Purification of DNA vaccine to prevent or treat cervical cancer

Human Papillomavirus (HPV) is worldwide sexually transmitted and associated with 99.7% of cervical cancer. The cancer progression is due to the expression of the oncoproteins E6 and E7, which can alter the cell cycle and are responsible for the viral replication and transformation of host cells. The vaccines available are only preventive ones, it being necessary to develop therapeutic ones, to prevent and treat a pre-existent infection. Deoxyribonucleic acid (DNA) vaccination along with the use of plasmid DNA (pDNA) as a non-viral vector arises as a good strategy that can activate both humoral and cellular immune responses, allowing the prevention and treatment of HPV infections. The combination of the amino-acid affinity chromatography (AC) with the innovative monolithic supports appears as a promising approach to obtain highly purified supercoiled (sc) pDNA – the active biological conformation – with high purity and recovery. This allows the selective interaction of specific ligands to the target biomolecule adding to the higher capacity of monoliths when compared to conventional chromatographic supports. Monoliths also allow the use of high flow rates, which allows a fast purification procedure and decreases the retention time of the target biomolecule, avoiding its degradation. In the present work, different elution strategies (manipulation of sodium chloride (NaCl) concentrations and/or pH and competition) were explored, in order to purify the supercoiled HPV-16 E6/E7MUT pDNA, by using the arginine monolith with spacer arm. The best elution strategy applied on both laboratorial and preparative scales allowed the removal of impurities within the regulatory agency recommendations, with 93.3% and 98.5% of purity degree, respectively. This reinforces the applicability of this monolith for the sc pDNA purification. Moreover, only one in thousands naked plasmids presented to the cells reach the nucleus and are expressed. The use of nanoparticles is a valuable strategy that permits the protection of the pDNA by avoiding the enzymatic degradation and facilitates the specific delivery, enhancing the cellular transfection. Thus, different magnesium carbonate (MgCO3) systems were characterized regarding its encapsulation efficiency (around 87%), morphology (round shape), size (99.7-237.4 nm) and zeta potential (positive). These data suggest that the developed nanoparticles are suitable for cellular uptake and thus appropriate for therapeutic applications. Additionally, in vitro studies accompanied with confocal microscopy were performed, which revealed that all the formulated systems are able to transfect eukaryotic cells.A infeção causada pelo Vírus do Papiloma Humano (HPV) é uma doença sexualmente transmitida, que afeta tanto homens como mulheres a nível mundial. Em último caso, a infeção causada pelo HPV pode levar ao aparecimento de massas tumorais. De facto, o ácido desoxirribonucleico (DNA) do HPV foi encontrado em 99,7% dos casos de cancro do colo do útero, provocando mais de meio milhão de mortes. A progressão do cancro é devida à expressão das oncoproteínas E6 e E7, consideradas tumorogénicas pela sua capacidade de alterar o ciclo celular, sendo estas responsáveis pela replicação viral e transformação e imortalização das células hospedeiras. Atualmente, existem apenas duas vacinas comercializadas contra a infeção pelo HPV: a Gardasil® e a Cervarix®. Estas vacinas profiláticas ativam unicamente a imunidade humoral, pela geração de anticorpos contra o HPV e são somente preventivas, ou seja, apenas são efetivas antes de ocorrer a infeção. Assim, as vacinas terapêuticas têm a promissora vantagem de conseguir eliminar lesões pré-existentes e até tumores. Surgem então algumas estratégias terapêuticas inovadoras, como a terapia génica e as vacinas de DNA, que ativam tanto a resposta humoral como a celular, permitindo a prevenção e o tratamento de doenças como o cancro do colo do útero. Nas vacinas de DNA, o uso do DNA plasmídico (pDNA) como vetor não viral torna-se bastante apelativo, não só pela sua baixa toxicidade e elevada segurança, mas também pela simples produção e aplicação. A produção destas vacinas requer a purificação à escala preparativa do pDNA superenrolado (sc), considerada a isoforma biologicamente ativa. É, por isso, necessário explorar diversas estratégias de purificação de forma a obter o maior rendimento e pureza do pDNA sc. A cromatografia de afinidade com aminoácidos tem demonstrado ser uma abordagem promissora, pois permite a interação seletiva entre ligandos específicos e as biomoléculas de interesse, à semelhança de interações biológicas que ocorrem naturalmente entre proteínas a aminoácidos no organismo. Para além disso, o uso de monolitos como suporte cromatográfico tem vindo a demonstrar que estes suportes são uma excelente alternativa aos convencionais, visto terem uma maior capacidade de ligação para moléculas de grandes dimensões e que possibilitam a utilização