Neoadjuvant Intratumoral Immunotherapy with Cowpea Mosaic Virus Induces Local and Systemic Antitumor Efficacy in Canine Mammary Cancer Patients

The lack of optimal models to evaluate novel agents is delaying the development of effective immunotherapies against human breast cancer (BC). In this prospective open label study, we applied neoadjuvant intratumoral immunotherapy with empty cowpea mosaic virus-like particles (eCPMV) to 11 companion dogs diagnosed with canine mammary cancer (CMC), a spontaneous tumor resembling human BC. We found that two neoadjuvant intratumoral eCPMV injections resulted in tumor reduction in injected tumors in all patients and in noninjected tumors located in the ipsilateral and contralateral mammary chains of injected dogs. Tumor reduction was independent of clinical stage, tumor size, histopathologic grade, and tumor molecular subtype. RNA-seq-based analysis of injected tumors indicated a decrease in DNA replication activity and an increase in activated dendritic cell infiltration in the tumor microenvironment. Immunohistochemistry analysis demonstrated significant intratumoral increases in neutrophils, T and B lymphocytes, and plasma cells. eCPMV intratumoral immunotherapy demonstrated antitumor efficacy without any adverse effects. This novel immunotherapy has the potential for improving outcomes for human BC patients

COVID-19 vaccines based on viral nanoparticles displaying a conserved B-cell epitope show potent immunogenicity and a long-lasting antibody response

The COVID-19 pandemic caused by SARS-CoV-2 sparked intensive research into the development of effective vaccines, 50 of which have been approved thus far, including the novel mRNA-based vaccines developed by Pfizer and Moderna. Although limiting the severity of the disease, the mRNA-based vaccines presented drawbacks, such as the cold chain requirement. Moreover, antibody levels generated by these vaccines decline significantly after 6 months. These vaccines deliver mRNA encoding the full-length spike (S) glycoprotein of SARS-CoV-2, but must be updated as new strains and variants of concern emerge, creating a demand for adjusted formulations and booster campaigns. To overcome these challenges, we have developed COVID-19 vaccine candidates based on the highly conserved SARS CoV-2, 809-826 B-cell peptide epitope (denoted 826) conjugated to cowpea mosaic virus (CPMV) nanoparticles and bacteriophage Qβ virus-like particles, both platforms have exceptional thermal stability and facilitate epitope delivery with inbuilt adjuvant activity. We evaluated two administration methods: subcutaneous injection and an implantable polymeric scaffold. Mice received a prime–boost regimen of 100 μg per dose (2 weeks apart) or a single dose of 200 μg administered as a liquid formulation, or a polymer implant. Antibody titers were evaluated longitudinally over 50 weeks. The vaccine candidates generally elicited an early Th2-biased immune response, which stimulates the production of SARS-CoV-2 neutralizing antibodies, followed by a switch to a Th1-biased response for most formulations. Exceptionally, vaccine candidate 826-CPMV (administered as prime-boost, soluble injection) elicited a balanced Th1/Th2 immune response, which is necessary to prevent pulmonary immunopathology associated with Th2 bias extremes. While the Qβ-based vaccine elicited overall higher antibody titers, the CPMV-induced antibodies had higher avidity. Regardless of the administration route and formulation, our vaccine candidates maintained high antibody titers for more than 50 weeks, confirming a potent and durable immune response against SARS-CoV-2 even after a single dose

Synthesis of mesoporous silica nanoparticles functionalized with monoclonal antibodies : an alternative for Hodgkin (HL) and Anaplastic Large Cell CD30+ (ALCL-CD30+) lymphomas treatment

