BIOSYNTHESIS AND PURIFICATION OF MINICIRCLE DNA FOR APPLICATION IN DIABETES CELL-SPECIFIC GENE THERAPY

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Co-delivery of Sildenafil (Viagra®) and Crizotinib for Synergistic and Improved Anti-tumoral Therapy

Publication . Marques, João Filipe Gonçalves; Gaspar, Vítor Manuel Abreu; Oppolzer, David; Costa, Elisabete C.; Gallardo, Eugenia; Correia, Ilídio Joaquim Sobreira

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.

2014-03-13Journal article

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Biosynthesis, purification and delivery of minicircle DNA for gene therapy

Publication . Gaspar, Vítor Manuel Abreu; Sousa, Fani Pereira de; Correia, Ilídio Joaquim Sobreira; Pichon, Chantal

The prevalence of numerous diseases which are currently responsible for high mortality rates or that affect people well-being is one of the major challenges faced by nowadays society. To improve general health and population quality of life, tremendous efforts have been focused on speeding up the discovery of innovative pharmaceutics for treatment of particularly complex and incurable pathologies such as cancer. Currently, the generally applied anticancer treatments result only in a slight increase in patient survival rates and additional lifetime without disease recurrence. However, these improvements are often short-lived and are associated with severe systemic toxicity that debilitates patients health during treatment. This fact demonstrates the ineffectiveness and reduced safety of clinically administered therapies, and above all, emphasizes the urging necessity to discover more efficient approaches that can promote a better therapeutic outcome with fewer side-effects. From the numerous anticancer treatments currently under development, those based on nonviral gene transfer with nucleic acid-based pharmaceuticals have a great therapeutic potential since they are currently approved for human use by United States Food and Drug Administration (US-FDA) and European Medidicines Agency (EMA) regulatory agencies, can be produced at industrial scale and are highly versatile. In fact, unlike standard cytotoxic drugs, the original structure of DNA biopharmaceuticals can be precisely engineered to encode multiple, tumor suppressor genes which may simultaneously affect cancer cells proliferation, or induce their destruction without direct damage to healthy tissues. In this context, plasmid DNA (pDNA) gene expression cassettes remain the gold standard biopharmaceuticals for gene therapy. However, despite being a promising tool, the widespread use of standard plasmid vectors is restricted by their short-term activity in vivo. Therefore, to realize the full potential of this therapeutic approach other alternatives for transgene expression in humans must be explored. In this context, a recently upgraded technology based on the use of minimalistic gene expression cassettes devoid of the bacterial backbone, so-termed DNA minicircles, has shown to provide the required biological efficiency. Due to the fact that this is a relatively new technology the parameters of mcDNA production in prokaryotic organisms have not yet been correctly explored. The optimization of this bioprocess assumes a critical importance from a manufacture and therapeutic point of view, since various factors may affect the productivity, final stability and purity of mcDNA preparations. A part from these necessary improvements, mcDNA gene transfer to diseased tissues also remains as one the most rate limiting steps in the translation of these therapies from bench-to-bedside, being necessary as well to improve the currently existing technologies for DNA transfer to eukaryotic cells. Taking this background into account, the main hypothesis of this work was to explore and optimize mcDNA biosynthesis, to screen ligands for purification of these vectors and develop biocompatible polymeric nanocarriers for minicircle gene delivery to different in vitro and in vivo cancer models. As an additional remark to this integrative study, the synthesis of stimuli-responsive delivery systems for co-administration of mcDNA-drug combinations to cancer cells was also investigated. Bioprocess optimization was initially performed by studying and manipulating the amplification of template parental plasmids (PP) in an Escherichia coli strain (ZYCY10P3S2T) genetically modified to produce mcDNA from plasmid templates with high efficiency. During these experiments it was hypothesized that the manipulation of bacterial growth temperature could provide important improvements in the quantity of mcDNA produced in later stages of the process. The obtained results indicated that an increase in bacterial growth temperature maximized the amount of template plasmids per biomass. In addition to this enhancement, a real-time monitoring of the PP–to-mcDNA intramolecular recombination process revealed that the time of mcDNA recovery significantly affects both the final yield and also the presence of residual PP and mini-plasmid (mP) species in minicircle preparations. These important findings demonstrate that maximum productivity and pharmaceutical-grade minicircle batches were obtained at specific time points during the induction phase. Having established optimal bioprocess parameters, the use of novel L-arginine dipeptide ligands for mcDNA biopharmaceuticals isolation or purification was investigated. These dipeptide ligands are particularly interesting as they take advantage of biomimetic interactions that are naturally established between amino acids and DNA in vivo. As a proof-of-concept, mcDNA vectors and PP template plasmids were injected into microfluidic chips containing chemically immobilized dipeptides. The sensitive screening obtained by surface plasmon resonance indicated that by manipulating temperature conditions and buffer type, different ligand-analyte interactions could be established. This dynamic binding-elution profile of mcDNA and PP species, under specific conditions, indicated these peptides could possibly be employed in the development of a platform for biopharmaceuticals isolation or purification. Alongside with these studies, the development of biocompatible nanosized gene delivery systems based on amino acid modified chitosan was explored. For this purpose L-histidine and L-arginine amino acids were selected for chitosan backbone functionalization through a two-step chemical