In Vitro and In Vivo Tumor Models for the Evaluation of Anticancer Nanoparticles.

Multiple studies about tumor biology have revealed the determinant role of the tumor microenvironment in cancer progression, resulting from the dynamic interactions between tumor cells and surrounding stromal cells within the extracellular matrix. This malignant microenvironment highly impacts the efficacy of anticancer nanoparticles by displaying drug resistance mechanisms, as well as intrinsic physical and biochemical barriers, which hamper their intratumoral accumulation and biological activity.Currently, two-dimensional cell cultures are used as the initial screening method in vitro for testing cytotoxic nanocarriers. However, this fails to mimic the tumor heterogeneity, as well as the three-dimensional tumor architecture and pathophysiological barriers, leading to an inaccurate pharmacological evaluation.Biomimetic 3D in vitro tumor models, on the other hand, are emerging as promising tools for more accurately assessing nanoparticle activity, owing to their ability to recapitulate certain features of the tumor microenvironment and thus provide mechanistic insights into nanocarrier intratumoral penetration and diffusion rates.Notwithstanding, in vivo validation of nanomedicines remains irreplaceable at the preclinical stage, and a vast variety of more advanced in vivo tumor models is currently available. Such complex animal models (e.g., genetically engineered mice and patient-derived xenografts) are capable of better predicting nanocarrier clinical efficiency, as they closely resemble the heterogeneity of the human tumor microenvironment.Herein, the development of physiologically more relevant in vitro and in vivo tumor models for the preclinical evaluation of anticancer nanoparticles will be discussed, as well as the current limitations and future challenges in clinical translation.

Using the quality by design (QbD) approach to optimize formulations of lipid nanoparticles and nanoemulsions: A review.

Quality-by-design (QbD) approach has been applied to optimize lipid-based nanosystems formulations, including solid lipid nanoparticles (SLN), nanostructured lipid carriers (NLC) and nanoemulsions, besides being increasingly requested by regulatory authorities. Different mathematical models and statistical tests have been used, with similar conclusions regarding the parameters that influence the physical features of the resulting nanosystems. These include, variations in composition (e.g. lipid(s) and/or emulsifier(s)) and manufacturing parameters (e.g. emulsification rate and/or time, sonication amplitude and/or time, and homogenization pressure and/or cycles). These are critical parameters that influence nanoparticle/globule mean size, polydispersity index, zeta potential, drug encapsulation efficiency and in vitro drug release. This review addresses the concepts and applications of QbD for the development of lipid-based nanosystems, reporting successful examples published in the last 2 years. Although, some limitations have been identified, it is expected that in the upcoming years the application of QbD in pharmaceutical development will be an established approach.

Nucleolin is expressed in patient-derived samples and glioblastoma cells, enabling improved intracellular drug delivery and cytotoxicity.

One of the major challenges in Glioblastoma (GBM) therapy relates with the existence of glioma stem-like cells (GSCs), known to be chemo- and radio-resistant. GSCs and non-stem GBM cells have the ability to interchange, emphasizing the importance of identifying common molecular targets among those cell sub-populations. Nucleolin overexpression has been recently associated with breast cancer sub-populations with different stem-like phenotype. The goal of this work was to evaluate the potential of cell surface nucleolin as a target in GBM cells. Different levels of nucleolin expression resulted in a 3.4-fold higher association of liposomes targeting nucleolin (functionalized with the nucleolin-binding F3 peptide) in U87, relative to GBM11 glioblastoma cells. Moreover, nucleolin was suggested as a potential marker in OCT4-, NANOG-positive GSC, and in the corresponding non-stem GBM cells, as well as in SOX2-positive GSC. Doxorubicin delivered by liposomes targeting nucleolin enabled a level of cytotoxicity that was 2.5- or 4.6-fold higher compared to the non-targeted counterparts. Importantly, an overexpression of nucleolin was also observed in cells of patient-derived samples, as compared with normal brain. Overall, these results suggested nucleolin as a therapeutic target in GBM.

