Nuno A. Fonseca

Lipid-based nanoparticles for siRNA delivery in cancer therapy: paradigms and challenges.

RNA interference (RNAi) is a specific gene-silencing mechanism that can be mediated by the delivery of chemical synthesized small-interfering RNA (siRNA). RNAi might constitute a novel therapeutic approach for cancer treatment because researchers can easily design siRNA molecules to inhibit, specifically and potently, the expression of any protein involved in tumor initiation and progression. Despite all the potential of siRNA as a novel class of drugs, the limited cellular uptake, low biological stability, and unfavorable pharmacokinetics of siRNAs have limited their application in the clinic. Indeed, blood nucleases easily degrade naked siRNAs, and the kidneys rapidly eliminate these molecules. Furthermore, at the level of target cells, the negative charge and hydrophilicity of siRNAs strongly impair their cellular internalization. Therefore, the translation of siRNA to the clinical setting is highly dependent on the development of an appropriate delivery system, able to ameliorate siRNA pharmacokinetic and biodistribution properties. In this regard, major advances have been achieved with lipid-based nanocarriers sterically stabilized by poly(ethylene glycol) (PEG), such as the stabilized nucleic acid lipid particles (SNALP). However, PEG has not solved all the major problems associated with siRNA delivery. In this Account, the major problems associated with PEGylated lipid-based nanoparticles, and the different strategies to overcome them are discussed. Although PEG has revolutionized the field of nanocarriers, cumulative experience has revealed that upon repeated administration, PEGylated liposomes lose their ability to circulate over long periods in the bloodstream, a phenomenon known as accelerated blood clearance. In addition, PEGylation impairs the internalization of the siRNA into the target cell and its subsequent escape from the endocytic pathway, which reduces biological activity. An interesting approach to overcome such limitations relies on the design of novel exchangeable PEG-derivatized lipids. After systemic administration, these lipids can be released from the nanoparticle surface. Moreover, the design and synthesis of novel cationic lipids that are more fusogenic and the use of internalizing targeting ligands have contributed to the emergence of novel lipid-based nanoparticles with remarkable transfection efficiency.

Antibacterial activity of chitosan nanofiber meshes with liposomes immobilized releasing gentamicin.

Chitsan (Ch) nanofiber mesh (NFM) is a material with natural characteristics favoring its use in human wound dressing. The present work proposes a gentamicin-loaded liposome immobilized at the surface of Ch NFMs to promote its antibacterial activity. To achieve this purpose, Ch NFMs were functionalized with thiol groups, and gentamicin-loaded liposomes were covalently immobilized by the reaction of the SH groups with maleimide. The maximum concentration of SH groups (55.52±11.19nmolcm(-2)) was obtained at pH 7. A fluorescent dye was covalently bound to the SH groups present at the surface of electrospun Ch NFMs. Their spatial distribution was uniform throughout the NFMs when analyzed by fluorescence microscopy. Gentamicin was successfully encapsulated into the liposomes with an efficiency of 17%. Gentamicin-loaded liposomes were uniformly distributed at the surface of the Ch NFMs and the drug release kinetic showed a sustained release of gentamicin during 16h, achieving a steady state at 24h. The in vitro susceptibility tests confirmed that the gentamicin released from the liposomes immobilized at the surface of electrospun Ch NFM has bactericidal activity against Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus. The results show that the developed system has promising performance for wound dressing applications, avoiding infections caused by these common pathogens.

