Influence of ClearT and ClearT2 Agitation Conditions in the Fluorescence Imaging of 3D Spheroids.

3D tumor spheroids have arisen in the last years as potent tools for the in vitro screening of novel anticancer therapeutics. Nevertheless, to increase the reproducibility and predictability of the data originated from the spheroids it is still necessary to develop or optimize the techniques used for spheroids' physical and biomolecular characterization. Fluorescence microscopy, such as confocal laser scanning microscopy (CLSM), is a tool commonly used by researchers to characterize spheroids structure and the antitumoral effect of novel therapeutics. However, its application in spheroids' analysis is hindered by the limited light penetration in thick samples. For this purpose, optical clearing solutions have been explored to increase the spheroids' transparency by reducing the light scattering. In this study, the influence of agitation conditions (i.e., static, horizontal agitation, and rotatory agitation) on the ClearT and ClearT2 methods' clearing efficacy and tumor spheroids' imaging by CLSM was characterized. The obtained results demonstrate that the ClearT method results in the improved imaging of the spheroids interior, whereas the ClearT2 resulted in an increased propidium iodide mean fluorescence intensity as well as a higher signal depth in the Z-axis. Additionally, for both methods, the best clearing results were obtained for the spheroids treated under the rotatory agitation. In general, this work provides new insights on the ClearT and ClearT2 clearing methodologies and their utilization for improving the reproducibility of the data obtained through the CLSM, such as the analysis of the cell death in response to therapeutics administration.

3D tumor spheroids as in vitro models to mimic in vivo human solid tumors resistance to therapeutic drugs.

Three-dimensional cell culture models, such as spheroids, can be used in the process of the development of new anticancer agents because they are able to closely mimic the main features of human solid tumors, namely their structural organization, cellular layered assembling, hypoxia, and nutrient gradients. These properties imprint to the spheroids an anticancer therapeutics resistance profile, which is similar to that displayed by human solid tumors. In this review, an overview of the drug resistance mechanisms observed in 3D tumor spheroids is provided. Furthermore, comparisons between the therapeutics resistance profile exhibited by spheroids, and 2D cell cultures are presented. Finally, examples of the therapeutic approaches that have been developed to surpass the drug resistance mechanisms exhibited by spheroids are described.

Optical clearing methods: An overview of the techniques used for the imaging of 3D spheroids.

Spheroids have emerged as in vitro models that reproduce in a great extent the architectural microenvironment found in human tissues. However, the imaging of 3D cell cultures is highly challenging due to its high thickness, which results in a light-scattering phenomenon that limits light penetration. Therefore, several optical clearing methods, widely used in the imaging of animal tissues, have been recently explored to render spheroids with enhanced transparency. These methods are aimed to homogenize the microtissue refractive index (RI) and can be grouped into four different categories, namely (a) simple immersion in an aqueous solution with high RI; (b) delipidation and dehydration followed by RI matching; (c) delipidation and hyperhydration followed by RI matching; and (d) hydrogel embedding followed by delipidation and RI matching. In this review, the main optical clearing methods, their mechanism of action, advantages, and disadvantages are described. Furthermore, the practical examples of the optical clearing methods application for the imaging of 3D spheroids are highlighted.

Microneedle-based delivery devices for cancer therapy: A review.

Macroscale delivery systems that can be locally implanted on the tumor tissue as well as avoid all the complications associated to the systemic delivery of therapeutics have captured researchers' attention, in recent years. Particularly, the microneedle-based devices can be used to efficiently deliver both small and macro-molecules, like chemotherapeutics, proteins, and genetic material, along with nanoparticle-based anticancer therapies. Such capacity prompted the application of microneedle devices for the development of new anticancer vaccines that can permeate the tumor tissue and simultaneously improve the effectiveness of therapeutic agents. Based on the promising results demonstrated by the microneedle systems in the local administration of anticancer therapeutics, this review summarizes the different microneedle formulations developed up to now aimed for application on cancer therapy (mphasizing those produced with polymers). Additionally, the microneedles' general properties, type of therapeutic approach and its main advantages are also highlighted.

Folate-targeted multifunctional amino acid-chitosan nanoparticles for improved cancer therapy.