Challenging the future of siRNA therapeutics against cancer: the crucial role of nanotechnology.

The identification of numerous deregulated signaling pathways on cancer cells and supportive stromal cells has revealed several molecular targets whose downregulation can elicit significant benefits for cancer treatment. In this respect, gene downregulation can be efficiently achieved by exploiting the RNA interference mechanism, particularly by the delivery of chemical synthesized small-interfering RNAs (siRNAs), which have the ability to mediate, in a specific manner, the degradation of any mRNA with complementary nucleotide sequence. However, several concerns regarding off-target effects and immune stimulation have been raised. Depending on their sequence, siRNAs can trigger an innate immune response, which might mediate undesirable side effects that ultimately compromise their clinical utility. This is a very relevant effect that will be discussed in the present manuscript. Moreover, the major drawback in the translation of siRNAs into the clinical practice is undoubtedly their inability to accumulate in tumor sites, particularly in organs other than the liver. In fact, upon systemic administration, owing to siRNAs physico-chemical features, they are rapidly cleared from the blood stream. Therefore, the development of a proper drug delivery system is of utmost importance. In this review, some of the latest advances on different nanotechnological platforms for siRNA delivery under clinical evaluation will be discussed. Along with this, targeting approaches towards cancer and/or endothelial cells will also be addressed, as these are some of the most promising strategies to enhance specific tumor accumulation while avoiding healthy tissues. Finally, clinical information on ongoing studies in patients with advanced solid tumors will be also provided.

Brain metastasis: An insight into novel molecular targets for theranostic approaches.

Brain metastases (BrM) are common malignant lesions in the central nervous system, and pose a significant threat in advanced-stage malignancies due to delayed diagnosis and limited therapeutic options. Their distinct genomic profiles underscore the need for molecular profiling to tailor effective treatments. Recent advances in cancer biology have uncovered molecular drivers underlying tumor initiation, progression, and metastasis. This, coupled with the advances in molecular imaging technology and radiotracer synthesis, has paved the way for the development of innovative radiopharmaceuticals with enhanced specificity and affinity for BrM specific targets. Despite the challenges posed by the blood-brain barrier to effective drug delivery, several radiolabeled compounds have shown promise in detecting and targeting BrM. This manuscript provides an overview of the recent advances in molecular biomarkers used in nuclear imaging and targeted radionuclide therapy in both clinical and preclinical settings. Additionally, it explores potential theranostic applications addressing the unique challenges posed by BrM.

Current Insights on Lipid-Based Nanosystems 2023.

Among the different types of nanosystems that have been investigated for therapeutic use, lipid-based ones are the most explored, as they have advantages over non-lipid nanosystems, especially for improving the transport and efficacy of drugs through different routes of administration, such as ocular, cutaneous, intranasal, and intravenous [...].

Preclinical validation of a new hybrid molecule loaded in liposomes for melanoma management.

The aggressiveness of melanoma and lack of effective therapies incite the discovery of novel strategies. Recently, a new dual acting hybrid molecule (HM), combining a triazene and a ʟ-tyrosine analogue, was synthesized. HM was designed to specifically be activated by tyrosinase, the enzyme involved in melanin biosynthesis and overexpressed in melanoma. HM displayed remarkable superior antiproliferative activity towards various cancer cell lines compared with temozolomide (TMZ), a triazene drug in clinical use, that acts through DNA alkylation. In B16-F10 cells, HM induced a cell cycle arrest at phase G0/G1 with a 2.8-fold decrease in cell proliferation index. Also, compared to control cells, HM led to a concentration-dependent reduction in tyrosinase activity and increase in caspase 3/7 activity. To maximize the therapeutic performance of HM in vivo, its incorporation in long blood circulating liposomes, containing poly(ethylene glycol) (PEG) at their surface, was performed for passively targeting tumour sites. HM liposomes (LIP HM) exhibited high stability in biological fluids. Preclinical studies demonstrated its safety for systemic administration and in a subcutaneous murine melanoma model, significantly reduced tumour progression. In a metastatic murine melanoma model, a superior antitumour effect was also observed for mice receiving LIP HM, with markedly reduction of lung metastases compared to positive control group (TMZ). Biodistribution studies using 111In-labelled LIP HM demonstrated its ability for passively targeting tumour sites, thus correlating with the high therapeutic effect observed in the two experimental murine melanoma models. Overall, our proposed nanotherapeutic strategy was validated as an effective and safe alternative against melanoma.