Bridging cancer biology and the patients' needs with nanotechnology-based approaches

Cancer remains as stressful condition and a leading cause of death in the western world. Actual cornerstone treatments of cancer disease rest as an elusive alternative, offering limited efficacy with extensive secondary effects as a result of severe cytotoxic effects in healthy tissues. The advent of nanotechnology brought the promise to revolutionize many fields including oncology, proposing advanced systems for cancer treatment. Drug delivery systems rest among the most successful examples of nanotechnology. Throughout time they have been able to evolve as a function of an increased understanding from cancer biology and the tumor microenvironment. Marketing of Doxil® unleashed a remarkable impulse in the development of drug delivery systems. Since then, several nanocarriers have been introduced, with aspirations to overrule previous technologies, demonstrating increased therapeutic efficacy besides decreased toxicity. Spatial and temporal targeting to cancer cells has been explored, as well as the use of drug combinations co-encapsulated in the same particle as a mean to take advantage of synergistic interactions in vivo. Importantly, targeted delivery of siRNA for gene silencing therapy has made its way to the clinic for a first in man trial using lipid-polymeric-based particles. Focusing in state-of-the-art technology, this review will provide an insightful vision on nanotechnology-based strategies for cancer treatment, approaching them from a tumor biology-driven perspective, since their early EPR-based dawn to the ones that have truly the potential to address unmet medical needs in the field of oncology, upon targeting key cell subpopulations from the tumor microenvironment.

Immobilization of bioactive factor-loaded liposomes on the surface of electrospun nanofibers targeting tissue engineering

Electrospun nanofiber meshes (NFM), due to their morphology and fibrous structure, are extensively proposed as biomedical devices, for tissue engineering on scaffolds and also as drug delivery systems. Liposomes are nanoparticles prepared from a biologically derived material (phospholipid), which are already in clinical use as a drug release device. Liposomes may be combined with biomaterial scaffolds to promote a local and sustained delivery of loaded bioactive agents. The main objective of the present study is to evaluate the efficacy of dexamethasone (Dex)-loaded liposomes immobilized on the surface of electrospun polycaprolactone (PCL) NFM for promoting the osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hBMSCs). The in vitro release profile demonstrates a sustained release of Dex over 21 days, after an initial burst release over 12 h. Biological assays show that Dex-loaded liposomes immobilized on the surface of electrospun PCL NFMs do not exhibit any cytotoxic effect, being able to successfully promote the osteogenic differentiation of hBMSCs. We herein validate the concept of using liposomes immobilized on the surface of a nanostructured fibrous system to be used as an advanced cell carrier device with autonomous release of growth/differentiation factors relevant for tissue engineering and regenerative medicine strategies.

On the use of dexamethasone-loaded liposomes to induce the osteogenic differentiation of human mesenchymal stem cells

Stem cells have received considerable attention by the scientific community because of their potential for tissue engineering and regenerative medicine. The most frequently used method to promote their differentiation is supplementation of the in vitro culture medium with growth/differentiation factors (GDFs). The limitations of that strategy caused by the short half‐life of GDFs limit its efficacy in vivo and consequently its clinical use. Thus, the development of new concepts that enable the bioactivity and bioavailability of GDFs to be protected, both in vitro and in vivo, is very relevant. Nanoparticle‐based drug delivery systems can be injected, protect the GDFs and enable spatiotemporal release kinetics to be controlled. Liposomes are well‐established nanodelivery devices presenting significant advantages, viz. a high load‐carrying capacity, relative safety and easy production, and a versatile nature in terms of possible formulations and surface functionalization. The main objective of the present study was to optimize the formulation of liposomes to encapsulate dexamethasone (Dex). Our results showed that the optimized Dex‐loaded liposomes do not have any cytotoxic effect on human bone marrow‐derived mesenchymal stem cells (hBMSCs). More importantly, they were able to promote an earlier induction of differentiation of hBMSCs into the osteogenic lineage, as demonstrated by the expression of osteoblastic markers, both phenotypically and genotypically. We concluded that Dex‐loaded liposomes represent a viable nanoparticle strategy with enhanced safety and efficacy for tissue engineering and regenerative medicine. Copyright

Nucleolin overexpression in breast cancer cell sub-populations with different stem-like phenotype enables targeted intracellular delivery of synergistic drug combination.