Orientadores: Mateus Borba Cardoso, Edvaldo SabadiniTese (doutorado) - Universidade Estadual de Campinas, Instituto de QuímicaResumo: O presente trabalho descreve a síntese de nanopartículas de sílica (SiO2NPs) utilizadas no encapsulamento do fármaco antitumoral doxorrubicina (DOX) e posterior funcionalização com o anticorpo monoclonal Ki-1, para o tratamento de células de linfoma CD30+. Devido às suas propriedades físico-químicas e ao eficiente transporte de moléculas biologicamente ativas, as SiO2NPs têm sido consideradas promissores sistemas de entrega de fármacos. No entanto, quando SiO2NPs estão em contato com fluidos biológicos, proteínas tendem adsorver em sua superfície, formando um recobrimento conhecido como coroa de proteínas (do inglês protein corona), consequentemente afetando sua interação biológica. Nesse contexto, o primeiro desafio a ser transposto é prevenir a adsorção inespecífica de proteínas na superfície das SiO2NPs. Assim, propusemos uma modificação na superfície das mesmas de forma a obter dupla funcionalização, de maneira a modular seu comportamento in vitro. Portanto, a funcionalização das SiO2NPs foi realizada com a sulfobetaína zwitteriônica, que fornece estabilidade coloidal, e com o 3-aminopropiltrietóxisilano (APTES), cujas as aminas primárias (-NH2) presentes em sua estrutura química possibilitam posteriores reações de acoplamento. Verificamos que a porção zwitteriônica foi bastante efetiva na prevenção da adsorção de proteínas, mesmo em soluções com elevada concentração das mesmas. Isso permitiu a manutenção da identidade biológica dos materiais sintetizados, possibilitando sua posterior interação com as células de interesse. Além disso, o segundo desafio desse trabalho está relacionado à avaliação do comportamento biológico e especificidade das SiO2NPs funcionalizadas com anticorpo monoclonal Ki-1 em ensaios celulares. Ki-1 foi escolhido devido à sua especificidade no reconhecimento da proteína transmembranar CD30, a qual é superexpressa na membrana celular de linfomas Hodgkin (LH) e anaplásico de grandes células (ALCL). Dessa forma, ensaios de viabilidade celular foram realizados através de citometria de fluxo com as linhagens celulares L540 e Karpas 299, sendo ambas CD30+. Investigamos o direcionamento seletivo dos materiais funcionalizados com Ki-1, e pudemos observar que as SiO2NPs não funcionalizadas apresentam efeito citotóxico negligenciável, mantendo a viabilidade celular acima de 90%. Por outro lado, as nanopartículas funcionalizadas com Ki-1 apresentaram redução de ~30% na viabilidade celular de ambas L540 e Karpas 299. Acreditamos que o efeito citotóxico das SiO2NPs só é observado quando as mesmas são internalizadas. Assim, é possível que a funcionalização com o Ki-1 favoreça a entrada das nanopartículas, por meio da interação do anticorpo com a proteína CD30 da membrana das células estudadas, resultando em internalização mediada por receptor celular. Ensaios de microscopia confocal possibilitaram validar a hipótese citada, porém ainda há a necessidade de investigações que justifiquem plenamente a internalização e consequente citotoxicidade ocasionada pelas SiO2NPs. Dessa maneira, o presente trabalho é de fundamental contribuição para o avanço das pesquisas relacionadas ao direcionamento biológico e seletivo utilizando nanopartículas. Apresentamos uma estratégia de modificação de superfície que, ao mesmo tempo que mantém a identidade biológica dos materiais, favorece o acoplamento com moléculas direcionadoras. Essa metodologia pode contribuir para a estabilidade e