Development, characterization and in vitro evaluation of single or co-loaded imatinib mesylate liposomal formulations.

Mitoxantrone-based combinations are a standard palliative treatment in hormone-refractory prostate cancer (HRPC) but with no survival benefit. Imatinib has shown preclinical activity against HRPC although minimal clinical therapeutic efficacy. Our previous in vitro studies demonstrated that simultaneous combination of imatinib with mitoxantrone yielded additive growth inhibition effects against PC-3 cell line. The main aim of the work was to develop novel liposomal formulations comprising imatinib co-encapsulated with mitoxantrone, by different loading methods and experimental conditions, in order to achieve the highest drug loading and maximum physical stability. In the optimized formulations, imatinib and mitoxantrone were actively co-loaded by means of a (NH4)2SO4 transmembrane gradient. Encapsulation efficiency, mean size diameter and drug retention in storage and in biological conditions were characterized. Our study presented for the first time an active loading method for imatinib and suggests that the optimized liposomal formulation co-encapsulates both drugs with high encapsulation efficiency (> 95%), shows enhanced drug retention under tested conditions and delivers a drug:drug ratio capable of improving tumor cell growth inhibition with a mitoxantrone dose reduction of 2.6-fold as compared to single liposomal formulation. Therefore, our nanotechnology-based drug combined platform may constitute a promising strategy in prostate cancer therapy.

New advances in exosome-based targeted drug delivery systems.

In recent years, various drug nano-delivery platforms have emerged to enhance drug effectiveness in cancer treatment. However, their successful translation to clinics have been hampered by unwanted side effects, as well as associated toxicity. Therefore, there is an imperative need for drug delivery vehicles capable of surpassing cellular barriers and also efficiently transfer therapeutic payloads to tumor cells. Exosomes, a class of small extracellular vesicles naturally released from all cells, have been exploited as a favorable delivery vehicle due to their natural role in intracellular communication and biocompatibility. In this review, information on exosome biogenesis, contents, forms of isolation and their natural functions is discussed, further complemented with the various successful methodologies for therapeutic payloads encapsulation, including distinct loading approaches. In addition, grafting of molecules to improve pharmacokinetics, tumor homing-ligands, as well as stimuli-responsive elements to enhance cell specificity are also debated. In the end, the current status of clinical-grade exosome-based therapies is outlined.

Immunotherapy in triple-negative breast cancer: Insights into tumor immune landscape and therapeutic opportunities.

