André F. Moreira

3D tumor spheroids: an overview on the tools and techniques used for their analysis

In comparison with 2D cell culture models, 3D spheroids are able to accurately mimic some features of solid tumors, such as their spatial architecture, physiological responses, secretion of soluble mediators, gene expression patterns and drug resistance mechanisms. These unique characteristics highlight the potential of 3D cellular aggregates to be used as in vitro models for screening new anticancer therapeutics, both at a small and large scale. Nevertheless, few reports have focused on describing the tools and techniques currently available to extract significant biological data from these models. Such information will be fundamental to drug and therapeutic discovery process using 3D cell culture models. The present review provides an overview of the techniques that can be employed to characterize and evaluate the efficacy of anticancer therapeutics in 3D tumor spheroids.

Chitosan/arginine–chitosan polymer blends for assembly of nanofibrous membranes for wound regeneration

Frequently, skin is subjected to damaging events, such as deep cuts, burns or ulcers, which may compromise the integrity of this organ. To overcome such lesions, different strategies have been employed. Among them, wound dressings aimed to re-establish skin native properties and decreased patient pain have been pursued for a long time. Herein, an electrospun membrane comprised by deacetylated/arginine modified chitosan (CH-A) was produced to be used as a wound dressing. The obtained results showed that the membrane has a highly hydrophilic and porous three-dimensional nanofibrous network similar to that found in human native extracellular matrix. In vitro data indicate that human fibroblasts adhere and proliferate in contact with membranes, thus corroborating their biocompatibility. This nanofiber-based biomaterial also demonstrated bactericidal activity for two bacterial strains. In vivo application of CH-A nanofibers in full thickness wounds resulted in an improved tissue regeneration and faster wound closure, when compared to non-modified membranes. Such findings support the suitability of using this membrane as a wound dressing in a near future.

Combinatorial delivery of Crizotinib–Palbociclib–Sildenafil using TPGS-PLA micelles for improved cancer treatment

The co-delivery of multiple chemotherapeutics by micellar delivery systems is a valuable approach to improve cancer treatment since various disease hallmarks can be targeted simultaneously. However, the delivery of multiple drugs requires a nanocarrier structure that can encapsulate various bioactive molecules. In this study, we evaluate the simultaneous encapsulation of a novel triple drug combination in D-α-tocopheryl polyethylene glycol 1000 succinate-poly(lactic acid) (TPGS-PLA) amphiphilic micelles for cancer therapy. The drug mixture involves two anti-tumoral drugs, Crizotinib and Palbociclib combined with Sildenafil, a compound that is capable of increasing drug accumulation in the intracellular compartment. Such combination aims to achieve an enhanced cytotoxic effect in cancer cells. Our results demonstrated that TPGS-PLA copolymers self-assembled into stable nanosized micelles (158.3nm) capable of co-encapsulating the three drugs with high loading efficiency. Triple drug loaded TPGS-PLA micelles were internalized in A549 non-small lung cancer cells and exhibited an improved cytotoxic effect in comparison with single (Crizotinib) or dual (Crizotinib-Palbociclib) drug loaded micelles, indicating the therapeutic potential of the triple co-delivery strategy. These findings demonstrate that TPGS-PLA micelles are suitable carriers for multiple drug delivery and also that this particular drug combination may have potential to improve cancer treatment.

Minicircle DNA vectors for gene therapy: advances and applications

Introduction: Nucleic-acid-based biopharmaceuticals enclose a remarkable potential for treating debilitating or life-threatening diseases that currently remain incurable. This promising area of research envisages the creation of state-of-the-art DNA vaccines, pluripotent cells or gene-based therapies, which can be used to overcome current issues. To achieve this goal, DNA minicircles are emerging as ideal nonviral vectors due to their safety and persistent transgene expression in either quiescent or actively dividing cells. Areas covered: This review focuses on the characteristics of minicircle DNA (mcDNA) technology and the current advances in their production. The possible modifications to further improve minicircle efficacy are also emphasized and discussed in light of recent advances. As a final point, the main therapeutic applications of mcDNA are summarized, with a special focus on pluripotent stem cells production and cancer therapy. Expert opinion: Achieving in-target and persistent transgene expression is a challenging issue that is of critical importance for a successful therapeutic outcome. The use of miniaturized mcDNA cassettes with additional modifications that increase and prolong expression may contribute to an improved generation of biopharmaceuticals. The unique features of mcDNA render it an attractive alternative to overcome current technical issues and to bridge the significant gap that exists between basic research and clinical applications.