Breast cancer stem cells (CSC) are thought responsible for tumor growth and relapse, metastization and active evasion to standard chemotherapy. The recognition that CSC may originate from non-stem cancer cells (non-SCC) through plastic epithelial-to-mesenchymal transition turned these into relevant cell targets. Of crucial importance for successful therapeutic intervention is the identification of surface receptors overexpressed in both CSC and non-SCC. Cell surface nucleolin has been described as overexpressed in cancer cells as well as a tumor angiogenic marker. Herein we have addressed the questions on whether nucleolin was a common receptor among breast CSC and non-SCC and whether it could be exploited for targeting purposes. Liposomes functionalized with the nucleolin-binding F3 peptide, targeted simultaneously, nucleolin-overexpressing putative breast CSC and non-SCC, which was paralleled by OCT4 and NANOG mRNA levels in cells from triple negative breast cancer (TNBC) origin. In murine embryonic stem cells, both nucleolin mRNA levels and F3 peptide-targeted liposomes cellular association were dependent on the stemness status. An inxa0vivo tumorigenic assay suggested that surface nucleolin overexpression per se, could be associated with the identification of highly tumorigenic TNBC cells. This proposed link between nucleolin expression and the stem-like phenotype in TNBC, enabled 100% cell death mediated by F3 peptide-targeted synergistic drug combination, suggesting the potential to abrogate the plasticity and adaptability associated with CSC and non-SCC. Ultimately, nucleolin-specific therapeutic tools capable of simultaneous debulk multiple cellular compartments of the tumor microenvironment may pave the way towards a specific treatment for TNBC patient care.

Simultaneous active intracellular delivery of doxorubicin and C6-ceramide shifts the additive/antagonistic drug interaction of non-encapsulated combination.

Drug resistance remains the Achilles tendon undermining the success of chemotherapy. It has been recognized that success requires the identification of compounds that, when combined, lead to synergistic tumor inhibition while simultaneously minimizing systemic toxicity. However, in vivo application of such protocols is dependent on the ability to deliver the appropriate drug ratio at the tumor level. In this respect, nanotechnology-based delivery platforms, like liposomes, offer an elegant solution for the in vivo translation of such strategy. In this work, we propose the active intracellular delivery of combinations of doxorubicin and the pro-apoptotic sphingolipid, C6-ceramide, using our previously described cytosolic triggered release-enabling liposomes, targeting nucleolin with the F3 peptide. Combination of doxorubicin (DXR):C6-ceramide (C6-Cer) at 1:2 molar ratio interacted synergistically against drug resistant/triple negative MDA-MB-231 breast cancer cells, as well as drug sensitive MDA-MB-435S melanoma cells. Cell viability studies indicated that F3-targeted liposomes encapsulating DXR:C6-Cer 1:2 molar ratio (p[F3]DC12) performed similarly as targeted liposomal DXR (p[F3]SL), encapsulating twice the amount of DXR, at the IC50, for an incubation time of 24 h. Importantly, F3-targeted liposomes encapsulating DXR:C6-Cer 1:2 molar ratio (p[F3]DC12) enabled a cell death above 90% at 24 h of treatment against both DXR-resistant and sensitive cells, unattainable by the F3-targeted liposomal doxorubicin. Furthermore, a F3-targeted formulation encapsulating a mildly additive/antagonistic DXR:C6-Cer 1:1 molar ratio (p[F3]DC11) enabled an effect above 90% for an incubation period as short as 4 h, suggesting that the delivery route at the cell level may shift the nature of drug interaction. Such activity, including the one for p[F3]DC12, induced a marked cell and nucleus swelling at similar extent, consistent with necrotic cell death. Overall, these results demonstrated that F3-targeted intracellular delivery of different DXR/C6-Cer ratios, with diversed drug interactions, enabled a highly relevant increased efficacy against chemotherapy resistant cells.