Triple-negative breast cancer (TNBC) is a clinically aggressive subtype of breast cancer that represents 15-20% of breast tumors and is more prevalent in young pre-menopausal women. It is the subtype of breast cancers with the highest metastatic potential and recurrence at the first 5 years after diagnosis. In addition, mortality increases when a complete pathological response is not achieved. As TNBC cells lack estrogen, progesterone, and HER2 receptors, patients do not respond well to hormone and anti-HER2 therapies, and conventional chemotherapy remains the standard treatment. Despite efforts to develop targeted therapies, this disease continues to have a high unmet medical need, and there is an urgent demand for customized diagnosis and therapeutics. As immunotherapy is changing the paradigm of anticancer treatment, it arises as an alternative treatment for TNBC patients. TNBC is classified as an immunogenic subtype of breast cancer due to its high levels of tumor mutational burden and presence of immune cell infiltrates. This review addresses the implications of these characteristics for the diagnosis, treatment, and prognosis of the disease. Herein, the role of immune gene signatures and tumor-infiltrating lymphocytes as biomarkers in TNBC is reviewed, identifying their application in patient diagnosis and stratification, as well as predictors of efficacy. The expression of PD-L1 expression is already considered to be predictive of response to checkpoint inhibitor therapy, but the challenges regarding its value as biomarker are described. Moreover, the rationales for different formats of immunotherapy against TNBC currently under clinical research are discussed, and major clinical trials are highlighted. Immune checkpoint inhibitors have demonstrated clinical benefit, particularly in early-stage tumors and when administered in combination with chemotherapy, with several regimens approved by the regulatory authorities. The success of antibody-drug conjugates and research on other emerging approaches, such as vaccines and cell therapies, will also be addressed. These advances give hope on the development of personalized, more effective, and safe treatments, which will improve the survival and quality of life of patients with TNBC.

Editorial-Current Insights on Lipid-Based Nanosystems.

Lipid-based nanosystems, including solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC), cationic lipid nanoparticles, nanoemulsions and liposomes, have been extensively studied to improve drug delivery through different administration routes [...].

Antagonist G-targeted liposomes for improved delivery of anticancer drugs in small cell lung carcinoma.

Ligand-mediated targeted liposomes have the potential to increase therapeutic efficacy of anticancer drugs. This work aimed to evaluate the ability of antagonist G, a peptide targeting agent capable of blocking the action of multiple neuropeptides, to selectivity improve targeting and internalization of liposomal formulations (long circulating liposomes, LCL, and stabilized antisense lipid particles containing ionizable amino lipid, SALP) to H69 and H82 small cell lung carcinoma (SCLC) cell lines. Antagonist G-targeted LCL and SALP were prepared by two different methods (either by direct covalent linkage at activated PEG grafted onto the liposomal surface or by post-insertion of DSPE-PEG-antagonist-G-conjugates into pre-formed liposomes). Association of the liposomal formulations with target SCLC cells was studied by fluorescence microscopy using fluorescence-labelled liposomes and confirmed quantitatively with [3H]-CHE-labelled liposomes. An antisense oligodeoxynucleotide against the overexpressed oncogene c-myc(as(c-myc)) was efficiently loaded into SALP formulations, the encapsulation efficiency decreased due to the inclusion of the targeting ligand. Also, liposome size was affected by as(c-myc) physical chemical properties. The amount of antagonist G linked to the surface of the liposomal formulations was dependent on the coupling method and lipid composition used. Covalent attachment of antagonist G increased liposomes cellular association and internalization via receptor-mediated and clathrin-dependent endocytosis, as assessed in SCLC cell lines. Biodistribution studies in healthy mice revealed a preferential lung accumulation of antagonist G-targeted SALP as compared to the non-targeted counterpart. Lung levels of the former were up to 3-fold higher 24 h after administration, highlighting their potential to be used as delivery vectors for SCLC treatment.

Targeted liposomal doxorubicin/ceramides combinations: The importance to assess the nature of drug interaction beyond bulk tumor cells.

