Onychocolone A produced by the fungus Onychocola sp. targets cancer stem cells and stops pancreatic cancer progression by inhibiting MEK2-dependent cell signaling.

Pancreatic cancer (PC) shows a high fatality rate that can only be faced with a combination of surgery and chemotherapy or palliative treatment in the case of advanced patients. Besides, PC tumors are enriched with subpopulations of cancer stem cells (CSCs) that are resistant to the existing chemotherapeutic agents, which raises an important need for the identification of new drugs. To fill this gap, we have tested the anti-tumoral activity of microbial extracts, which chemical diversity offers a broad spectrum of potential new bioactive compounds. Extracts derived from the fungus Onychocola sp. CF-107644 were assayed via high throughput screening followed by bioassay-guided fractionation and resulted in the identification and isolation of six benzophenone derivatives with antitumoral activity: onychocolones A-F (#1-6). The structures of the compounds were established by spectroscopic methods, including ESI-TOF MS, 1D and 2D NMR analyses and X-ray diffraction. Compounds #1-4 significantly inhibited the growth of the pancreas tumoral cell lines, with low-micromolar Median Effective Doses (ED50s). Compound #1 (onychocolone A) was prioritized for further profiling due to its pro-apoptotic effect, which was further validated on 3D spheroids and pancreatic CSCs. Protein expression assays showed that the effect was mechanistically linked to the inhibition of MEK onco-signaling pathway. The efficacy of onychocolone A was also demonstrated in vivo by the reduction of tumor growth in a pancreatic xenograft mouse model generated by CSCs. Altogether, the data support that onychocolone A is a promising new small molecule for hit-to-lead development of a new treatment for PC.

Autologous patient-derived exhausted nano T-cells exploit tumor immune evasion to engage an effective cancer therapy.

BACKGROUND: Active targeting by surface-modified nanoplatforms enables a more precise and elevated accumulation of nanoparticles within the tumor, thereby enhancing drug delivery and efficacy for a successful cancer treatment. However, surface functionalization involves complex procedures that increase costs and timelines, presenting challenges for clinical implementation. Biomimetic nanoparticles (BNPs) have emerged as unique drug delivery platforms that overcome the limitations of actively targeted nanoparticles. Nevertheless, BNPs coated with unmodified cells show reduced functionalities such as specific tumor targeting, decreasing the therapeutic efficacy. Those challenges can be overcome by engineering non-patient-derived cells for BNP coating, but these are complex and cost-effective approaches that hinder their wider clinical application. Here we present an immune-driven strategy to improve nanotherapeutic delivery to tumors. Our unique perspective harnesses T-cell exhaustion and tumor immune evasion to develop a groundbreaking new class of BNPs crafted from exhausted T-cells (NExT) of triple-negative breast cancer (TNBC) patients by specific culture methods without sophisticated engineering. METHODS: NExT were generated by coating PLGA (poly(lactic-co-glycolic acid)) nanoparticles with TNBC-derived T-cells exhausted in vitro by acute activation. Physicochemical characterization of NExT was made by dynamic light scattering, electrophoretic light scattering and transmission electron microscopy, and preservation and orientation of immune checkpoint receptors by flow cytometry. The efficacy of chemotherapy-loaded NExT was assessed in TNBC cell lines in vitro. In vivo toxicity was made in CD1 mice. Biodistribution and therapeutic activity of NExT were determined in cell-line- and autologous patient-derived xenografts in immunodeficient mice. RESULTS: We report a cost-effective approach with a good performance that provides NExT naturally endowed with immune checkpoint receptors (PD1, LAG3, TIM3), augmenting specific tumor targeting by engaging cognate ligands, enhancing the therapeutic efficacy of chemotherapy, and disrupting the PD1/PDL1 axis in an immunotherapy-like way. Autologous patient-derived NExT revealed exceptional intratumor accumulation, heightened chemotherapeutic index and efficiency, and targeted the tumor stroma in a PDL1+ patient-derived xenograft model of triple-negative breast cancer. CONCLUSIONS: These advantages underline the potential of autologous patient-derived NExT to revolutionize tailored adoptive cancer nanotherapy and chemoimmunotherapy, which endorses their widespread clinical application of autologous patient-derived NExT.