The cancer stem cell phenotype as a determinant factor of the heterotypic nature of breast tumors

Gathering evidence supports the existence of a population of cells with stem-like characteristics, named cancer stem cells (CSC), which is involved not only in tumor recurrence but also in tumorigenicity, metastization and drug resistance. Several markers have been used to identify putative CSC sub-populations in different cancers. Notwithstanding, it has been acknowledged that breast CSC may originate from non-stem cancer cells (non-SCC), interconverting through an epithelial-to-mesenchymal transition-mediated process, and presenting several deregulated canonical and developmental signaling pathways. These support the heterogeneity that, directly or indirectly, influences fundamental biological features supporting breast tumor development. Accordingly, CSC have increasingly become highly relevant cellular targets. In this review, we will address the stemness concept in cancer, setting the perspective on CSC and their origin, by exploring their relation and regulation within the tumor microenvironment, in the context of emerging therapeutic targets. Within this framework, we will discuss nucleolin, a protein that has been associated with angiogenesis and, more recently, with the stemness phenotype, becoming a common denominator between CSC and non-SCC for multicellular targeting.

Inoculated Cell Density as a Determinant Factor of the Growth Dynamics and Metastatic Efficiency of a Breast Cancer Murine Model

4T1 metastatic breast cancer model have been widely used to study stage IV human breast cancer. However, the frequent inoculation of a large number of cells, gives rise to fast growing tumors, as well as to a surprisingly low metastatic take rate. The present work aimed at establishing the conditions enabling high metastatic take rate of the triple-negative murine 4T1 syngeneic breast cancer model. An 87% 4T1 tumor incidence was observed when as few as 500 cancer cells were implanted. 4T1 cancer cells colonized primarily the lungs with 100% efficiency, and distant lesions were also commonly identified in the mesentery and pancreas. The drastic reduction of the number of inoculated cells resulted in increased tumor doubling times and decreased specific growth rates, following a Gompertzian tumor expansion. The established conditions for the 4T1 mouse model were further validated in a therapeutic study with peguilated liposomal doxorubicin, in clinical used in the setting of metastatic breast cancer. Inoculated cell density was proven to be a key methodological aspect towards the reproducible development of macrometastases in the 4T1 mouse model and a more reliable pre-clinical assessment of antimetastatic therapies.

Dual release of a hydrophilic and a hydrophobic osteogenic factor from a single liposome

Delivery systems may be designed to protect and control the release kinetics of growth/differentiation factors in a spatiotemporal manner. Liposomes are examples of biological-based bioactive agent delivery systems. In this work, ascorbic acid (AscA) was encapsulated in the inner compartment of the liposome and dexamethasone (Dex) was encapsulated within the lipid bilayer in order to develop a dual release system of these bioactive agents involved in the osteogenic differentiation of mesenchymal stem cells (MSCs). The particle size (∼150 nm) of the prepared liposomes showed a monodisperse distribution. The bioactive agent release study showed that Dex was released more rapidly from the liposomes than AscA. The Dex release profile showed an initial burst release within 12 h; afterwards, a slower and sustained release was observed until 21 days. The release of AscA from the liposomes was not detected until 6 h; afterwards, a linear release was observed from 24 h until 21 days. The effect of Dex–AscA-loaded liposomes on the viability, proliferation and osteogenic differentiation of human bone marrow-derived MSCs (hBMSCs) were assessed. The cell culture results showed that the Dex–AscA-loaded liposomes (in a single dose or in repeated doses) do not have any cytotoxic effect. Dex–AscA-loaded liposomes given once did not promote induction of hBMSCs differentiation into the osteogenic lineage. However, Dex–AscA-loaded liposomes given repeatedly promoted the hBMSCs differentiation into the osteogenic lineage, both in basal medium and complete osteogenic medium. These results were genotypically demonstrated by the expression of osteoblastic markers. In conclusion, Dex–AscA-loaded liposomes represent a biological nanoparticle strategy with potential safety and efficacy for bone tissue engineering approaches.