One of the major assets of anticancer nanomedicine is the ability to co-deliver drug combinations, as it enables targeting of different cellular populations and/or signaling pathways implicated in tumorigenesis and thus tackling tumor heterogeneity. Moreover, drug resistance can be circumvented, for example, upon co-encapsulation and delivery of doxorubicin and sphingolipids, as ceramides. Herein, the impact of short (C6) and long (C18) alkyl chain length ceramides on the nature of drug interaction, within the scope of combination with doxorubicin, was performed in bulk triple-negative breast cancer (TNBC) cells, as well as on the density of putative cancer stem cells and phenotype, including live single-cell tracking. C6- or C18-ceramide enabled a synergistic drug interaction in all conditions and (bulk) cell lines tested. However, differentiation among these two ceramides was reflected on the migratory potential of cancer cells, particularly significant against the highly motile MDA-MB-231 cells. This effect was supported by the downregulation of the PI3K/Akt pathway enabled by C6-ceramide and in contrast with C18-ceramide. The decrease of the migratory potential enabled by the targeted liposomal combinations is of high relevance in the context of TNBC, due to the underlying metastatic potential. Surprisingly, the nature of the drug interaction assessed at the level of bulk cancer cells revealed to be insufficient to predict whether a drug combination enables a decrease in the percentage of the master regulators of tumor relapse as ALDH+/high putative TNBC cancer stem cells, suggesting, for the first time, that it should be extended further down to this level.

Nucleolin expression has prognostic value in neuroblastoma patients.

BACKGROUND: Neuroblastoma (NB) represents the most frequent form of extra-cranial solid tumour of infants, responsible for 15% of childhood cancer deaths. Nucleolin (NCL) prognostic value in NB was investigated. METHODS: NCL protein expression was retrospectively evaluated in tumour samples of NB patients at diagnosis and after chemotherapy. NCL prognostic value at mRNA level was assessed in a cohort of 20 patients with stage 4 NB (qPCR20, n=20, discovery dataset) and in the MultiPlatform786 including 786 patients of all stages (validation dataset). Overall and event-free survival curves were plotted by Kaplan-Meier method and compared by log-rank test. FINDINGS: NCL protein, down-modulated after chemotherapy in association with features of neuroblastic differentiation,resulted statistically significantly overexpressed in NB tumours and higher in stage 4 compared to stage 1,2,3 patients. In the stage 4 patients cohort qPCR20, patients with high NCLmRNA expression revealed a statisticallysignificant lower survival probability than those with low NCL expression (OS: HR 4.1 95%CI 1.2-13.8;p=0.0215[Log-rank test], EFS: HR 4.1 95%CI 1.2-14.0, p=0.0197[Log-rank test]). In the MultiPlatform786 (n=786), multivariate analysis suggested thatNCL expression has a statistically significant prognostic value even in the model adjusted for established prognostic markers. NCL expression significantly stratified also patients with >18 months and stage 4 tumour (OS: HR 1.8 95%CI 1.2-2.7, p=0.0009[Log-rank test]; EFS: HR 1.7 95%CI 1.1-2.5, p=0.002[Log-rank test]), patients with>18 months stage 4 with MYCN non amplified tumour[EFS: HR 2.3 95%CI 1.2-4.7, p=0.01[Log-rank test]), and patients with MYCN non amplified and MYC high [OS: HR 11.9 95%CI 2.3-62.4, p=0.003[Log-rank test]; EFS: HR 7.2 95%CI 1.6-33.4, p=0.01[Log-rank test]). A statistically significant correlation between NCL and MYCN, MYC, and TERT was found in independent datasets (MultiPlatform786 (n=786) and Agilent394 (n=394). Gene set enrichment analysis revealed a statisticallysignificant positive enrichment of MYC target genes and genes involved in telomerase maintenance. INTERPRETATION: NCL is a novel and independent (adjusting for age, INSS stage, and MYCN status) prognostic marker for NB. FUNDING: IMH-EuroNanoMed II-2015 and AIRC-IG.

Liposomal imatinib-mitoxantrone combination: formulation development and therapeutic evaluation in an animal model of prostate cancer.