Human mesenchymal stromal cells-laden crosslinked hyaluronic acid-alginate bioink for 3D bioprinting applications in tissue engineering.

Three-dimensional (3D) bioprinting is considered one of the most advanced tools to build up materials for tissue engineering. The aim of this work was the design, development and characterization of a bioink composed of human mesenchymal stromal cells (hMSC) for extrusion through nozzles to create these 3D structures that might potentially be apply to replace the function of damaged natural tissue. In this study, we focused on the advantages and the wide potential of biocompatible biomaterials, such as hyaluronic acid and alginate for the inclusion of hMSC. The bioink was characterized for its physical (pH, osmolality, degradation, swelling, porosity, surface electrical properties, conductivity, and surface structure), mechanical (rheology and printability) and biological (viability and proliferation) properties. The developed bioink showed high porosity and high swelling capacity, while the degradation rate was dependent on the temperature. The bioink also showed negative electrical surface and appropriate rheological properties required for bioprinting. Moreover, stress-stability studies did not show any sign of physical instability. The developed bioink provided an excellent environment for the promotion of the viability and growth of hMSC cells. Our work reports the first-time study of the effect of storage temperature on the cell viability of bioinks, besides showing that our bioink promoted a high cell viability after being extruded by the bioprinter. These results support the suggestion that the developed hMSC-composed bioink fulfills all the requirements for tissue engineering and can be proposed as a biological tool with potential applications in regenerative medicine and tissue engineering.

Unlocking the effective alliance of ß-lapachone and hydroxytyrosol against triple-negative breast cancer cells.

Triple-negative breast cancer (TNBC) is characterised by its aggressiveness and resistance to chemotherapy, demanding the development of effective strategies against its unique characteristics. Derived from lapacho tree bark, ß-lapachone (ß-LP) selectively targets cancer cells with elevated levels of the detoxifying enzyme NQO1. Hydroxytyrosol (HT) is a phenolic compound derived from olive trees with important anticancer properties that include the inhibition of cancer stem cells (CSCs) and metastatic features in TNBC, as well as relevant antioxidant activities by mechanisms such as the induction of NQO1. We aimed to study whether these compounds could have synergistic anticancer activity in TNBC cells and the possible role of NQO1. For this pourpose, we assessed the impact of ß-LP (0.5 or 1.5 µM) and HT (50 and 100 µM) on five TNBC cell lines. We demonstrated that the combination of ß-LP and HT exhibits anti-proliferative, pro-apoptotic, and cell cycle arrest effects in several TNBC cells, including docetaxel-resistant TNBC cells. Additionally, it effectively inhibits the self-renewal and clonogenicity of CSCs, modifying their aggressive phenotype. However, the notable impact of the ß-LP-HT combination does not appear to be solely associated with the levels of the NQO1 protein and ROS. RNA-Seq analysis revealed that the combination's anticancer activity is linked to a strong induction of endoplasmic reticulum stress and apoptosis through the unfolded protein response. In conclusion, in this study, we demonstrated how the combination of ß-LP and HT could offer an affordable, safe, and effective approach against TNBC.

Exploring the Impact of Nanoparticle Stealth Coatings in Cancer Models: From PEGylation to Cell Membrane-Coating Nanotechnology.