Gas-generating TPGS-PLGA microspheres loaded with nanoparticles (NIMPS) for co-delivery of minicircle DNA and anti-tumoral drugs

Drug-DNA combination therapies are receiving an ever growing focus due to their potential for improving cancer treatment. However, such approaches are still limited by the lack of multipurpose delivery systems that encapsulate drugs and condense DNA simultaneously. In this study, we describe the successful formulation of gas-generating pH-responsive D-α-tocopherol PEG succinate-poly(D,L-lactic-co-glycolic acid) (TPGS-PLGA) hollow microspheres loaded with both Doxorubicin (Dox) and minicircle DNA (mcDNA) nanoparticles as a strategy to co-deliver these therapeutics. For this study mcDNA vectors were chosen due to their increased therapeutic efficiency in comparison to standard plasmid DNA. The results demonstrate that TPGS-PLGA microcarriers can encapsulate Dox and chitosan nanoparticles completely condense mcDNA. The loading of mcDNA-nanoparticles into microspheres was confirmed by 3D confocal microscopy and co-localization analysis. The resulting TPGS-PLGA-Dox-mcDNA nanoparticle-in-microsphere hybrid carriers exhibit a well-defined spherical shape and neutral surface charge. Microcarriers incubation in acidic pH produced a gas-mediated Dox release, corroborating the microcarriers stimuli-responsive character. Also, the dual-loaded TPGS-PLGA particles achieved 5.2-fold higher cellular internalization in comparison with non-pegylated microspheres. This increased intracellular concentration resulted in a higher cytotoxic effect. Successful transgene expression was obtained after nanoparticle-mcDNA co-delivery in the microspheres. Overall these findings support the concept of using nanoparticle-microsphere multipart systems to achieve efficient co-delivery of various drug-mcDNA combinations.

Preparation of end-capped pH-sensitive mesoporous silica nanocarriers for on-demand drug delivery

Nanocarriers with a pH responsive behavior are receiving an ever growing attention due to their potential for promoting on-demand drug release and thus increase the therapeutic effectiveness of anti-tumoral pharmaceutics. However, the majority of these systems require costly, time-consuming and complex chemical modifications of materials or drugs to synthesize nanoparticles with pH triggered release. Herein, the development of dual drug loaded pH-responsive mesoporous silica nanoparticles (MSNs) with a calcium carbonate-based coating is presented as an effective alternative. This innovative approach allowed the loading of a non-steroidal anti-inflammatory drug (Ibuprofen) and Doxorubicin, with high efficiency. The resulting dual drug loaded MSNs have spherical morphology and a mean size of 171nm. Our results indicate that under acidic conditions the coating disassembles and the drugs are rapidly released, whereas at physiologic pH the release is slower and gradually increases with time. Furthermore, an improved cytotoxic effect was obtained for Doxorubicin-Ibuprofen MSNs coated with CaCO3 in comparison with non-coated particles. The cytotoxic effect of dual loaded carbonate coated particles, was similar to that of Doxorubicin+Ibuprofen free drug administration at 72h, even with the delivery of a significantly lower amount of drug by MSNs-CaCO3. These results suggest that the carbonate coating of MSNs is a promising approach to create a pH-sensitive template for a delivery system with application in cancer therapy.