BACKGROUND: Mitoxantrone plus prednisone is a palliative treatment for hormone-refractory prostate cancer (HRPC) but without survival benefit. Imatinib has shown activity against HRPC but only in the preclinical setting. Our previous in vitro cytotoxicity screening study established that their free combination act additively to inhibit proliferation of PC-3 cells. We aim to develop a liposomal imatinib-mitoxantrone (LIM) formulation with improved in vivo therapeutic activity. METHODS: Imatinib and mitoxantrone were simultaneously co-loaded into DSPC/Chol liposomes by means of a (NH4)2SO4-generated proton gradient method. The optimized formulation was characterized in terms of mean size diameter, loading parameters and drug retention in human serum. In vivo antitumor activity of developed LIM formulation was evaluated in a nude mice bearing subcutaneous PC-3 xenograft model. RESULTS: LIM formulation exhibited maximal encapsulation efficiency (>95%) and enhanced drug retention for both drugs. Additionally, this LIM formulation, administered at a low mitoxantrone dose (0.5 mg/kg), showed a tumor inhibition activity (TGI = 66.7% and 4.0-fold tumor volume increase) slightly superior to that of liposomal mitoxantrone (LM) at 2 mg/kg (TGI = 53.0% and 4.2-fold volume increase). Therefore, therapeutic activity of mitoxantrone was significantly improved by co-loading with imatinib since a four times lower dose was needed to achieve an equivalent growth inhibition effect. CONCLUSIONS: The loading parameters and drug retention properties of our LIM formulation, combined with its in vivo antitumor activity, make this formulation an excellent strategy to improve the therapeutic index of mitoxantrone and a promising candidate for clinical development in prostate cancer therapy.

Supramolecular protamine/Gd-loaded liposomes adducts as relaxometric protease responsive probes.

A new approach to enzyme-responsive MRI agents based on the use of liposomes loaded with a high number of paramagnetic metal complexes (Gd-HPDO3A) is presented. It relies on the disruption of low relaxivity aggregates formed by liposomes and a macromolecular substrate that is selectively cleaved by the enzyme of interest. The interaction of anionic liposomes composed of POPC:CHOL:DPGS and the cationic protein protamine yields a poorly soluble supramolecular assembly endowed with a low relaxivity. The action of the serine protease trypsin causes the digestion of protamine and the consequent de-assembly of the supramolecular adduct. The process is accompanied by an overall relaxation enhancement of solvent water protons as consequence of the dissolution of the aggregated liposomes. The observed increase of relaxivity is linearly dependent on the enzyme concentration. An illustrative example of the possible use of the herein presented responsive agent has been reported. It consists of the entrapment of the supramolecular assembly in alginate microcapsules that have often been used as envelopes for in vivo applications of stem cells and pancreatic islets. The change in the observed longitudinal relaxation rate R(1) (leading to an hyperintense signal in the corresponding MR images) may act as a sensor of the protease activity in the biological environment in which the capsules is located.

Cancer Stem Cells and Nucleolin as Drivers of Carcinogenesis.

Cancer, one of the most mortal diseases worldwide, is characterized by the gain of specific features and cellular heterogeneity. Clonal evolution is an established theory to explain heterogeneity, but the discovery of cancer stem cells expanded the concept to include the hierarchical growth and plasticity of cancer cells. The activation of epithelial-to-mesenchymal transition and its molecular players are widely correlated with the presence of cancer stem cells in tumors. Moreover, the acquisition of certain oncological features may be partially attributed to alterations in the levels, location or function of nucleolin, a multifunctional protein involved in several cellular processes. This review aims at integrating the established hallmarks of cancer with the plasticity of cancer cells as an emerging hallmark; responsible for tumor heterogeneity; therapy resistance and relapse. The discussion will contextualize the involvement of nucleolin in the establishment of cancer hallmarks and its application as a marker protein for targeted anticancer therapies.

Intranasal delivery of nanostructured lipid carriers, solid lipid nanoparticles and nanoemulsions: A current overview of in vivo studies.

