Browsing by Author "Pichon, Chantal"
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- Bioreducible poly(2-ethyl-2-oxazoline)–PLA–PEI-SS triblock copolymer micelles for co-delivery of DNA minicircles and DoxorubicinPublication . Gaspar, Vítor Manuel Abreu; Baril, Patrick; Costa, Elisabete C.; Diogo, Duarte Miguel de Melo; Foucher, Frédéric; Queiroz, João; Sousa, Fani; Pichon, Chantal; Correia, I.J.The co-delivery of minicircle DNA (mcDNA) and small anti-cancer drugs via stimuli-sensitive nanocarriers is a promising approach for combinatorial cancer therapy. However, the simultaneous loading of drugs and DNA in nanosized delivery systems is remarkably challenging. In this study we describe the synthesis of triblock copolymer micelles based on poly(2-ethyl-2-oxazoline)–poly(L-lactide) grafted with bioreducible polyethylenimine (PEOz–PLA-g–PEI-SS) for co-delivery of supercoiled (sc) mcDNA vectors and Doxorubicin (Dox). These amphiphilic carriers take advantage of non-fouling oxazolines to confer biological stability, of PLA to provide a hydrophobic core for drug encapsulation and of bioreducible PEI-SS to provide mcDNA complexation and an on-demand stimuli-responsive release. The obtained results show that mcDNA-loaded micelleplexes penetrate into in vitro tumor spheroid models with specific kinetics and exhibit a higher gene expression when compared to non-bioreducible nanocarriers. Moreover, in vivo bioluminescence imaging showed that gene expression is detected up to 8 days following mcDNA-micelles intratumoral administration. Furthermore, drug–gene co-delivery in PEOz–PLA-g–PEI-SS carriers was verified by successful encapsulation of both Dox and mcDNA with high efficacy. Moreover, dual-loaded micelleplexes presented significant uptake and a cytotoxic effect in 2D cultures of cancer cells. The co-delivery of mcDNA-Dox to B16F10-Luciferase tumor bearing mice resulted in a reduction in tumor volume and cancer cells viability. Overall, such findings indicate that bioreducible triblock micelles are efficient for focal delivery in vivo and have potential for future application in combinatorial DNA-drug therapy.
- Folate-Targeted Multifunctional Amino Acid-Chitosan Nanoparticles for Improved Cancer TherapyPublication . Gaspar, Vítor Manuel Abreu; Costa, Elisabete C.; Queiroz, João; Pichon, Chantal; Sousa, Fani; Correia, Ilídio Joaquim SobreiraPurpose 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, 1H 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.
- Gas-generating TPGS-PLGA microspheres loaded with nanoparticles (NIMPS) for co-delivery of minicircle DNA and anti-tumoral drugsPublication . Gaspar, Vítor Manuel Abreu; Moreira, André; Costa, Elisabete C.; Queiroz, João; Sousa, Fani; Pichon, Chantal; Correia, Ilídio Joaquim SobreiraDrug-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.
- Highly selective capture of minicircle DNA biopharmaceuticals by a novel zinc-histidine peptide conjugatePublication . Gaspar, Vítor Manuel Abreu; Cruz, Carla Patrícia Alves Freire Madeira; Queiroz, João; Pichon, Chantal; Correia, Ilídio; Sousa, FaniThe use of minicircle DNA (mcDNA) biomolecules as a pharmaceutical product holds remarkable potential due to their improved therapeutic efficacy in comparison with standard non-viral gene expression vectors. However, mcDNA translation into clinical application is still highly restricted due to the lack of robust technologies for minicircles detection and purification. In this study, the potential of a zinc-binding histidine-based peptide to function as a novel ligand for mcDNA recovery was investigated by using high-throughput surface plasmon resonance (SPR) analysis. The histidine-based peptide successfully bound zinc cationic ions and had affinity towards mcDNA biomolecules as confirmed by the dynamic binding responses obtained in SPR experiments. Notably, the obtained results indicate that not only zinc-peptide ligands are able to bind mcDNA with very high affinity (KD = 4.21 × 10−10 M), but also that this interaction is mostly dependent on buffer type. In general, the findings indicated that Zn2+ bound peptide has high affinity to mcDNA in low ionic strength buffers, whereas with high salt buffers no binding is detected. Overall, the novel zinc-binding peptide has shown to have suitable properties for mcDNA binding and recovery under experimental conditions that assure genetic material stability. More importantly, the straightforward approach of employing simple biomimetic ligands for mcDNA capture will contribute for development of new technologies to purify DNA biopharmaceuticals.