Thermo- and pH-responsive nano-in-micro particles for combinatorial drug delivery to cancer cells

Abstract Drug combinatorial therapy has been gaining the scientific community attention as a suitable approach to increase treatments efficacy and promote cancer eradication. In this study, a new pH‐ and thermo‐ responsive carrier was developed by combining doxorubicin‐loaded gold‐core silica shell nanorods with salicylic acid loaded poly (lactic‐co‐glycolic acid) based microparticles (NIMPS). The obtained results showed that the drugs and nanorods release could be triggered by the near‐infrared (NIR) laser irradiation or by the exposition to an acidic environment. The in vitro 2D cell studies showed that the NIMPS are biocompatible and easily uptaken by HeLa cells. In addition, 3D cell culture models revealed that the NIMPS administration, combined with the NIR laser irradiation, was capable of reducing the size of the HeLa spheroids up to 48%. Overall, the attained data support the application of the nano‐in‐micro spheres as a dual stimuli responsive drug carrier system for the local administration of combined therapies to cervical cancer cells. Graphical abstract Figure. No Caption available.

The effect of the shape of gold core–mesoporous silica shell nanoparticles on the cellular behavior and tumor spheroid penetration

Size, surface charge, and shape have a huge influence on the behavior, cellular uptake, and cytotoxic profile of nanoparticles. Herein, gold core and silica shell based nanoparticles (Au-MSSs) with spherical or rod-like shape were produced, in order to disclose the effect of the shape of nanomaterials on the cellular uptake, reactive oxygen species (ROS) production, 3D tumor spheroid penetration and cytotoxicity towards cancer cells. The Au-MSS spheres induced greater reduction of the ROS content of cancer cells and also displayed a more homogeneous distribution and penetration in 3D tumor spheroids. However, the Au-MSS rods show enhanced cellular migration and uptake in 2D cell cultures, which results in a higher drug delivering capacity. Furthermore, the Au-MSS rods displayed an enhanced cellular cytotoxicity upon laser irradiation (808 nm, 1.7 W cm-2, 10 min), where less than 10% of cells remained viable. In addition, both Au-MSSs have the potential to be used as imaging agents, which further expands their applicability as theranostic agents in the biomedical area. In summary, the obtained results show that the shape of Au-MSSs is crucial for their biological performance, which will have a great influence on their therapeutic outcome. Therefore, our findings may provide useful information for the development and design of new drug delivery systems towards a more effective therapy.

Spheroids formation on non-adhesive surfaces by Liquid Overlay Technique: considerations and practical approaches†

Scalable and reproducible production of 3D cellular spheroids is highly demanded, by pharmaceutical companies, for drug screening purposes during the pre‐clinical evaluation phase. These 3D cellular constructs, unlike the monolayer culture of cells, can mimic different features of human tissues, including cellular organization, cell–cell and cell‐extracellular matrix (ECM) interactions. Up to now, different techniques (scaffold‐based and ‐free) have been used for spheroids formation, being the Liquid Overlay Technique (LOT) one of the most explored methodologies, due to its low cost and easy handling. Additionally, during the last few decades, this technique has been widely investigated in order to enhance its potential for being applied in high‐throughput analysis. Herein, an overview of the LOT advances, practical approaches, and troubleshooting is provided for those researchers that intend to produce spheroids using LOT, for drug screening purposes. Moreover, the advantages of the LOT over the other scaffold‐free techniques used for the spheroids formation are also addressed.

Chapter 6 – Multifunctional nanocarriers for codelivery of nucleic acids and chemotherapeutics to cancer cells

Combinatorial therapies established on codelivery of drugs and nucleic acids are receiving increased attention due to their outstanding potential for improving cancer therapy in comparison to standalone treatments. This encouraging approach gathers the anticancer activity of chemotherapeutics and nucleic acid capacity to repair deregulated signaling pathways, as a joint strategy to achieve a beneficial anticancer effect. Such coadministration of drugs and genes is, however, remarkably challenging as these therapeutics exhibit distinct physicochemical properties. This chapter outlines the concepts underlying combinatorial therapy and the development of multifunctional nanocarriers specifically designed for codelivery of drug–gene combinations to cancer cells. A particular emphasis is given to key nanocarrier physicochemical properties required for drug–nucleic acid loading, release, and delivery in target organs. Various examples of multifunctional nanobiomaterials employed in multifunctional particle assembly are also discussed. As a final point the perspective of future improvements toward clinical applications are discussed in light of recent advances.