Vera Moura

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.

Toward a siRNA-containing nanoparticle targeted to breast cancer cells and the tumor microenvironment

The present work aimed at designing a lipid-based nanocarrier for siRNA delivery toward two cell sub-populations within breast tumors, the cancer and the endothelial cells from angiogenic tumor blood vessels. To achieve such goal, the F3 peptide, which is specifically internalized by nucleolin overexpressed on both those sub-populations, was used as a targeting moiety. The developed F3-targeted stable nucleic acid lipid particles presented adequate features for systemic administration. In addition, the attachment of the F3 peptide onto the liposomal surface enabled an internalization by both cancer and endothelial cells from angiogenic blood vessels that was significantly higher than the one observed with non-cancer cells. Sequence-specific downregulation of enhanced green fluorescent protein (eGFP) in eGFP-overexpressing human cancer cell lines, both at the protein and mRNA levels, was further observed upon delivery of anti-eGFP siRNA by F3-targeted liposomes, in contrast with the non-targeted counterpart. This effect was highly dependent on the content of poly(ethylene glycol) (PEG), as evidenced by the co-localization studies between the siRNA and the lysosomes. Overall, the present work represents an important contribution toward a nanoparticle with multi-targeting capabilities in breast cancer, both at the cellular and molecular level.

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.

Anticancer activity and antibody-dependent cell-mediated cytotoxicity of novel anti-nucleolin antibodies

Nucleolin arises as a relevant target for cancer therapy, as it is overexpressed at the surface of cancer and angiogenic endothelial cells thus enabling a dual cellular targeting strategy. Immunotherapeutic strategies, albeit of proven therapeutic relevance, have been scarcely explored against this target. Therefore, this work aimed at engineering an anti-nucleolin VHH-based antibody capable of triggering anticancer immune responses. Herein, anti-nucleolin VHHs have been generated upon grafting F3 peptide-derived nucleolin-binding sequences onto a VHH CDR1 or CDR3. One of these nucleolin-binding CDR3-grafted VHH was subsequently fused to a human IgG1 Fc region, enabling a significant antibody-dependent cell-mediated cytotoxicity (ADCC). The generated anti-nucleolin VHH revealed increased binding and antiproliferative effects against cancer cells, relative to the parental VHH, while the VHH-Fc counterpart presented increased cytotoxicity relative to the corresponding VHH. This VHH-Fc also triggered an ADCC effect, in the nanomolar range, against a nucleolin-overexpressing cancer cell line. This effect was evidenced by a 2 or 1.7-fold increase of cell death, in the presence of PBMCs, relative to the parental VHH-Fc or the VHH counterpart, respectively. Overall, these formats represent the first anti-nucleolin VHHs and the first anti-nucleolin antibody with ADCC activity that have been successfully developed.

Lipid-Based Nanosystems for the Delivery of siRNA: Challenges and Trends

Abstract The introduction of the RNA interference (RNAi) mechanism by small-interference RNA (siRNA)-mediated gene silencing had an extensive impact on the study of cell biology, enabling novel therapeutic solutions. Nevertheless, their development has faced several challenges based on the prospect of systemic administration of siRNAs. Overcoming the challenging pharmacokinetics of siRNAs using different strategies, including lipid-based delivery systems, has required an enormous effort, which has shaped the current landscape of RNAi medicines progressing through clinical development, focusing on the liver as the major target organ. Still, there is promising preliminary clinical data on human gene silencing working against different cancers. Accordingly, this chapter explores RNAi based on siRNA as a therapeutic tool in oncology, in the context of lipid-based delivery. Herein, the challenges in achieving siRNA-mediated gene silencing upon systemic administration are addressed, as well as the plethora of lipid-based technologies currently in clinical trials against different cancers that have emerged from the effort in overcoming those hurdles. In addition, novel strategies and concepts, such as ligand-mediated targeted delivery, are explored in the context of the tumor microenvironment.

Moving Liposome Technology from the Bench to the Oncological Patient: Towards Performance-by-Design

A liposome is defined as a spherical vesicle having multiple or a single lipid bilayer surrounding an aqueous compartment enabling the entrapment of hydrophobic or water-soluble substances respectively, and thus suitable as a drug delivery system. Different methods for preparation of liposomes at small scale were developed throughout time, establishing the principles for the introduction of modern large scale industrial production processes. Associated with the adaptation and/or introduction of powerful characterization techniques, such as dynamic light scattering for particle size distribution analysis, novel therapeutic drug delivery systems were developed aiming at the oncology setting, many reaching the clinics, such as Doxil/Caelyx (liposomal PEGylated doxorubicin). Nevertheless, the approval of novel liposome-based medicines has not fulfilled the expectations, owing to the biological barriers they face, before reaching the cancer cell in the tumor microenvironment. Accordingly, the increased understanding on cancer biology, subsequently shaping the design of the delivery system, has become paramount for the successful development of novel generations of liposomes. Thus, herein we will address the technological accomplishments associated with the development of nanoliposomal formulations, focusing on their preparation and characterization as part of the design and quality control. We will further address the impact of liposome properties on their interaction with living organisms, upon systemic administration, and how this knowledge has benefited the oncological patient.

Targeting Cancer Stem Cells and Non-Stem Cancer Cells: The Potential of Lipid- Based Nanoparticles

Background Cancer stem cells (CSCs) have been described as a relevant contributor to tumorigenicity, metastasis, tumor recurrence and drug resistance, making this cell population a relevant target in solid tumors. METHODS This has stimulated the development of different therapeutic strategies often targeting surface markers (CD44, epithelial cell adhesion molecule (EpCAM), aldehyde dehydrogenase (ALDH) and nucleolin) and/or signaling pathways that are aberrantly activated and contribute to CSCs proliferation and survival. RESULTS There are a variety of signaling pathways often involved in physiological processes of cell function that aberrantly regulate CSCs, including Notch, Hedgehog, Wnt, PI3K/Akt, JAK/STAT and Ras/ERK signaling pathways. The inhibition of these pathways usually depletes CSC population and increases tumor sensitivity to chemotherapy. However, the recognition of the potential of cells to interconvert in response to environmental stimulus, turned both CSCs and non-stem cancer cells into two relevant therapeutic targets. Therefore, the use of drug combinations is increasingly needed. These drugs with different mechanisms of action often characterized by distinct pharmacokinetics profiles and, as such, will present distinct biodistribution patterns, following systemic administration. To synchronize pharmacokinetics, one can encapsulate synergistic drug combinations into lipid-based nanoparticles, assuring tumor delivery of the selected drug ratio. CONCLUSION This review will focus on the multiple strategies to target CSCs, as well as on the potential of lipid-based nanoparticles to target both CSCs and non-stem cancer cells.

Advances on nucleic acid delivery with nonviral vectors

Abstract Oligonucleotide delivery is currently considered a promising strategy against a wide variety of diseases, either acutely acquired or genetically inherited, by modulating their genetic causes. However, the success of such a strategy is highly dependent on the efficient delivery of intact therapeutic genetic material to the target tissues or cells. The degradation by nucleases in biological medium, as well as the large molecular size and negative charge of the genetic material to be delivered represent major challenges to the in vivo application of gene therapy. In this chapter, the major challenges faced by oligonucleotide delivery will be discussed, as well as the most recent and promising advances in the field, highlighting the use of innovative nonviral vectors. New promising tools for gene therapy, such as miRNA and gene editing technologies (eg, TALENs or CRISPR), will also be explored.