de fluxos mais elevados, diminuindo o tempo de retenção da biomolécula de interesse, evitando assim a sua degradação. Assim, o presente trabalho teve como primeiro objetivo explorar diferentes estratégias de eluição cromatográficas, utilizando um monolito de arginina com um braço espaçador, no sentido de purificar o pDNA sc a usar numa vacina de DNA contra o cancro do colo do útero. Inicialmente, foram realizados vários ensaios, quer em condições de eluição iónicas quer hidrofóbicas, para avaliar o comportamento cromatográfico e a influência dos diferentes grupos imobilizados no monolito de epóxi. Depois, o monolito de arginina com um braço espaçador foi caracterizado em termos de capacidade dinâmica de ligação (2.53 mg/mL obtido a 10% da curva “breakthrough”), confirmando que este suporte apresenta maior capacidade de ligação do que um suporte convencional (0.133 mg/mL), modificados com o mesmo ligando (arginina). Por outro lado, este valor é menor que o valor de capacidade de ligação obtido com o monolito de arginina (3.55 mg/mL), provavelmente devido à eletronegatividade do braço espaçador que promove repulsão pelo pDNA. Para avaliar a seletividade do suporte, vários ensaios foram realizados utilizando amostras de plasmídeo pré-purificado com o kit comercial (isoformas circular aberta, linear e sc), manipulando a concentração de cloreto de sódio (NaCl) e o pH do tampão de eluição. Os resultados comprovaram que é possível obter a isoforma sc purificada, apesar da sua recuperação ser ligeiramente sacrificada. Posteriormente, prosseguiu-se para a purificação do pDNA sc a partir de uma amostra mais complexa de lisado de Escherichia coli (E. coli). Diferentes estratégias de eluição foram abordadas, incluindo a manipulação de NaCl e pH, assim como a adição de arginina no tampão de eluição como agente de competição. Após várias otimizações, a estratégia que melhor resultou na purificação da isoforma de interesse foi a de um gradiente por passos com o tampão de equilíbrio a 680 mM de NaCl em tampão 10 mM tris e 10 mM EDTA (Tris-EDTA), pH 7 e o tampão de eluição a 649 mM e 1 M de NaCl em Tris-EDTA, pH 7,5. Esta estratégia cromatográfica permitiu obter o plasmídeo sc com 93,3% de pureza e 72% de recuperação. A aplicabilidade do monolito de arginina com um braço espaçador na purificação do plasmídeo à escala preparativa também foi avaliada, tendo-se recuperado o plasmídeo com 98,5% de pureza. As impurezas (DNA genómico, proteínas e endotoxinas) das frações recolhidas de pDNA sc, tanto na escala laboratorial como na preparativa, foram quantificadas, estando os resultados dentro dos valores recomendados pelas agências reguladoras. Assim sendo, o monolito de arginina com um braço espaçador permitiu uma rápida e eficaz separação do pDNA sc, recorrendo a baixas concentrações de sal, tanto numa escala laboratorial como preparativa. Por outro lado, sabe-se que apenas um em mil plasmídeos apresentados às células eucarióticas conseguem alcançar o núcleo e levar à expressão do gene de interesse. Desta forma, torna-se crucial desenvolver estratégias que permitam a proteção do pDNA e que facilitem a sua entrada no núcleo. O uso de nanopartículas tem revelado ser uma valiosa solução, pois além de protegerem o pDNA da degradação enzimática, permitem uma entrega específica e, consequentemente, um aumento na transfeção celular. Assim sendo, este trabalho teve como segundo objetivo a formulação de nanopartículas de carbonato de magnésio (MgCO3) e gelatina, funcionalizadas com os ligandos de manose e galactose para direcionar as nanopartículas para as células alvo (células dendríticas). Em termos da morfologia, as imagens obtidas na microscopia eletrónica de varrimento (SEM) e na microscopia eletrónica de transmissão (TEM) permitiram concluir que todos os sistemas adquirem uma forma arredondada. Foi também calculada a eficiência de encapsulação (EE) dos diferentes sistemas com diferentes quantidades de pDNA, constatando-se que o sistema com 5 µg de pDNA possibilitou uma melhor encapsulação (cerca de 87%). Para além disso, a gelatina permitiu diminuir o tamanho médio das nanopartículas e a funcionalização com os ligandos de manose e galactose não aumentou significativamente o tamanho das nanopartículas de gelatina, estando os valores entre 99,7 nm e 237,4 nm. Por fim, os valores do potencial zeta foram positivos, o que sugere uma interação facilitada das nanopartículas com a membrana celular que é carregada negativamente, possibilitando uma transfeção mais eficiente. Todos os sistemas estudados apresentam características promissoras para um uptake celular adequado, o que foi comprovado pela transfecção de células HeLa. Em conclusão, o presente trabalho mostrou que o monolito de arginina com braço espaçador permitiu a purificação do pDNA sc com um bom grau de pureza e recuperação e as nanopartículas de MgCO3 provaram ser um sistema de entrega eficiente, sendo uma estratégia promissora para o desenvolvimento de uma vacina de DNA eficaz contra infeções provocadas pelo HPV