PURPOSE: Tumor targeting nanomaterials have potential for improving the efficiency of anti-tumoral therapeutics. However, the evaluation of their biological performance remains highly challenging. In this study we describe the synthesis of multifunctional nanoparticles decorated with folic acid-PEG and dual amino acid-modified chitosan (CM-PFA) complexed with DNA and their evaluation in organotypic 2D co-cultures of cancer-normal cells and also on 3D multicellular tumor spheroids models. METHODS: The physicochemical characterization of CM-PFA multifunctional carriers was performed by FTIR, (1)H NMR and DLS. 2D co-culture models were established by using a 1:2 cancer-to-normal cell ratio. 3D organotypic tumor spheroids were assembled using micromolding technology for high throughput screening. Nanoparticle efficiency was evaluated by flow cytometry and confocal microscopy. RESULTS: The CM-PFA nanocarriers (126-176 nm) showed hemocompatibility and were internalized by target cells, achieving a 3.7 fold increase in gene expression. In vivo-mimicking 2D co-cultures confirmed a real affinity towards cancer cells and a negligible uptake in normal cells. The targeted nanoparticles penetrated into 3D spheroids to a higher extent than non-targeted nanocarriers. Also, CM-PFA-mediated delivery of p53 tumor suppressor promoted a decrease in tumor-spheroids volume. CONCLUSION: These findings corroborate the improved efficiency of this delivery system and demonstrate its potential for application in cancer therapy.

Optimization of liquid overlay technique to formulate heterogenic 3D co-cultures models.

Three-dimensional (3D) cell culture models of solid tumors are currently having a tremendous impact in the in vitro screening of candidate anti-tumoral therapies. These 3D models provide more reliable results than those provided by standard 2D in vitro cell cultures. However, 3D manufacturing techniques need to be further optimized in order to increase the robustness of these models and provide data that can be properly correlated with the in vivo situation. Therefore, in the present study the parameters used for producing multicellular tumor spheroids (MCTS) by liquid overlay technique (LOT) were optimized in order to produce heterogeneous cellular agglomerates comprised of cancer cells and stromal cells, during long periods. Spheroids were produced under highly controlled conditions, namely: (i) agarose coatings; (ii) horizontal stirring, and (iii) a known initial cell number. The simultaneous optimization of these parameters promoted the assembly of 3D characteristic cellular organization similar to that found in the in vivo solid tumors. Such improvements in the LOT technique promoted the assembly of highly reproducible, individual 3D spheroids, with a low cost of production and that can be used for future in vitro drug screening assays.

Co-delivery of Sildenafil (Viagra(®)) and Crizotinib for synergistic and improved anti-tumoral therapy.

PURPOSE: Cancer multi-drug resistance is a major issue associated with current anti-tumoral therapeutics. In this work, Crizotinib an anti-tumoral drug approved for the treatment of non-small lung cancer in humans, and Sildenafil (Viagra(®)), were loaded into micellar carriers to evaluate the establishment of a possible synergistic anti-tumoral effect in breast cancer cells. METHODS: Micellar carriers comprised by PEG-PLA block co-polymers were formulated by the solvent displacement method in which the simultaneous encapsulation of Crizotinib and Sildenafil was promoted. Encapsulation efficiency was analyzed by a new UPLC method validated for this combination of compounds. Micelle physicochemical characterization and cellular uptake were characterized by light scattering and confocal microscopy. The bio- and hemocompatibility of the carriers was also evaluated. MCF-7 breast cancer cells were used to investigate the synergistic anti-tumoral effect. RESULTS: Our results demonstrate that this particular combination induces massive apoptosis of breast cancer cells. The co-delivery of Crizotinib and Sildenafil was only possible due to the high encapsulation efficiency of the micellar systems (>70%). The micelles with size ranging between 93 and 127 nm were internalized by breast cancer cells and subsequently released their payload in the intracellular compartment. The results obtained demonstrated that the delivery of both drugs by micellar carriers led to a 2.7 fold increase in the anti-tumoral effect, when using only half of the concentration that is required when free drugs are administered. CONCLUSIONS: Altogether, co-delivery promoted a synergistic effect and demonstrated for the first time the potential of PEG-PLA-Crizotinib-Sildenafil combination for application in cancer therapy.

Nanoformulations for the Delivery of Dietary Anthocyanins for the Prevention and Treatment of Diabetes Mellitus and Its Complications.

Diabetes mellitus (DM) is a metabolic disease characterized by abnormal blood glucose levels-hyperglycemia, caused by a lack of insulin secretion, impaired insulin action, or a combination of both. The incidence of DM is increasing, resulting in billions of dollars in annual healthcare costs worldwide. Current therapeutics aim to control hyperglycemia and reduce blood glucose levels to normal. However, most modern drugs have numerous side effects, some of which cause severe kidney and liver problems. On the other hand, natural compounds rich in anthocyanidins (cyanidin, delphinidin, malvidin, pelargonidin, peonidin, and petunidin) have also been used for the prevention and treatment of DM. However, lack of standardization, poor stability, unpleasant taste, and decreased absorption leading to low bioavailability have hindered the application of anthocyanins as therapeutics. Therefore, nanotechnology has been used for more successful delivery of these bioactive compounds. This review summarizes the potential of anthocyanins for the prevention and treatment of DM and its complications, as well as the strategies and advances in the delivery of anthocyanins using nanoformulations.

