaping of the micro-guide wire into a wide curve. Catheterization of the PHA was a challenge and was achieved by reshaping of the micro-guide wire or by looping technique. TACE was done after super selective catheterization of the tumor feeding artery using a mixture of 50 mg of adriamycin, 7cc of lipiodol and gelfoam. RESULTS: In the eleven patients with celiac artery occlusion, DSA showed complete celiac axis occlusion in all patients. Collateral arteries supplying the liver were readily evident via PDA and GDA from SMA. Successful catheterization of the PHA was achieved in all patients. Chemoembolization was performed to all patients after super selective catheterization of the feeding artery. Follow-up triphasic CT was performed in all patients, 9 patients showed good lipiodol trapping with no residual tumor enhancement. Two patients required another session of TACE. CONCLUSION: Chemoembolization of HCC through the PDA and the GDA using micro-guide wire preshaping technique and the microcatheter looping technique in patients with celiac axis occlusion is a challenging but effective treatment for HCC.

The Effect of Bone Marrow-Derived Mesenchymal Stem Cells and Their Conditioned Media Topically Delivered in Fibrin Glue on Chronic Wound Healing in Rats.

Bone marrow-derived mesenchymal stem cells (BM-MSCs) represent a modern approach for management of chronic skin injuries. In this work, we describe BM-MSCs application versus their conditioned media (CM) when delivered topically admixed with fibrin glue to enhance the healing of chronic excisional wounds in rats. Fifty-two adult male rats were classified into four groups after induction of large-sized full-thickness skin wound: control group (CG), fibrin only group (FG), fibrin + MSCs group (FG + SCs), and fibrin + CM group (FG + CM). Healing wounds were evaluated functionally and microscopically. Eight days after injury, number of CD68+ macrophages infiltrating granulation tissue was considerably higher in the latter two groups. Although--later--none of the groups depicted a substantially different healing rate, the quality of regenerated skin was significantly boosted by the application of either BM-MSCs or their CM both (1) structurally as demonstrated by the obviously increased mean area percent of collagen fibers in Masson's trichrome-stained skin biopsies and (2) functionally as supported by the interestingly improved epidermal barrier as well as dermal tensile strength. Thus, we conclude that topically applied BM-MSCs and their CM-via fibrin vehicle--could effectively improve the quality of healed skin in chronic excisional wounds in rats, albeit without true acceleration of wound closure.

Behaviour and ultrastructure of human bone marrow-derived mesenchymal stem cells immobilised in alginate-poly-l-lysine-alginate microcapsules.

CONTEXT: Human bone marrow mesenchymal stem cells (hBM-MSCs) show a great promise for the treatment of a variety of diseases. Despite the previous trials to encapsulate hBM-MSCs in alginate-poly-l-lysine-alginate (APA) systems, the various changes that follow immobilisation have not been ascertained yet. OBJECTIVE: Determine the various consequences derived from entrapment on cell behaviour, putting special emphasis on the ultrastructure. METHODS: hBM-MSCs were immobilised in APA microcapsules to further characterise their viability, metabolic activity, proliferation, VEGF-secretability, and morphology. RESULTS: The VEGF produced by monolayer hBM-MSCs increased significantly 1 d post-encapsulation, and was maintained for at least 4 weeks. TEM imaging of cells revealed well preserved ultrastructure indicating protein synthesis and high metabolic activity. CONCLUSION: Although APA microencapsulation did not support 100% of fully viable hBM-MSCs for long-term cultures, it was conceived to enhance both VEGF secretion and metabolic activity while not losing their stemness characteristics.