Nanocarriers for simultaneous delivery of structurally different polynucleotides encoding antigens and adjuvants

The rapid approval of mRNA-based vaccines for human use following the Covid-19 pandemic established their public health value. Yet, room for improvement to reduce number of required booster doses still exists. This study aimed to investigate the development of a nanocarrier to co-deliver NA-encoded antigens and adjuvants and achieve their time-resolved expression, such that adjuvant expression follows target cell (APC) priming with antigen expression. Hence, a core-shell system was constructed based on a plasmid DNA (pDNA)-gelatin coacervate core, thermally stabilized into a nanogel, coated with protamine (P-TS-CoAc), and surface loaded with mRNA. The system showed unique co-transfectional ability for two such NAs encoding fluorescent reporter proteins when compared to several established controls in dendritic murine cell line (DC2.4). The system also showed time-resolved expression of the two NAs, with a rapid and transient expression of mRNA on the shell and a delayed, prolonged-expression of pDNA in the core. NA-encoded adjuvant candidates were selected to assess P-TS-CoAc’s capacity to transfect them into DC2.4, namely, CCL4 and CCR7, involved in mobilizing immune cells towards inflammation sites or draining lymph nodes, respectively. CCL4 encoding NAs induced CCL4 release from DC2.4 following electroporation, yet, not upon DC2.4 treatment with P-TS-CoAc loaded with CCL4 encoding-NAs. Further studies could still be conducted to improve system performance.Die rasche Zulassung von mRNA-basierten Impfstoffen für den menschlichen Gebrauch nach der Covid-19-Pandemie hat ihren Wert für die öffentliche Gesundheit bewiesen. Dennoch gibt es Raum für Verbesserungen, um die Anzahl der erforderlichen Auffrischungsdosen zu verringern. Ziel dieser Studie war es, einen Nanoträger zu entwickeln, der Nukleotid (NT)-kodierte Antigene und Adjuvantien gemeinsam abgibt und deren zeitversetzte Expression ermöglicht, so dass die Adjuvantexpression dem Priming der Zielzellen (APC) mit der Antigenexpression folgt. Daher wurde ein Kern-Schale-System konstruiert, das auf einem Plasmid-DNA (pDNA)- Gelatine-Koazervat-Kern basiert, der thermisch zu einem Nanogel stabilisiert, mit Protamin (P-TS-CoAc) beschichtet und an der Oberfläche mit mRNA beladen wurde. Das System zeigte eine einzigartige Co-Transfektion für beide NT, die für fluoreszierende Reporterproteine kodieren, im Vergleich zu mehreren etablierten Kontrollen in einer dendritischen Mäusezelllinie (DC2.4). Ebenso zeigte das System eine zeitversetzte Expression der beiden NT, mit einer schnellen,vorübergehenden Expression der mRNA und einer verzögerten, längeren Expression der pDNA. Es wurden NT-kodierte Adjuvans-Kandidaten ausgewählt, um die Transfektionsfähigkeit von P-TS-CoAc in DC2.4 zu prüfen. Diese waren CCL4 und CCR7, die an der Mobilisierung von Immunzellen zu Entzündungsherden bzw. drainierenden Lymphknoten beteiligt sind. CCL4-kodierende NT induzierten die Freisetzung von CCL4 aus DC2.4 nach Elektroporation, jedoch nicht nach Behandlung von DC2.4 mit entsprechend beladenen PTS- CoAc. Weitere Studien könnten durchgeführt werden, um die Leistung des Systems zu verbessern