Animal-derived products in science and current alternatives.

More than fifty years after the 3Rs definition and despite the continuous implementation of regulatory measures, animals continue to be widely used in basic research. Their use comprises not only in vivo experiments with animal models, but also the production of a variety of supplements and products of animal origin for cell and tissue culture, cell-based assays, and therapeutics. The animal-derived products most used in basic research are fetal bovine serum (FBS), extracellular matrix proteins such as Matrigel™, and antibodies. However, their production raises several ethical issues regarding animal welfare. Additionally, their biological origin is associated with a high risk of contamination, resulting, frequently, in poor scientific data for clinical translation. These issues support the search for new animal-free products able to replace FBS, Matrigel™, and antibodies in basic research. In addition, in silico methodologies play an important role in the reduction of animal use in research by refining the data previously to in vitro and in vivo experiments. In this review, we depicted the current available animal-free alternatives in in vitro research.

Silk Sericin: A Promising Sustainable Biomaterial for Biomedical and Pharmaceutical Applications.

Silk is a natural composite fiber composed mainly of hydrophobic fibroin and hydrophilic sericin, produced by the silkworm Bombyx mori. In the textile industry, the cocoons of B. mori are processed into silk fabric, where the sericin is substantially removed and usually discarded in wastewater. This wastewater pollutes the environment and water sources. However, sericin has been recognized as a potential biomaterial due to its biocompatibility, immunocompatibility, biodegradability, anti-inflammatory, antibacterial, antioxidant and photoprotective properties. Moreover, sericin can produce hydrogels, films, sponges, foams, dressings, particles, fibers, etc., for various biomedical and pharmaceutical applications (e.g., tissue engineering, wound healing, drug delivery, cosmetics). Given the severe environmental pollution caused by the disposal of sericin and its beneficial properties, there has been growing interest in upcycling this biomaterial, which could have a strong and positive economic, social and environmental impact.

Poly (vinyl alcohol)/chitosan layer-by-layer microneedles for cancer chemo-photothermal therapy.

The combination of photothermal and chemo- therapies displays a high potential to increase the efficacy of the cancer treatments or even promote their eradication. In this study, the micromoulding and electrospraying techniques were combined to produce polyvinylpyrrolidone microneedles coated with chitosan and poly (vinyl alcohol) for mediating the delivery of doxorubicin and AuMSS nanorods (Dox@MicroN) to cancer cells. The microneedles' physicochemical characterization demonstrated that the electrospraying technique can be used to produce a layer-by-layer coating consisting of layers of doxorubicin-loaded chitosan and AuMSS enriched poly (vinyl alcohol). Further, the Dox@MicroN patches presented a good photothermal capacity leading to a temperature increase of 12 °C under near-infrared irradiation (808 nm, 1.7 W/cm-2 for 5 min), which in conjugation with the chitosan' pH sensitivity could be used to control the doxorubicin release. Moreover, the microneedles were able to penetrate the tumor-mimicking agarose gel and promote a layer dependent drug release. Additionally, the Dox@MicroN patches' capacity to simultaneously mediate the chemo- and photothermal-therapies rendered a superior cytotoxic effect against the cervical cancer cells. Overall, the Dox@MicroN patches demonstrated to be a simple macroscale delivery device that can be used to mediate the local administration of new drug-photothermal combinations, avoiding all the issues related to the systemic administration of anti-cancer therapeutics.

Hyaluronic acid and vitamin E polyethylene glycol succinate functionalized gold-core silica shell nanorods for cancer targeted photothermal therapy.

Gold-core mesoporous silica shell (AuMSS) nanorods unique physicochemical properties makes them versatile and promising nanomedicines for cancer photothermal therapy. Nevertheless, these nanomaterials present a reduced half-life in the blood and poor specificity towards the tumor tissue. Herein, d-α-Tocopherol polyethylene glycol 1000 succinate (TPGS) and Hyaluronic Acid (HA) were combined for the first time to improve the AuMSS nanorods biological performance. The obtained results revealed that AuMSS surface functionalization induced the surface charge neutralization, from -28 ±â€¯10 mV to -3 ±â€¯5 mV and -10 ±â€¯4 mV for AuMSS-TPGS-HA (1:1) and (4:1) formulations, without impacting on nanomaterials' photothermal capacity. Moreover, the AuMSS functionalization improved the nanomaterials hemocompatibility and selectivity towards the cancer cells, particularly in the AuMSS-TPGS-HA (4:1) formulation. Furthermore, both formulations were able to mediate an on-demand photothermal effect, that induced the HeLa cancer cells death, confirming its potential for being applied as targeted multifunctional theragnostic nanomedicines.