- Improved Minicircle DNA Biosynthesis for Gene Therapy ApplicationsPublication . Gaspar, V. M.; Maia, C J; Queiroz, João; Pichon, Chantal; Correia, Ilídio; Sousa, FaniMinicircular DNA (mcDNA) biopharmaceuticals have recently risen as a valuable alternative for the development of a next generation of bioactive therapeutics because they are more efficient and safer than standard plasmid DNA (pDNA). To date, the relatively insufficient knowledge regarding mcDNA biosynthesis is currently hindering its manufacture in suitable amounts for clinical trial evaluations. Addressing this limitation is therefore mandatory to bring forth the full therapeutic potential of this cutting-edge technology. Herein, we describe for the first time new processing parameters that improve the overall yield of mcDNA obtained from bacterial fermentations. We provide details for further in-line monitoring and optimization in view of the current good manufacturing guidelines. Our results show that by rising growth temperature to 42°C, an increase in the overall minicircle producer plasmid yield is attained, while biomass amounts are reduced. Moreover, by monitoring in real time the dynamic recombination of parental plasmids to mcDNA, we found that this event is more efficient at specific time points, regardless of the growth temperature and inductor concentration used. These are important findings since mcDNA can be recovered with higher yields at these determined key stages. Indeed, the manipulation of these parameters resulted in a 2.21-fold increase in mcDNA production compared with the established growth temperatures for this technology. Overall, our findings highlight that to achieve maximum productivity while attaining pharmaceutical-grade mcDNA preparations, process design and biosynthesis optimization must take into account key parameters such as temperature, inductor concentration, and recovery time.
- In Vivo bone tissue induction by freeze-dried collagen-nanohydroxyapatite matrix loaded with BMP2/NS1 mRNAs lipopolyplexesPublication . Wang, Pinpin; Perche, Federico; Midoux, Patrick; Cabral, Cátia S. D.; Malard, Virginie; Correia, I.J.; EI-Hafci, Hanane; Petite, Hervé; Logeart-Avramoglou, Delphine; Pichon, ChantalMessenger RNA (mRNA) activated matrices (RAMs) are interesting to orchestrate tissue and organ regeneration due to the in-situ and sustained production of functional proteins. However, the immunogenicity of in vitro transcribed mRNA and the paucity of proper in vivo mRNA delivery vector need to be overcome to exert the therapeutic potential of RAM. We developed a dual mRNAs system for in vitro osteogenesis by co-delivering NS1 mRNA with BMP2 mRNA to inhibit RNA sensors and enhance BMP-2 expression. Next, we evaluated a lipopolyplex (LPR) formulation platform for in vivo mRNA delivery and adapted the LPRs for RAM preparation. The LPR formulated BMP2/NS1 mRNAs were incorporated into an optimized collagen-nanohydroxyapatite scaffold and freeze-dried to prepare ready-to-use RAMs. The loaded BMP2/NS1 mRNAs lipopolyplexes maintained their spherical morphology in the RAM, thanks to the core-shell structure of LPR. The mRNAs release from RAMs lasted for 16 days resulting in an enhanced prolonged transgene expression period compared to direct cell transfection. Once subcutaneously implanted in mice, the BMP2/NS1 mRNAs LPRs containing RAMs (RAM-BMP2/NS1) induced significant new bone tissue than those without NS1 mRNA, eight weeks post implantation. Overall, our results demonstrate that the BMP2/NS1 dual mRNAs system is suitable for osteogenic engagement, and the freeze-dried RAM-BMP2/NS1 could be promising off-the-shelf products for clinical orthopedic practice.