Nanotechnological platforms offer advantages over conventional therapeutic and diagnostic modalities. However, the efficient biointerfacing of nanomaterials for biomedical applications remains challenging. In recent years, nanoparticles (NPs) with different coatings have been developed to reduce nonspecific interactions, prolong circulation time, and improve therapeutic outcomes. This study aims to compare various NP coatings to enhance surface engineering for more effective nanomedicines. We prepared and characterized polystyrene NPs with different coatings of poly(ethylene glycol), bovine serum albumin, chitosan, and cell membranes from a human breast cancer cell line. The coating was found to affect the colloidal stability, adhesion, and elastic modulus of NPs. Protein corona formation and cellular uptake of NPs were also investigated, and a 3D tumor model was employed to provide a more realistic representation of the tumor microenvironment. The prepared NPs were found to reduce protein adsorption, and cell-membrane-coated NPs showed significantly higher cellular uptake. The secretion of proinflammatory cytokines in human monocytes after incubation with the prepared NPs was evaluated. Overall, the study demonstrates the importance of coatings in affecting the behavior and interaction of nanosystems with biological entities. The findings provide insight into bionano interactions and are important for the effective implementation of stealth surface engineering designs.

Development and characterization of a poloxamer hydrogel composed of human mesenchymal stromal cells (hMSCs) for reepithelization of skin injuries.

Wound healing is a natural physiological reaction to tissue injury. Hydrogels show attractive advantages in wound healing not only due to their biodegradability, biocompatibility and permeability but also because provide an excellent environment for cell migration and proliferation. The main objective of the present study was the design and characterization of a hydrogel loaded with human mesenchymal stromal cells (hMSCs) for use in would healing of superficial skin injures. Poloxamer 407® was used as biocompatible biomaterial to embed hMSCs. The developed hydrogel containing 20 % (w/w) of polymer resulted in the best formulation with respect to physical, mechanical, morphological and biological properties. Its high swelling capacity confirmed the hydrogel's capacity to absorb wounds' exudate. LIVE/DEAD® assay confirm that hMSCs remained viable for at least 48 h when loaded into the hydrogels. Adding increasing concentrations of hMSCs-loaded hydrogel to the epithelium did not affect keratinocytes' viability and healing capacity and all wound area was closed in less than one day. Our study opens opportunities to exploit poloxamer hydrogels as cell carriers for the treatment of skin superficial wound.

Interferon-Alpha Decreases Cancer Stem Cell Properties and Modulates Exosomes in Malignant Melanoma.

Malignant melanoma (MM) can spread to other organs and is resistant in part due to the presence of cancer stem cell subpopulations (CSCs). While a controversial high dose of interferon-alpha (IFN-α) has been used to treat non-metastatic high-risk melanoma, it comes with undesirable side effects. In this study, we evaluated the effect of low and high doses of IFN-α on CSCs by analyzing ALDH activity, side population and specific surface markers in established and patient-derived primary cell lines. We also assessed the clonogenicity, migration and tumor initiation capacities of IFN-α treated CSCs. Additionally, we investigated genomic modulations related to stemness properties using microRNA sequencing and microarrays. The effect of IFN-α on CSCs-derived exosomes was also analyzed using NanoSight and liquid chromatography (LC-HRMS)-based metabolomic analysis, among others. Our results showed that even low doses of IFN-α reduced CSC formation and stemness properties, and led to a significant decrease in the ability to form tumors in mice xenotransplants. IFN-α also modulated the expression of genes and microRNAs involved in several cancer processes and metabolomics of released exosomes. Our work suggests the utility of low doses of interferon, combined with the analysis of metabolic biomarkers, as a potential clinical approach against the aggressiveness of CSCs in melanoma.

Ratiometric Two-Photon Near-Infrared Probe to Detect DPP IV in Human Plasma, Living Cells, Human Tissues, and Whole Organisms Using Zebrafish.