Hyaluronic acid functionalized nanoparticles loaded with IR780 and DOX for cancer chemo-photothermal therapy.

IR780 is a near infrared (NIR) dye with a huge potential to be applied in cancer phototherapy and imaging. However, IR780 poor water solubility and acute cytotoxicity limit its direct use in cancer theragnostic. Herein, a novel Hyaluronic acid (HA)-based amphiphilic polymer was used, for the first time, in the preparation of polymeric nanoparticles (HPN) encapsulating IR780 aimed to be applied in breast cancer therapy. Furthermore, HPN co-encapsulating IR780 and Doxorubicin (DOX) were also produced in order to further enhance the therapeutic effectiveness of this nanoformulation. The results revealed that HPN were able to successfully encapsulate IR780 (IR-HPN) and the IR780-DOX combination (IR/DOX-HPN). Furthermore, the encapsulation of IR780 in HPN improved its absorption at 808 nm by about 2.2-fold, thereby enhancing its photothermal potential, as well as its cytocompatibility. The 2D in vitro cell uptake studies demonstrated that the nanostructures displayed a higher internalization by breast cancer cells than by normal cells. In addition, the assays performed in 3D in vitro models of breast cancer revealed that HPN can penetrate into spheroids. Furthermore, the 3D in vitro studies also demonstrated that the combined application of IR-HPN and NIR light was unable to induce cytotoxicity on spheroids. In contrast, IR/DOX-HPN produced a decrease on spheroids cells' viability, and their combination with NIR light induced an even stronger therapeutic effect, thus revealing the potential of these nanoparticles for cancer chemo-phototherapy.

Establishment of 2D Cell Cultures Derived From 3D MCF-7 Spheroids Displaying a Doxorubicin Resistant Profile.

In vitro 3D cancer spheroids generally exhibit a drug resistance profile similar to that found in solid tumors. Due to this property, these models are an appealing for anticancer compounds screening. Nevertheless, the techniques and methods aimed for drug discovery are mostly standardized for cells cultured in 2D. The development of 2D cell culture models displaying a drug resistant profile is required to mimic the in vivo tumors, while the equipment, techniques, and methodologies established for conventional 2D cell cultures can continue to be employed in compound screening. In this work, the response of 3D-derived MCF-7 cells subsequently cultured in 2D in medium supplemented with glutathione (GSH) (antioxidant agent found in high levels in breast cancer tissues and a promoter of cancer cells resistance) to Doxorubicin (DOX) is evaluated. These cells demonstrated a resistance toward DOX closer to that displayed by 3D spheroids, which is higher than that exhibited by standard 2D cell cultures. In fact, the 50% inhibitory concentration (IC50 ) of DOX in 3D-derived MCF-7 cell cultures supplemented with GSH is about eight-times higher than that obtained for conventional 2D cell cultures (cultured without GSH), and is only about two-times lower than that attained for 3D MCF-7 spheroids (cultured without GSH). Further investigation revealed that this improved resistance of 3D-derived MCF-7 cells may result from their increased P-glycoprotein (P-gp) activity and reduced production of intracellular reactive oxygen species (ROS).

Polyethylene glycol molecular weight influences the ClearT2 optical clearing method for spheroids imaging by confocal laser scanning microscopy.

Some fluorescence microscopy techniques, such as confocal laser scanning microscopy (CLSM), have a limited penetration depth. Consequently, the visualization and imaging of three-dimensional (3-D) cell cultures, such as spheroids, using these methods can be a significant challenge. Therefore, to improve the imaging of 3-D tissues, optical clearing methods have been optimized to render transparency to the opaque spheroids. The influence of the polyethylene glycol (PEG) molecular weight (MW) used in the ClearT2 method for the imaging of propidium iodide (PI)-stained spheroids was investigated. The results demonstrated that the ClearT2 clearing method contributes to spheroids transparency and to the preservation of PI fluorescence intensity for all the PEG MW used (4000, 8000, and 10,000 Da). Furthermore, the ClearT2 method performed using PEG 4000 Da allowed a better PI signal penetration depth and cross-section depth. Overall, the optimization of PEG MW can improve the imaging of intact spheroids by CLSM. Furthermore, this work may also contribute to increase the application of 3-D cell culture models by the pharmaceutical industry for the high-throughput screening of therapeutics.

