Isolation and Purification of Plasmid DNA for Cancer Therapy

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A bi-layer electrospun nanofiber membrane for plasmid DNA recovery from fermentation broths

Publication . Correia, Tiago R.; Antunes, Bernardo Paiva; Castilho, Pedro Henrique Barata; Nunes, José; Amorim, Maria T. Pessoa de; Escobar, Isabel; Queiroz, João; Correia, I.J.; Morão, António

The demanding ever-increasing quantities of highly purified biomolecules by bio-industries, has triggered the development of new, more efficient, purification techniques. The application of membrane-based technologies has become very attractive in this field, for their high throughput capability, simplicity of operation and scale-up. Herein we report the production of a bi-layer membrane by electrospinning (ES), in which a support of poly ε-caprolactone nanofibers was coated with a polyethylene oxide/sodium alginate layer, and subsequently cross-linked with calcium chloride. The membranes were characterized by SEM, ATR-FTIR, contact angle measurements, and were applied in the recovery process of a plasmid. The results show that membranes retained the suspended solids while allowing the permeation of plasmid DNA, with high recovery yields and improved RNA retention. Moreover, they also showed a very low fouling tendency. To the best of our knowledge it is the first time that ES membranes are applied in this type of bioprocess.

2013-04-06Journal article

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In vitro characterization of 3D printed scaffolds aimed at bone tissue regeneration

Publication . Boga, João Miguel Carvalho Freire; Miguel, Sónia P.; Diogo, Duarte Miguel de Melo; Mendonça, António; Louro, Ricardo; Correia, Ilídio Joaquim Sobreira

The incidence of fractures and bone-related diseases like osteoporosis has been increasing due to aging of the world’s population. Up to now, grafts and titanium implants have been the principal therapeutic approaches used for bone repair/regeneration. However, these types of treatment have several shortcomings, like limited availability, risk of donor-to-recipient infection and tissue morbidity. To overcome these handicaps, new 3D templates, capable of replicating the features of the native tissue, are currently being developed by researchers from the area of tissue engineering. These 3D constructs are able to provide a temporary matrix on which host cells can adhere, proliferate and differentiate. Herein, 3D cylindrical scaffolds were designed to mimic the natural architecture of hollow bones, and to allow nutrient exchange and bone neovascularization. 3D scaffolds were produced with tricalcium phosphate (TCP)/alginic acid (AA) using a Fab@home 3D printer. Furthermore, graphene oxide (GO) was incorporated into the structure of some scaffolds to further enhance their mechanical properties. The results revealed that the scaffolds incorporating GO displayed greater porosity, without impairing their mechanical properties. These scaffolds also presented a controlled swelling profile, enhanced biomineralization capacity and were able to increase the Alkaline Phosphatase (ALP) activity. Such characteristics make TCP/AA scaffolds functionalized with GO promising 3D constructs for bone tissue engineering applications.

2018-02-21Journal 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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Biofunctionalized nanoparticles with pH-responsive and cell penetrating blocks for gene delivery

Publication . Gaspar, Vítor Manuel Abreu; Marques, João Filipe Gonçalves; Sousa, Fani; Louro, Ricardo; Queiroz, João; Correia, I.J.

Bridging the gap between nanoparticulate delivery systems and translational gene therapy is a long sought after requirement in nanomedicine-based applications. However, recent developments regarding nanoparticle functionalization have brought forward the ability to synthesize materials with biofunctional moieties that mimic the evolved features of viral particles. Herein we report the versatile conjugation of both cell penetrating arginine and pH-responsive histidine moieties into the chitosan polymeric backbone, to improve the physicochemical characteristics of the native material. Amino acid coupling was confirmed by 2D TOCSY NMR and Fourier transform infrared spectroscopy. The synthesized chitosan–histidine–arginine (CH–H–R) polymer complexed plasmid DNA biopharmaceuticals, and spontaneously assembled into stable 105 nm nanoparticles with spherical morphology and positive surface charge. The functionalized delivery systems were efficiently internalized into the intracellular compartment, and exhibited remarkably higher transfection efficiency than unmodified chitosan without causing any cytotoxic effect. Additional findings regarding intracellular trafficking events reveal their preferential escape from degradative lysosomal pathways and nuclear localization. Overall, this assembly of nanocarriers with bioinspired moieties provides the foundations for the design of efficient and customizable materials for cancer gene therapy.

2013-06-13Journal article

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miRNA-29 bioseparation and target delivery strategies for Alzheimer's disease

Publication . Pereira, Patrícia Alexandra Nunes; Sousa, Fani Pereira de; Figueiras, Ana Rita; Correia, Ilídio Joaquim Sobreira