DPP IV, otherwise known as CD26 lymphocyte T surface antigen, is a transmembrane glycoprotein also found in circulation in the blood. It plays an important role in several processes like glucose metabolism and T-cell stimulation. Moreover, it is overexpressed in renal, colon, prostate, and thyroid human carcinoma tissues. It can also serve as a diagnostic in patients with lysosomal storage diseases. The biological and clinical importance of having readouts for the activity of this enzyme, in physiological and disease conditions, has led us to design a near-infrared (NIR) fluorimetric probe that also has the characteristics of being ratiometric and excitable by two simultaneous NIR photons. The probe consists of assembling an enzyme recognition group (Gly-Pro) (Mentlein, 1999; Klemann et al., 2016) on the two-photon (TP) fluorophore (derivative of dicyanomethylene-4H-pyran, DCM-NH2) disturbing its NIR characteristic internal charge transfer (ICT) emission spectrum. When the dipeptide group is released by the DPP IV-specific enzymatic action, the donor-acceptor DCM-NH2 is restored, forming a system that shows high ratiometric fluorescence output. With this new probe, we have been able to detect, quickly and efficiently, the enzymatic activity of DPP IV in living cells, human tissues, and whole organisms, using zebrafish. In addition, due to the possibility of being excited by two photons, we can avoid the autofluorescence and subsequent photobleaching that the raw plasma has when it is excited by visible light, achieving detection of the activity of DPP IV in that medium without interference.

Biogeneration of silver nanoparticles from Cuphea procumbens for biomedical and environmental applications.

Nanotechnology is one of the most important and relevant disciplines today due to the specific electrical, optical, magnetic, chemical, mechanical and biomedical properties of nanoparticles. In the present study we demonstrate the efficacy of Cuphea procumbens to biogenerate silver nanoparticles (AgNPs) with antibacterial and antitumor activity. These nanoparticles were synthesized using the aqueous extract of C. procumbens as reducing agent and silver nitrate as oxidizing agent. The Transmission Electron Microscopy demonstrated that the biogenic AgNPs were predominantly quasi-spherical with an average particle size of 23.45 nm. The surface plasmonic resonance was analyzed by ultraviolet visible spectroscopy (UV-Vis) observing a maximum absorption band at 441 nm and Infrared Spectroscopy (FT IR) was used in order to structurally identify the functional groups of some compounds involved in the formation of nanoparticles. The AgNPs demonstrated to have antibacterial activity against the pathogenic bacteria Escherichia coli and Staphylococcus aureus, identifying the maximum zone of inhibition at the concentration of 0.225 and 0.158 µg/mL respectively. Moreover, compared to the extract, AgNPs exhibited better antitumor activity and higher therapeutic index (TI) against several tumor cell lines such as human breast carcinoma MCF-7 (IC50 of 2.56 µg/mL, TI of 27.65 µg/mL), MDA-MB-468 (IC50 of 2.25 µg/mL, TI of 31.53 µg/mL), human colon carcinoma HCT-116 (IC50 of 1.38 µg/mL, TI of 51.07 µg/mL) and melanoma A-375 (IC50 of 6.51 µg/mL, TI of 10.89 µg/mL). This fact is of great since it will reduce the side effects derived from the treatment. In addition, AgNPs revealed to have a photocatalytic activity of the dyes congo red (10-3 M) in 5 min and malachite green (10-3 M) in 7 min. Additionally, the degradation percentages were obtained, which were 86.61% for congo red and 82.11% for malachite green. Overall, our results demonstrated for the first time that C. procumbens biogenerated nanoparticles are excellent candidates for several biomedical and environmental applications.

TGFß Governs the Pleiotropic Activity of NDRG1 in Triple-Negative Breast Cancer Progression.