melhor biodisponibilidade de fármacos encapsulados, assim como reduzir possíveis efeitos adversos apresentados pelos tratamentos quimioterápicos convencionaisAbstract: The present work describes the synthesis of silica nanoparticles (SiO2NPs) used on the antitumor drug doxorubicin (DOX) encapsulation and subsequent functionalization with the monoclonal antibody Ki-1, for CD30+ lymphoma cells treatment. Due to their physico-chemical properties and the efficient transport of biologically active molecules, SiO2NPs have been considered promising drug delivery systems. However, when SiO2NPs are in contact with biological fluids, proteins tend to adsorb on their surface, forming a coating known as the protein corona, consequently affecting their biological interaction. In this context, the first challenge to be transposed is to prevent nonspecific proteins adsorption on the SiO2NPs surface. Thus, we propose a surface modification to obtain dual functionalization, in order to modulate their behavior in vitro. Therefore, the functionalization of SiO2NPs was performed with the zwitterionic sulfobetaine, which provides colloidal stability and with 3-aminopropyltriethoxysilane (APTES), whose primary amines (-NH2) present in their chemical structure enable subsequent coupling reactions. We verified that the zwitterionic portion was very effective in the protein adsorption prevention, even in solutions with high protein concentration. This allowed the maintenance of the biological identity of the synthesized materials, allowing their subsequent interaction with the targeted cells. In addition, the second challenge of this work is related to the evaluation of the biological behavior and specificity of SiO2NPs functionalized with Ki-1 monoclonal antibody in cell assays. Ki-1 was chosen because of its specificity in the recognition of CD30 transmembrane protein, which is overexpressed in the cell membrane of Hodgkin's (LH) and anaplastic large cell (ALCL) lymphomas. Thus, cell viability assays were performed through flow cytometry with L540 and Karpas 299 cell lines, both CD30+. We investigated the selective targeting of Ki-1 functionalized materials, and we could observe that the non-functionalized SiO2NPs show negligible cytotoxic effect, maintaining cell viability above 90%. On the other hand, Ki-1 functionalized nanoparticles showed ~ 30% reduction in the cellular viability for both L540 and Karpas 299. We believe that the cytotoxic effect of SiO2NPs is only observed when they are internalized. Thus, it is possible that the functionalization with Ki-1 favors the entry of the nanoparticles, through the interaction of the antibody with the membrane CD30 protein of the cells studied, resulting in cell receptor-mediated internalization. Confocal microscopy assays allowed to validate the hypothesis cited, but there is still a need for investigations that fully justify the internalization and consequent cytotoxicity caused by SiO2NPs. In this way, the present work is of fundamental contribution for the advancement of research related to the biological and selective targeting using nanoparticles. We present a surface modification strategy that, while maintaining the biological identity of the materials, favors the coupling with targeting molecules. This methodology can contribute to the stability and better bioavailability of encapsulated drugs, as well as reduce possible side effects presented by conventional chemotherapeutic treatmentsDoutoradoFísico-QuímicaDoutora em Ciências300142/2014-5, 301298/2014-92013/22429-9CAPESFAPES