modification. In this study it was proposed that amino acid biocompatible moieties could improve the polymer physicochemical structure and its capacity to condense and deliver genetic material to cancer cells. The chemical modification of chitosan with both amino acid moieties through zero-length crosslinkers was successful and resulted in the development of a novel biofunctionalized material with pH-responsive character and positive charge. These characteristics allowed the formulation of DNA-nanocarriers through attractive electrostatic interactions with model pDNA vectors, under mild conditions. These nanoparticles achieved an improved cellular uptake and higher transgene expression efficiency in cancer cells when compared to their non-modified counterparts. As a further attempt to improve the selectivity of these delivery systems towards target cancer cells, in a subsequent study, the hybrid polymer was chemically grafted with poly(ethylene glycol)-folic acid blocks via Michael type thiol-maleimide coupling. This chemical grafting promoted a selective inclusion of folic acid cell targeting moieties into amino acid modified chitosan. The targeting specificity of the formulated DNA-loaded multifunctional carriers was confirmed in in vitro 2D co-culture models comprised by folic acid positive cancer cells and normal human fibroblasts. In addition, the results obtained in 3D multicellular spheroids indicated that the targeted particles penetrated into these in vitro models of solid tumors and accomplished significant transgene expression. A time-course, high-throughput analysis, performed after nanocarriers containing the p53 tumor suppressor DNA were administered, revealed a reduction in spheroids volume along time, thereby supporting the possible use of this technology for cell-selective gene transfer. From this standpoint, it was hypothesized that polymeric delivery systems could also be used for simultaneous co-delivery of chemotherapeutic drugs and DNA biopharmaceuticals. It was anticipated that such combinatorial approach could provide a superior anticancer effect and contribute for the development of more efficient treatments. To materialize this challenging concept, beyond-state-of-the-art triblock copolymers were chemically synthesized since biofunctional chitosan nanocarriers required complex chemical modifications to co-encapsulate mcDNA and drugs. The new synthetic nanomaterials for co-delivery were produced in an application-oriented, safe-by-design, approach that took into account the necessity to assure materials biocompatibility, biological performance and also the physicochemical properties required for simultaneous encapsulation of drug and genes in a single nanocarrier. The obtained results demonstrate that the triblock copolymers self-assembled into nanosized biocompatible micelles with core-shell structure in aqueous environment and condensed mcDNA vectors with high efficacy. In vivo administration of mcDNA-loaded micelleplexes to solid tumors also originated significant transgene expression, which shows the therapeutic potential of this delivery system. The co-delivery concept was also demonstrated with the simultaneous encapsulation of an anticancer drug (Doxorubicin), and mcDNA, in the micellar carriers. As revealed by confocal microscopy and metabolic assays, the dual-loaded micelleplexes presented significant cellular uptake and cytotoxic activity in cancer cells when compared to free drug. After demonstrating the potential associated with drug-gene co-delivery, the formulation of stimuli-sensitive co-delivery systems was investigated. The production of carriers with dynamic response to precise biological cues was expected to provide a new level of therapeutic efficiency since the release of bioactive molecules could be controlled in a spatiotemporal mode. To explore the manufacture of such “smart” nanomaterials two different approaches based on the formerly developed nanocarriers were explored. In a first study, mcDNA-loaded biofunctionalized chitosan nanocarriers were encapsulated in gas-generating poly(D,L-lactic-co-glycolic acid) (PLGA) biodegradable microspheres, previously loaded with an antitumoral drug and sodium bicarbonate. The assembled nanoparticle-in-microsphere hybrid systems were capable of generating carbon dioxide (CO2) bubbles in acidic environment due to bicarbonate presence. In turn, this gas production originated a rapid disassemble of microspheres shell, and consequent contents release. In vitro, the dual-loaded hybrid carrier demonstrated a higher cytotoxicity in cancer cells when compared to that of free drugs or single drug-loaded microspheres. In addition to this platform, in a second study the cationic block of the triblock copolymers was modified with disulfide linkages to allow a redox-responsive release of mcDNA vectors in intracellular compartments poly(2-ethyl-2-oxazoline)-poly(L-lactic acid)-g-polyethylenimine-disulfide (PEOz-PLA-g-PEI-SS). In this study an affinity chromatography monolith disk immobilized with previously investigated L-arginine dipeptide ligands was used to isolate mcDNA supercoiled isoform. The evaluation of bioreducible micelles in 3D in vitro models and orthotopic in vivo tumors indicated that this system has improved transgene expression efficacy and promotes tumor regression when it is used for co-delivery of Doxorubicin and mcDNA. Overall, the research performed throughout this Doctoral thesis described improvements in mcDNA production process and led to the discovery of potential ligands for the isolation of its supercoiled isoform. The original results obtained during this work provide an important body of knowledge in the applicability of the mcDNA technology at a larger scale. Furthermore, the pre-clinical evaluation performed on newly developed nanomedicines demonstrated that grafting multifunctional moieties, or imprinting a stimuli-sensitive character to nanocarriers has a positive effect on their biological performance. It is important to mention that the future inclusion of one or more tumor suppressor genes in mcDNA vectors may contribute to potentiate their therapeutic effect. In this context, and as a concluding remark, the particularly promising results obtained with the administration of PEOz-PLA-g-PEI-SS dual-loaded and stimuli-sensitive micelles demonstrated that these systems enclose an outstanding potential for medical applications in a foreseeable future.