Self-assembled hydrogel nanoparticles for drug delivery applications

Hydrogel nanoparticles—also referred to as polymeric nanogels or macromolecular micelles—are emerging as promising drug carriers for therapeutic applications. These nanostructures hold versatility and properties suitable for the delivery of bioactive molecules, namely of biopharmaceuticals. This article reviews the latest developments in the use of self-assembled polymeric nanogels for drug delivery applications, including small molecular weight drugs, proteins, peptides, oligosaccharides, vaccines and nucleic acids. The materials and techniques used in the development of self-assembling nanogels are also described

Editorial: biodegradable materials

This Special Issue “Biodegradable Materials” features research and review papers concerning recent advances on the development, synthesis, testing and characterisation of biomaterials. These biomaterials, derived from natural and renewable sources, offer a potential alternative to existing non-biodegradable materials with application to the food and biomedical industries amongst many others. In this Special Issue, the work is expanded to include the combined use of fillers that can enhance the properties of biomaterials prepared as films. The future application of these biomaterials could have an impact not only at the economic level, but also for the improvement of the environment

Novel amphiphilic block copolymers and their self-assembled injectable hydrogels for gene delivery

This work describes the development and investigation of a family of novel smart copolymers as non-viral gene delivery vectors. The copolymers have five blocks, and thus named pentablock, with a central block of a hydrophobic polymer, surrounded by two blocks of a hydrophilic polymer, and capped at each terminal end with cationic polymer blocks, arranged in an architecture to provide temperature and pH sensitivity to the copolymers. They are derived from commercially available triblock Pluronic copolymers. The cationic copolymers can efficiently condense negatively charged plasmid DNA in nanostructures with efficient cellular uptake. The amphiphilic nature of copolymers causes them to exist as micelles in aqueous solutions that help them traverse cellular membranes with minimal cell membrane damage. Intra-cellular trafficking of copolymer/DNA complexes revealed that they are up-taken by the cells predominately via endocytosis and are able to deliver the ferried gene into the nuclei. The copolymers efficiently protect the condensed DNA against degradation by nucleases while their protonation capability at low pH assists them in escape from endosomal vesicles into the cytoplasm. The efficiency of the copolymers to deliver condensed DNA into the cells in vitro was comparable to the commercially available polymeric transfection vectors, and they were also found to be significantly less cytotoxic. Adding non-ionic Pluronic copolymers to the formulation of pentablock copolymer/DNA complexes sterically shielded their surface charge and protected them against aggregation with serum proteins. These stabilized formulations were able to retain their ability to transfect cells even in complete growth media supplemented with serum proteins, warranting efficient transfection efficiency in an in vivo application. The amphiphilic nature of copolymers further permits copolymer/DNA complexes to form thermo-reversible hydrogels at physiological temperatures. At concentrations above 15 wt%, copolymer/DNA complexes existed as solutions at room temperature and formed elastic hydrogels at 37°C that dissolved over seven days in excess buffers to release colloidally stable polyplexes. The system thus permits an injectable aqueous pharmaceutical preparation at room temperature that can be injected subcutaneously in tissues/cavities to form a localized depot in situ, which provides a long-term sustained release of therapeutic genes well protected inside the copolymer/DNA complexes

Controlled Release Systems for DNA Delivery

Adapting controlled release technologies to the delivery of DNA has the potential to overcome extracellular barriers that limit gene therapy. Controlled release systems can enhance gene delivery and increase the extent and duration of transgene expression relative to more traditional delivery methods (e.g., injection). These systems typically deliver vectors locally, which can avoid distribution to distant tissues, decrease toxicity to nontarget cells, and reduce the immune response to the vector. Delivery vehicles for controlled release are fabricated from natural and synthetic polymers, which function either by releasing the vector into the local tissue environment or by maintaining the vector at the polymer surface. Vector release or binding is regulated by the effective affinity of the vector for the polymer, which depends upon the strength of molecular interactions. These interactions occur through nonspecific binding based on vector and polymer composition or through the incorporation of complementary binding sites (e.g., biotin–avidin). This review examines the delivery of nonviral and viral vectors from natural and synthetic polymers and presents opportunities for continuing developments to increase their applicability