Comparative study of the therapeutic effect of Doxorubicin and Resveratrol combination on 2D and 3D (spheroids) cell culture models.

The assessment of drug-combinations for pancreatic cancer treatment is usually performed in 2D cell cultures. In this study, the therapeutic effect and the synergistic potential of a particular drug-combination towards 2D and 3D cell cultures of pancreatic cancer were compared for the first time. Thus, the effect of Doxorubicin:Resveratrol (DOX:RES) combinations (at molar ratios ranging from 5:1 to 1:5) in the viability of PANC-1 cells cultured as 2D monolayers and as 3D spheroids was analyzed. The results showed that the cells' viability was more affected when DOX:RES combinations containing higher contents of RES (1:2-1:5 molar ratios) were used. This can be explained by the ability of RES to reduce the P-glycoprotein (P-gp)-mediated efflux of DOX. Further, it was also revealed that the synergic effect of this drug combination was different in 2D and in 3D cell cultures. In fact, despite of the 1:4 and 1:5 DOX:RES ratios being both synergistic for both types of PANC-1 cell cultures, their Combination Indexes (CI) in the monolayers were lower than those attained in spheroids. Overall, the obtained results revealed that the DOX:RES combination is promising for pancreatic cancer treatment and corroborate the emergent need to evaluate drug combinations in 3D cell cultures.

Development of poly-2-ethyl-2-oxazoline coated gold-core silica shell nanorods for cancer chemo-photothermal therapy.

AIM: Develop a new poly-2-ethyl-2-oxazoline (PEOZ)-based coating for doxorubicin-loaded gold-core mesoporous silica shell (AuMSS) nanorods application in cancer chemo-photothermal therapy. METHODS: PEOZ functionalized AuMSS nanorods were obtained through the chemical grafting on AuMSS of a PEOZ silane derivative. RESULTS: The PEOZ chemical grafting on the surface of AuMSS nanorods allowed the neutralization of nanodevices' surface charge, from -30 to -15 mV, which improved nanoparticles' biocompatibility, namely by decreasing the blood hemolysis to negligible levels. In vitro antitumoral studies revealed that the combined treatment mediated by the PEOZ-coated AuMSS nanorods result in a synergistic effect, allowing the complete eradication of cervical cancer cells. CONCLUSION: The application of the PEOZ coating improves the AuMSS nanorods performance as a multifunctional combinatorial therapy for cervical cancer.

Functionalization of graphene family nanomaterials for application in cancer therapy.

Graphene family nanomaterials' (GFN) ability to interact with near-infrared light has propelled their application in cancer photothermal therapy. Furthermore, the graphitic lattice of GFN can adsorb different types of molecules, which has motivated their use in cancer drug delivery. However, the direct application of GFN in cancer therapy is severely hindered by their poor colloidal stability, sub-optimal safety, inefficient tumor uptake and non-selectivity towards cancer cells. To overcome these limitations, GFN have been functionalized with different types of materials. This review is focused on the different functionalizations used in the design of GFN aimed for application in cancer therapy, disclosing their role on surpassing the critical issues related to GFN-based therapies.

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. Highlights • 2D cell culture drawbacks are summarized; • spheroids mimic the features of human tissues; • scaffold-based and scaffold-free technologies for spheroids production are discussed; • advantages of LOT over other scaffold-free techniques are highlighted; • LOT advances, practical approaches and troubleshooting are underlined.

Hyaluronic acid functionalized green reduced graphene oxide for targeted cancer photothermal therapy.

Reduced graphene oxide (rGO) nanomaterials display promising properties for application in cancer photothermal therapy (PTT). rGO is usually obtained by treating graphene oxide (GO) with hydrazine hydrate. However, this reducing agent contributes for the low cytocompatibility exhibited by rGO. Furthermore, rGO has a low water stability and does not show selectivity towards cancer cells. Herein, rGO attained using an environmentally-friendly method was functionalized with a novel hyaluronic acid (HA)-based amphiphilic polymer to be used in targeted cancer PTT. Initially, the green-reduction of GO with L-Ascorbic acid was optimized considering the near infrared absorption and the size distribution of the nanomaterials. Then, rGO was functionalized with the HA-based amphiphile. The functionalization of rGO improved its stability, cytocompatibility and internalization by CD44 overexpressing cells, which indicates the targeting capacity of this nanoformulation. Furthermore, the on-demand PTT mediated by HA-functionalized rGO induced cancer cells' ablation, thereby confirming its potential for targeted cancer therapy.