2015Doctoral thesis

Open access

Manufacture of β-TCP/alginate scaffolds through a Fab@home model for application in bone tissue engineering

Publication . Carlos, Gabriela Soares Diogo; Gaspar, Vítor Manuel Abreu; Serra, Inês Raquel Tavares; Fradique, Ricardo Gil; Correia, Ilídio Joaquim Sobreira

The growing need to treat bone-related diseases in an elderly population compels the development of novel bone substitutes to improve patient quality of life. In this context, the advent of affordable and effective rapid prototyping equipment, such as the Fab@home plotter, has contributed to the development of novel scaffolds for bone tissue engineering. In this study, we report for the first time the use of a Fab@home plotter for the production of 3D scaffolds composed by beta-tricalcium phosphate (β-TCP)/alginate hybrid materials. β-TCP/alginate mixtures were used in a proportion of 50/50% (w/w), 30/70% (w/w) and 20/80% (w/w). The printing parameters were optimized to a nozzle diameter of 20 Gauge for the production of rigid scaffolds with pre-defined architectures. We observed that, despite using similar printing parameters, both the precision and resolution of the scaffolds were significantly affected by the blend's viscosity. In particular, we demonstrate that the higher viscosity of 50/50 scaffolds (150.0 ± 3.91 mPa s) provides a higher precision in the extrusion process. The physicochemical and biological characterization of the samples demonstrated that the 50/50 scaffolds possessed a resistance to compression comparable to that of native trabecular bone. Moreover, this particular formulation also exhibited a Young's modulus that was higher than that of trabecular bone. Scanning electron microscopy and fluorescence microscopy analysis revealed that osteoblasts were able to adhere, proliferate and also penetrate into the scaffold's architecture. Altogether, our findings suggest that the Fab@home printer can be employed in the manufacture of reproducible scaffolds, using a formulation 50/50 alginate-β-TCP that has suitable properties to be applied as bone substitutes in the future.

2014-03-21Journal article

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Evaluation of Nanoparticle Uptake in Co-culture Cancer Models

Publication . Costa, Elisabete C.; Gaspar, Vítor Manuel Abreu; Marques, João Filipe Gonçalves; Coutinho, Paula; Correia, Ilídio Joaquim Sobreira

Co-culture models are currently bridging the gap between classical cultures and in vivo animal models. Exploring this novel approach unlocks the possibility to mimic the tumor microenvironment in vitro, through the establishment of cancer-stroma synergistic interactions. Notably, these organotypic models offer a perfect platform for the development and pre-clinical evaluation of candidate nanocarriers loaded with anti-tumoral drugs in a high throughput screening mode, with lower costs and absence of ethical issues. However, this evaluation was until now limited to co-culture systems established with precise cell ratios, not addressing the natural cell heterogeneity commonly found in different tumors. Therefore, herein the multifunctional nanocarriers efficiency was characterized in various fibroblast-MCF-7 co-culture systems containing different cell ratios, in order to unravel key design parameters that influence nanocarrier performance and the therapeutic outcome. The successful establishment of the co-culture models was confirmed by the tissue-like distribution of the different cells in culture. Nanoparticles incubation in the various co-culture systems reveals that these nanocarriers possess targeting specificity for cancer cells, indicating their suitability for being used in this illness therapy. Additionally, by using different co-culture ratios, different nanoparticle uptake profiles were obtained. These findings are of crucial importance for the future design and optimization of new drug delivery systems, since their real targeting capacity must be addressed in heterogenous cell populations, such as those found in tumors.

2013-07-26Journal article

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

Publication . Moreira, André; Gaspar, Vítor Manuel Abreu; Costa, Elisabete C.; Diogo, Duarte Miguel de Melo; Machado, Paulo Filipe Brito; Paquete, Catarina; Correia, Ilídio Joaquim Sobreira

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 171 nm. 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 72 h, 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.

2014-09-16Journal article

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