The management of the central nervous system (CNS) disorders is challenging, due to the need of drugs to cross the blood‒brain barrier (BBB) and reach the brain. Among the various strategies that have been studied to circumvent this challenge, the use of the intranasal route to transport drugs from the nose directly to the brain has been showing promising results. In addition, the encapsulation of the drugs in lipid-based nanocarriers, such as solid lipid nanoparticles (SLNs), nanostructured lipid carriers (NLCs) or nanoemulsions (NEs), can improve nose-to-brain transport by increasing the bioavailability and site-specific delivery. This review provides the state-of-the-art of in vivo studies with lipid-based nanocarriers (SLNs, NLCs and NEs) for nose-to-brain delivery. Based on the literature available from the past two years, we present an insight into the different mechanisms that drugs can follow to reach the brain after intranasal administration. The results of pharmacokinetic and pharmacodynamics studies are reported and a critical analysis of the differences between the anatomy of the nasal cavity of the different animal species used in in vivo studies is carried out. Although the exact mechanism of drug transport from the nose to the brain is not fully understood and its effectiveness in humans is unclear, it appears that the intranasal route together with the use of NLCs, SLNs or NEs is advantageous for targeting drugs to the brain. These systems have been shown to be more effective for nose-to-brain delivery than other routes or formulations with non-encapsulated drugs, so they are expected to be approved by regulatory authorities in the coming years.

In Vitro Studies on Nasal Formulations of Nanostructured Lipid Carriers (NLC) and Solid Lipid Nanoparticles (SLN).

The nasal route has been used for many years for the local treatment of nasal diseases. More recently, this route has been gaining momentum, due to the possibility of targeting the central nervous system (CNS) from the nasal cavity, avoiding the blood-brain barrier (BBB). In this area, the use of lipid nanoparticles, such as nanostructured lipid carriers (NLC) and solid lipid nanoparticles (SLN), in nasal formulations has shown promising outcomes on a wide array of indications such as brain diseases, including epilepsy, multiple sclerosis, Alzheimer's disease, Parkinson's disease and gliomas. Herein, the state of the art of the most recent literature available on in vitro studies with nasal formulations of lipid nanoparticles is discussed. Specific in vitro cell culture models are needed to assess the cytotoxicity of nasal formulations and to explore the underlying mechanism(s) of drug transport and absorption across the nasal mucosa. In addition, different studies with 3D nasal casts are reported, showing their ability to predict the drug deposition in the nasal cavity and evaluating the factors that interfere in this process, such as nasal cavity area, type of administration device and angle of application, inspiratory flow, presence of mucoadhesive agents, among others. Notwithstanding, they do not preclude the use of confirmatory in vivo studies, a significant impact on the 3R (replacement, reduction and refinement) principle within the scope of animal experiments is expected. The use of 3D nasal casts to test nasal formulations of lipid nanoparticles is still totally unexplored, to the authors best knowledge, thus constituting a wide open field of research.

Current challenges and emerging opportunities of CAR-T cell therapies.

Infusion of chimeric antigen receptor (CAR)-genetically modified T cells (CAR-T cells) have led to remarkable clinical responses and cancer remission in patients suffering from relapsed or refractory B-cell malignancies. This is a new form of adoptive T cell therapy (ACT), whereby the artificial CAR enables the redirection of T cells endogenous antitumor activity towards a predefined tumor-associated antigen, leading to the elimination of a specific tumor. The early success in blood cancers has prompted the US Food and Drug Administration (FDA) to approve the first CAR-T cell therapies for the treatment of CD19-positive leukemias and lymphomas in 2017. Despite the emergence of CAR-T cells as one of the latest breakthroughs of cancer immunotherapies, their wider application has been hampered by specific life-threatening toxicities, and a substantial lack of efficacy in the treatment of solid tumors, owing to the strong immunosuppressive tumor microenvironment and the paucity of reliable tumor-specific targets. Herein, besides providing an overview of the emerging CAR-technologies and current clinical applications, the major hurdles of CAR-T cell therapies will be discussed, namely treatment-related life-threatening toxicities and the obstacles posed by the immunosupressive tumor-microenvironment of solid tumors, as well as the next-generation strategies currently designed to simultaneously improve safety and efficacy of CAR-T cell therapies in vivo.