The possibility of selectively alter the expression pattern of a particular gene has been sought by scientists and clinicians for a long time. Nowadays, RNA interference (RNAi)-based technology has become a novel tool for silencing gene expression in cells. In addition, this strategy encloses an enormous therapeutic potential that could change the course of the currently applied treatments in several life threatening pathologies and it is expected that this technology can be translated onto clinical applications in a near future. MicroRNA (miRNA) has become a commonly employed tool for gene silencing, since it prevents protein synthesis by inducing the messenger RNA (mRNA) degradation, with a high specificity degree. Consequently, in the last years, the miRNAs have emerged as biopharmaceuticals to regulate several pathways involved in the insurgence and progression of the Alzheimer’s disease (AD), since they might have key regulatory roles in many neuronal functions, such as differentiation, synaptic plasticity and memory formation, and typically they are down-regulated in disease conditions. In the literature there are some studies describing a causal relationship between miR-29 expression and AD, since a loss of miR-29 cluster can contribute to increased beta-amyloid precursor protein-converting enzyme 1 (BACE1) and Amyloid-β (Aβ) levels in sporadic AD patients. Thus, this evidence supports the possibility to use miR-29 as a potential therapeutic target for AD therapy. In general, miRNA-based therapy relies on the use of synthetic microRNAs. However, these synthesized formulations typically present contaminants that can lead to non-targeted gene silencing, which still restricts the pre-clinical or clinical application of these RNAs. Thus, considering this therapeutic purpose and the global distribution of novel biopharmaceuticals it is necessary to develop efficient processes for their preparation. The development of new strategies for microRNA production with high purity degree and biologically active is extremely required. One of the strategies might be the use of the recombinant production of biomolecules using prokaryotic hosts. Hence, the present work intends to develop and establish an integrative biotechnological platform to biosynthesize and purify a recombinant miRNA precursor (pre-miR-29b) to act in the selective silencing of endogenous pathways directly related with AD, in particular BACE1 and Aβ. In addition, the success of these therapies also depends upon the ability to selectively and efficiently deliver the pre-miR-29b in the cytoplasmic compartment of neuronal cells, the location where their function is exerted; therefore the development of miRNA delivery systems was also envisioned. The expression system Rhodovulum sulfidophilum (R. sulfidophilum) DSM 1374 allowed, for the first time, the production of human pre-miR-29b with a straightforward recuperation of pre-miR-29b in a single step, maintaining its biological active form. The application of this recombinant bacterial microorganism is innovative and is supported by the unusual capacity of secreting the nucleic acids to the extracellular space and the absence of host ribonucleases in the culture medium. Therefore, it is expected that the secreted miRNA will be devoid of main bacterial associated impurities. Regarding the growth conditions, and conversely to what was previously described for this bacterium, our results showed to be possible to develop an original approach for the aerobic growth of the R. sulfidophilum, which results in a cell growth improvement followed by an enhanced production of human pre-miR-29b. The extracellular pre-miR-29b concentration was approximately 182 μg/L, after 40 hours of bacterial growth and the total intracellular pre-miR-29b was of about 358 μg/L, at 32 hours of cell growth. To further develop a potential therapeutic application, the major interest is not only to produce high quantities of RNA but also to obtain and preserve its biological active form, fulfilling the requirements of regulatory agencies. Hence, to assure that this prerequisite is met it was used a novel and effective purification strategy, based on affinity chromatography, to purify the pre-miR-29b. Therefore, in order to achieve the selectivity towards the target pre-miRNA and the maximum resolution between the pre-miR-29b and other host biomolecules (transfer RNAs and proteins) it was used an affinity support that exploits the same biological interactions that are established within the cell, by using immobilized amino acids (L-lysine and L-arginine), as specific ligands. The recognition of the pre-miR-29b achieved with these supports, allowed its selective recovery from a complex mixture with high efficiency and high purity. In parallel, the binding of pre-miRNA to these different amino acids was studied by Surface Plasmon Resonance. This information brings important insights concerning the characterization of the pre-miRNA binding onto chromatographic supports. Moreover, it was possible to determine some particular conditions enabling the improvement of the binding specificity of the amino acid ligands used to purify miRNA, preserving the RNA integrity. Taking into account that the structure of the chromatographic supports has been continuously developed to afford rapid and efficient separations, namely for the purification of nucleic acids, it was also tested a monolithic support to purify the pre-miR-29b. The association of the high capacity of these supports with the specificity conferred by the agmatine ligand (a derivative of L-arginine) represented a novelty and an advantage to obtain highly pure pre-miR-29b (90%) with a high recovery yield (95%). The establishment of an effective application of miRNAs is usually constrained by different phenomena, namely their easy degradation when in contact with the body fluids. To overcome this limitation, delivery systems, such as polymeric systems (polyplexes), were developed and characterized in order to encapsulate and protect the pre-miR-29b biopharmaceuticals from degradation, allowing their sustained and targeted release. The formulations prepared with chitosan and polyethylenimine demonstrated high loading capacity, small sizes and exhibited a strong positive charge on their surface. In addition, considering the application field of this work, the delivery systems should also have the ability to penetrate the Blood-Brain Barrier (BBB), causing an increase of the pre-miRNAs concentration in the brain and, consequently the improvement of the therapeutic effect. Actually, BBB is an intrinsic barrier limiting miRNA therapeutic effect on the central nervous system. Thus, to improve the delivery of pre-miRNA therapeutics in the brain, the polyplexes were functionalized with specific ligands, namely lactoferrin and stearic acid which are recognized by cell surface receptors of BBB. Finally, it was evaluated the biological activity of the recombinant pre-miR-29b by measuring the efficiency on human BACE1 knockdown, using in vitro neuronal cell lines. The effect of recombinant pre-miR-29b administration was verified by both assessing the mRNA and protein human BACE1 levels, by using RT-qPCR, Western blot and Imunocytochemistry. Results suggest that recombinant pre-miR-29b can represent a novel biopharmaceutical product for the therapeutic modulation of human BACE1 levels, because high levels of inhibition were achieved, namely 80% of reduction for BACE1 protein expression and 45% for Aβ42 levels. Globally, the implementation of these cutting-edge technologies can have a great impact on the biopharmaceutical industry, providing the basis for the implementation of novel miRNA-based therapeutics, not only for neurological disorders but also for future therapeutic targets that can be of potential interest.

2016Doctoral thesis

Open access