Amino acids, peptides, and proteins:Implications for nanotechnological applications in biosensing and drug/gene delivery

Over various scientific fields in biochemistry, amino acids have been highlighted in research works. Protein, peptide- and amino acid-based drug delivery systems have proficiently transformed nanotechnology via immense flexibility in their features for attaching various drug molecules and biodegradable polymers. In this regard, novel nanostructures including carbon nanotubes, electrospun carbon nanofibers, gold nanoislands, and metal-based nanoparticles have been introduced as nanosensors for accurate detection of these organic compounds. These nanostructures can bind the biological receptor to the sensor surface and increase the surface area of the working electrode, significantly enhancing the biosensor performance. Interestingly, protein-based nanocarriers have also emerged as useful drug and gene delivery platforms. This is important since, despite recent advancements, there are still biological barriers and other obstacles limiting gene and drug delivery efficacy. Currently available strategies for gene therapy are not cost-effective, and they do not deliver the genetic cargo effectively to target sites. With rapid advancements in nanotechnology, novel gene delivery systems are introduced as nonviral vectors such as protein, peptide, and amino acid-based nanostructures. These nano-based delivery platforms can be tailored into functional transformation using proteins and peptides ligands based nanocarriers, usually overexpressed in the specified diseases. The purpose of this review is to shed light on traditional and nanotechnology-based methods to detect amino acids, peptides, and proteins. Furthermore, new insights into the potential of amino protein-based nanoassemblies for targeted drug delivery or gene transfer are presented

Electrospun polyvinyl alcohol/carbon dioxide modified polyethyleneimine composite nanofiber scaffolds

A novel biocompatible polyvinyl alcohol/carbon dioxide modified polyethyleneimine (PVA/PEI-CO2) composite nanofiber was fabricated by a green and facile protocol, which reduces the cytotoxicity of PEI through the surface modification of the PEI with CO2. The 13C NMR spectrum, elemental analysis, and TGA show that CO2 has been incorporated in the PEI surface resulting in a relatively stable structure. The resulting PVA/PEI-CO2 composite nanofibers have been characterized by attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR), contact angle, and scanning electron microscopy (SEM). The results show that the average diameters of the nanofibers range from 265 ± 53 nm to 423 ± 80 nm. The cytotoxicity of PVA/PEI-CO2 composite nanofibers was assessed by cytotoxicity evaluation using the growth and cell proliferation of normal mice Schwann cells. SEM and the MTT assay demonstrated the promotion of cell growth and proliferation on the PVA/PEI-CO2 composite scaffold. It suggests that PEI-CO2 can have tremendous potential applications in biological material research

Aptamer-functionalized natural protein-based polymers as innovative biomaterials

Producción CientíficaBiomaterials science is one of the most rapidly evolving fields in biomedicine. However, although novel biomaterials have achieved well-defined goals, such as the production of devices with improved biocompatibility and mechanical properties, their development could be more ambitious. Indeed, the integration of active targeting strategies has been shown to allow spatiotemporal control of cell–material interactions, thus leading to more specific and better-performing devices. This manuscript reviews recent advances that have led to enhanced biomaterials resulting from the use of natural structural macromolecules. In this regard, several structural macromolecules have been adapted or modified using biohybrid approaches for use in both regenerative medicine and therapeutic delivery. The integration of structural and functional features and aptamer targeting, although still incipient, has already shown its ability and wide-reaching potential. In this review, we discuss aptamer-functionalized hybrid protein-based or polymeric biomaterials derived from structural macromolecules, with a focus on bioresponsive/bioactive systems.Ministerio de Economía, Industria y Competitividad - Fondo Europeo de Desarrollo Regional - Fondo Social Europeo (Proyects MAT2016-79435-R, DTS19/00162, and PID2019-106386RB-I00)Junta de Castilla y León (Project VA317P18