In triple-negative breast cancer (TNBC), the pleiotropic NDRG1 (N-Myc downstream regulated gene 1) promotes progression and worse survival, yet contradictory results were documented, and the mechanisms remain unknown. Phosphorylation and localization could drive NDRG1 pleiotropy, nonetheless, their role in TNBC progression and clinical outcome was not investigated. We found enhanced p-NDRG1 (Thr346) by TGFß1 and explored whether it drives NDRG1 pleiotropy and TNBC progression. In tissue microarrays of 81 TNBC patients, we identified that staining and localization of NDRG1 and p-NDRG1 (Thr346) are biomarkers and risk factors associated with shorter overall survival. We found that TGFß1 leads NDRG1, downstream of GSK3ß, and upstream of NF-κB, to differentially regulate migration, invasion, epithelial-mesenchymal transition, tumor initiation, and maintenance of different populations of cancer stem cells (CSCs), depending on the progression stage of tumor cells, and the combination of TGFß and GSK3ß inhibitors impaired CSCs. The present study revealed the striking importance to assess both total NDRG1 and p-NDRG1 (Thr346) positiveness and subcellular localization to evaluate patient prognosis and their stratification. NDRG1 pleiotropy is driven by TGFß to differentially promote metastasis and/or maintenance of CSCs at different stages of tumor progression, which could be abrogated by the inhibition of TGFß and GSK3ß.

Generation of a H9 Clonal Cell Line With Inducible Expression of NUP98-KDM5A Fusion Gene in the AAVS1 Safe Harbor Locus.

Pediatric acute myeloid leukemia (AML) is a rare and heterogeneous disease that remains the major cause of mortality in children with leukemia. To improve the outcome of pediatric AML we need to gain knowledge on the biological bases of this disease. NUP98-KDM5A (NK5A) fusion protein is present in a particular subgroup of young pediatric patients with poor outcome. We report the generation and characterization of human Embryonic Stem Cell (hESC) clonal lines with inducible expression of NK5A. Temporal control of NK5A expression during hematopoietic differentiation from hESC will be critical for elucidating its participation during the leukemogenic process.

Nanomedicine as a Promising Tool to Overcome Immune Escape in Breast Cancer.

Breast cancer is the most common type of malignancy and leading cause of cancer death among women worldwide. Despite the current revolutionary advances in the field of cancer immunotherapy, clinical response in breast cancer is frequently below expectations, in part due to various mechanisms of cancer immune escape that produce tumor variants that are resistant to treatment. Thus, a further understanding of the molecular events underlying immune evasion in breast cancer may guarantee a significant improvement in the clinical success of immunotherapy. Furthermore, nanomedicine provides a promising opportunity to enhance the efficacy of cancer immunotherapy by improving the delivery, retention and release of immunostimulatory agents in targeted cells and tumor tissues. Hence, it can be used to overcome tumor immune escape and increase tumor rejection in numerous malignancies, including breast cancer. In this review, we summarize the current status and emerging trends in nanomedicine-based strategies targeting cancer immune evasion and modulating the immunosuppressive tumor microenvironment, including the inhibition of immunosuppressive cells in the tumor area, the activation of dendritic cells and the stimulation of the specific antitumor T-cell response.

Design, synthesis, HER2 inhibition and anticancer evaluation of new substituted 1,5-dihydro-4,1-benzoxazepines.

A series of 11 new substituted 1,5-dihydro-4,1-benzoxazepine derivatives was synthesised to study the influence of the methyl group in the 1-(benzenesulphonyl) moiety, the replacement of the purine by the benzotriazole bioisosteric analogue, and the introduction of a bulky substituent at position 6 of the purine, on the biological effects. Their inhibition against isolated HER2 was studied and the structure-activity relationships have been confirmed by molecular modelling studies. The most potent compound against isolated HER2 is 9a with an IC50 of 7.31 µM. We have investigated the effects of the target compounds on cell proliferation. The most active compound (7c) against all the tumour cell lines studied (IC50 0.42-0.86 µM) does not produce any modification in the expression of pro-caspase 3, but increases the caspase 1 expression, and promotes pyroptosis.

Silver Nanoparticles from Annona muricata Peel and Leaf Extracts as a Potential Potent, Biocompatible and Low Cost Antitumor Tool.