- Minicircle DNA vectors for gene therapy: advances and applicationsPublication . Gaspar, Vítor Manuel Abreu; Diogo, Duarte Miguel de Melo; Costa, Elisabete C.; Moreira, André; Queiroz, João; Pichon, Chantal; Correia, Ilídio Joaquim Sobreira; Sousa, FaniIntroduction: 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.
- Multifunctional Nanocarriers for Co-Delivery of Nucleic Acids and Chemotherapeutics to Cancer CellsPublication . Gaspar, Vítor Manuel Abreu; Moreira, André; Diogo, Duarte Miguel de Melo; Costa, Elisabete C.; Queiroz, João; Sousa, Fani; Pichon, Chantal; Correia, Ilídio Joaquim SobreiraCombinatorial 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.
- Non-coding RNAs: Emerging from the discovery to therapeutic applicationsPublication . Baptista, Bruno; Riscado, Micaela; Queiroz, João; Pichon, Chantal; Sousa, F.The knowledge about non-coding RNAs (ncRNAs) is rapidly increasing with new data continuously emerging, regarding their diverse types, applications, and roles. Particular attention has been given to ncRNA with regulatory functions, which may have a critical role both in biological and pathological conditions. As a result of the diversity of ncRNAs and their ubiquitous involvement in several biologic processes, ncRNA started to be considered in the biomedical field, with immense potential to be exploited either as biomarkers or as therapeutic agents in certain pathologies. Indeed, ncRNA-based therapeutics have been proposed in many disorders and some even reached clinical trials. However, to prepare an RNA product suitable for pharmacological applications, certain criteria must be fulfilled, and it has to be guaranteed RNA purity, stability, and bioactivity. So, in this review, the different types of ncRNAs are identified and characterized, by describing their biogenesis, functions, and applications. A perspective on the main challenges and innovative approaches for the future and broad therapeutic application of RNA is also presented.
- Poly(2-ethyl-2-oxazoline)–PLA-g–PEI amphiphilic triblock micelles for co-delivery of minicircle DNA and chemotherapeuticsPublication . Gaspar, Vítor Manuel Abreu; Gonçalves, Cristine; Diogo, Duarte Miguel de Melo; Costa, Elisabete C.; Queiroz, João; Pichon, Chantal; Sousa, Fani; Correia, Ilídio Joaquim SobreiraThe design of nanocarriers for the delivery of drugs and nucleic-acids remains a very challenging goal due to their physicochemical differences. In addition, the reported accelerated clearance and immune response of pegylated nanomedicines highlight the necessity to develop carriers using new materials. Herein, we describe the synthesis of amphiphilic triblock poly(2-ethyl-2-oxazoline)–PLA-g–PEI (PEOz–PLA-g–PEI) micelles for the delivery of minicircle DNA (mcDNA) vectors. In this copolymer the generally used PEG moieties are replaced by the biocompatible PEOz polymer backbone that assembles the hydrophilic shell. The obtained results show that amphiphilic micelles have low critical micellar concentration, are hemocompatible and exhibit stability upon incubation in serum. The uptake in MCF-7 cells was efficient and the nanocarriers achieved 2.7 fold higher expression than control particles. Moreover, mcDNA-loaded micelleplexes penetrated into 3D multicellular spheroids and promoted widespread gene expression. Additionally, to prove the concept of co-delivery, mcDNA and doxorubicin (Dox) were simultaneously encapsulated in PEOz–PLA-g–PEI carriers, with high efficiency. Dox–mcDNA micelleplexes exhibited extensive cellular uptake and demonstrated anti-tumoral activity. These findings led us to conclude that this system has a potential not only for the delivery of novel mcDNA vectors, but also for the co-delivery of drug–mcDNA combinations without PEG functionalization.