Cancer is one of the most prevalent diseases in the world and requires new therapies for its treatment. In this context, the biosynthesis of silver nanoparticles (AgNPs) has been developed to treat different types of tumors. The Annona muricata plant is known for having anticancer activity. Its main compounds present in the leaves, stems and skin, allowing for its use as reducing agents. In this manuscript, AgNPs with leaf extract (AgNPs-LE) and fruit peel extract (AgNPs-PE) of A. muricata were biosynthesized obtaining an average nanoparticle diameter sizes smaller than 50 nm, being 19.63 ± 3.7 nm and 16.56 ± 4.1 nm, and with a surface plasmonic resonance (SPR) at 447 and 448 nm, respectively. The lactone functional group present in the LE and PE extracts was identified by the FTIR technique. The behavior and antiproliferation activity of AgNPs-LE and AgNPs-PE were evaluated in breast, colon and melanoma cancer cell lines. Our results showed that Annona muricata fruit peel, which is a waste product, has an antitumor effect more potent than leaf extract. This difference is maintained with AgNPs where the destruction of cancer cells was, for the first time, achieved using concentrations that do not exceed 3 µg/mL with a better therapeutic index in the different tumor strains. In conclusion, we present a low-cost one-step experimental setup to generate AgNPs-PE whose in-vitro biocompatibility and powerful therapeutic effect make it a very attractive tool worth exploiting.

Development of a Biomimetic Hydrogel Based on Predifferentiated Mesenchymal Stem-Cell-Derived ECM for Cartilage Tissue Engineering.

The use of decellularized extracellular matrix (dECM) as a biomaterial has been an important step forward for the development of functional tissue constructs. In addition to tissues and organs, cell cultures are gaining a lot of attention as an alternative source of dECM. In this work, a novel biomimetic hydrogel is developed based on dECM obtained from mesenchymal stem cells (mdECM) for cartilage tissue engineering. To this end, cells are seeded under specific culture conditions to generate an early chondrogenic extracellular matrix (ECM) providing cues and elements necessary for cartilage development. The composition is determined by quantitative, histological, and mass spectrometry techniques. Moreover, the decellularization process is evaluated by measuring the DNA content and compositional analyses, and the hydrogel is formulated at different concentrations (3% and 6% w/v). Results show that mdECM derived hydrogels possess excellent biocompatibility and suitable physicochemical and mechanical properties for their injectability. Furthermore, it is evidenced that this hydrogel is able to induce chondrogenesis of mesenchymal stem cells (MSCs) without supplemental factors and, furthermore, to form hyaline cartilage-like tissue after in vivo implantation. These findings demonstrate for the first time the potential of this hydrogel based on mdECM for applications in cartilage repair and regeneration.

Antioxidants for the Treatment of Breast Cancer: Are We There Yet?

Breast cancer is the most frequent cancer and the leading cause of cancer death in women. Oxidative stress and the generation of reactive oxygen species (ROS) have been related to cancer progression. Compared to their normal counterparts, tumor cells show higher ROS levels and tight regulation of REDOX homeostasis to maintain a low degree of oxidative stress. Traditionally antioxidants have been extensively investigated to counteract breast carcinogenesis and tumor progression as chemopreventive agents; however, there is growing evidence indicating their potential as adjuvants for the treatment of breast cancer. Aimed to elucidate whether antioxidants could be a reality in the management of breast cancer patients, this review focuses on the latest investigations regarding the ambivalent role of antioxidants in the development of breast cancer, with special attention to the results derived from clinical trials, as well as their potential use as plausible agents in combination therapy and their power to ameliorate the side effects attributed to standard therapeutics. Data retrieved herein suggest that antioxidants play an important role in breast cancer prevention and the improvement of therapeutic efficacy; nevertheless, appropriate patient stratification based on "redoxidomics" or tumor subtype is mandatory in order to define the dosage for future standardized and personalized treatments of patients.

Metabolomic profile of cancer stem cell-derived exosomes from patients with malignant melanoma.

Malignant melanoma (MM) is the most aggressive and life-threatening form of skin cancer. It is characterized by an extraordinary metastasis capacity and chemotherapy resistance, mainly due to melanoma cancer stem cells (CSCs). To date, there are no suitable clinical diagnostic, prognostic or predictive biomarkers for this neoplasia. Therefore, there is an urgent need for new MM biomarkers that enable early diagnosis and effective disease monitoring. Exosomes represent a novel source of biomarkers since they can be easily isolated from different body fluids. In this work, a primary patient-derived MM cell line enriched in CSCs was characterized by assessing the expression of specific markers and their stem-like properties. Exosomes derived from CSCs and serums from patients with MM were characterized, and their metabolomic profile was analysed by high-resolution mass spectrometry (HRMS) following an untargeted approach and applying univariate and multivariate statistical analyses. The aim of this study was to search potential biomarkers for the diagnosis of this disease. Our results showed significant metabolomic differences in exosomes derived from MM CSCs compared with those from differentiated tumour cells and also in serum-derived exosomes from patients with MM compared to those from healthy controls. Interestingly, we identified similarities between structural lipids differentially expressed in CSC-derived exosomes and those derived from patients with MM such as the glycerophosphocholine PC 16:0/0:0. To our knowledge, this is the first metabolomic-based study aimed at characterizing exosomes derived from melanoma CSCs and patients' serum in order to identify potential biomarkers for MM diagnosis. We conclude that metabolomic characterization of CSC-derived exosomes sets an open door to the discovery of clinically useful biomarkers in this neoplasia.

Obtaining Human Breast Adipose Cells for Breast Cancer Cell Co-culture Studies.

Primary human breast cancers invade surrounding fat and contact adipocytes, inflammatory infiltrates, and fibrous stroma. This tissue niche influences breast tumor progression. Here, we present a protocol to enable the in vitro study of the complex interactions that occur between breast cancer cells and adipose cells. We describe how to obtain different adipose cell populations, including adipose-derived stem cells, immature adipocytes, and mature adipocytes, from human breast fat tissue and detail the application for co-culture assays with breast cancer cells. For complete details on the use and execution of this protocol, please refer to Picon-Ruiz et al. (2016) and Qureshi et al. (2020).

Large-Scale Production of Lentiviral Vectors: Current Perspectives and Challenges.

Lentiviral vectors (LVs) have gained value over recent years as gene carriers in gene therapy. These viral vectors are safer than what was previously being used for gene transfer and are capable of infecting both dividing and nondividing cells with a long-term expression. This characteristic makes LVs ideal for clinical research, as has been demonstrated with the approval of lentivirus-based gene therapies from the Food and Drug Administration and the European Agency for Medicine. A large number of functional lentiviral particles are required for clinical trials, and large-scale production has been challenging. Therefore, efforts are focused on solving the drawbacks associated with the production and purification of LVsunder current good manufacturing practice. In recent years, we have witnessed the development and optimization of new protocols, packaging cell lines, and culture devices that are very close to reaching the target production level. Here, we review the most recent, efficient, and promising methods for the clinical-scale production ofLVs.

Clinical failure of nanoparticles in cancer: mimicking nature's solutions.

The use of nanotechnology has become a promising approach in the treatment of cancer. However, most intravenously injected nanoparticles (NPs) do not effectively reach the tumor mass due to the biological barriers in the body. In an attempt to unify clinical criteria and basic research, we have collected the latest studies and described novel alternatives such as the use of NPs covered with cell membranes to increase NP delivery efficiency. Furthermore, we focus on the prospect of using the cell's natural messengers, exosomes, as vehicles to transport anti-cancer agents and we discuss the technical complications involved. Finally, we propose novel approaches to produce engineered exosomes which may overcome such technical limitations in order to achieve a proper anti-cancer nanotherapy.