Functionaliztion of silver nanoparticles with 'beta'-lactam antibiotics : an alternative to bacterial resistance

Orientadores: Mateus Borba Cardoso, Edvaldo SabadinDissertação (mestrado) - Universidade Estadual de Campinas, Instituto de QuímicaResumo: O trabalho aqui descrito teve como objetivo a obtenção de um sistema com duplo poder de inibição bacteriana baseado em nanopartículas de prata (AgNPs) recobertas com sílica mesoporosa (SM) para subsequente funcionalização com antibióticos b-lactâmicos. O agente b-lactâmico utilizado durante o desenvolvimento do projeto foi a ampicilina. Além disso, o trabalho teve como foco: (1) analisar o possível sinergismo da utilização das AgNPs e do antibiótico b-lactâmico contra bactérias suscetíveis à ampicilina e (2) investigar uma possível ação bactericida dos sistemas sintetizados contra Escherichia coli resistente ao agente b-lactâmico escolhido. Durante o trabalho, duas sínteses distintas para a obtenção das AgNPs foram testadas. A primeira delas é baseada na redução química dos íon Ag+ por boroidreto de sódio (NaBH4) utilizando citrato de sódio como agente protetor. Através desse método, não foi possível recobrir as AgNPs com sílica. No entanto, essa metodologia possibilitou estudar o fenômeno de crescimento e agregação parcial das nanopartículas sintetizadas. Dessa forma, a segunda alternativa para sintetizar as AgNPs foi baseada na redução química dos íons Ag+ em etileno glicol (EG), na presença de polivinilpirrolidona (PVP). Essa estratégia permitiu o recobrimento das AgNPs com sílica e essas nanopartículas foram então utilizadas para os procedimentos de funcionalização com a ampicilina. Foram realizados experimentos de atividade biológica com as bactérias Escherichia coli e Escherichia coli resistente à ampicilina (Gram-negativas) e Staphylococcus aureus (Gram-positiva), através da incubação do material juntamente com as bactérias, seguida pela cultura das mesmas em placas de ágar. Todos os materiais sintetizados apresentaram propriedades antibacterianas, que aumentam com o aumento da concentração dos materiais utilizados. A partir dos ensaios biológicos, foi possível observar que as nanopartículas de prata recobertas com sílica mesoporosa (Ag@SiO2) possuem elevado efeito antimicrobiano, ocasionando 100% de redução do número de colônias para todas as bactérias estudadas. Além disso, é possível notar que a funcionalização com ampicilina aumentou as propriedades bactericidas das nanopartículas de sílica e, em altas concentrações, o sistema de prata funcionalizado com ampicilina (Ag@SiO2-Ampicilina), apresentou resultados semelhantes ao seu respectivo precursor sem funcionalização. Uma explicação viável para a menor eficiência do sistema Ag@SiO2-Ampicilina em baixas concentrações pode estar relacionada à oclusão dos poros da sílica mesoporosa, ocasionada pelo processo de funcionalização, impedindo assim a lixiviação da prata iônica. Apesar dos resultados satisfatórios obtidos para o sistema Ag@SiO2-Ampicilina, ainda é preciso investigar uma alternativa que não ocasione a obstrução dos poros da sílica e, dessa forma não reduza o efeito da prata presente no sistema estudadoAbstract: The work described here aimed to obtain a dual bacterial inhibition system based on silver nanoparticles (AgNPs) coated with mesoporous silica (MS) for further functionalization with b-lactams antibiotics. Ampicillin was the b-lactam chosen for the project development. Moreover, this work is focused on: (1) analyzing the possible synergism of using AgNPs combined with b-lactam antibiotic against ampicillin susceptible bacteria and (2) investigating a possible bactericidal action of the synthesized systems against Escherichia coli strain which is resistant to b-lactam. Two different methodologies for AgNPs synthesis were tested. The first one is based on the chemical reduction of Ag+ ions by sodium borohydride (NaBH4) in the presence of sodium citrate as capping agent, which did not allow the silica coating. However, it allowed us to study growth and partial aggregation of these nanoparticles. Therefore, the second alternative to synthesize AgNPs was based on the chemical reduction of Ag+ ions solubilized by ethylene glycol (EG) in the presence of polyvinylpyrrolidone (PVP). This strategy allowed us coating the AgNPs with silica and these nanoparticles were used for functionalization with ampicillin. Biological activity experiments were conducted with Escherichia coli and Escherichia coli resistant to ampicillin (Gram-negatives) and Staphylococcus aureus (Gram-positive), through nanoparticles incubation test in tubes containing the bacteria, followed by culturing on agar plates. All synthesized materials showed antibacterial properties, which increase with increasing concentration of the materials tested. From the biological tests, it is observed that silver nanoparticles coated with mesoporous silica (Ag@SiO2) have high antimicrobial effect, promoting 100% reduction in the number of colonies for all bacteria studied. Furthermore, it is noticed that the functionalization with ampicillin increased the bactericidal properties of silica nanoparticles, and in high concentrations, silver functionalized with ampicillin (Ag@SiO2-Ampicillin) presented similar results to its respective precursor without functionalization. One possible explanation for the lower efficiency of the Ag@SiO2-Ampicillin system at low concentrations may be related to the occluded pores of mesoporous silica, caused by the functionalization process, while preventing the leaching of ionic silver. Despite the satisfactory results obtained for Ag@SiO2-Ampicillin system, it is still necessary to investigate an alternative which does not cause occlusion of silica pores and, consequently, does not reduce the effect of silver in the studied systemMestradoFísico-QuímicaMestra em Químic

Partial Aggregation of Silver Nanoparticles Induced by Capping and Reducing Agents Competition

It is well known that nanomaterials properties and applications are dependent on the size, shape, and morphology of these structures. Among nanomaterials, silver nanoparticles (AgNPs) have attracted attention since they have considerably versatile properties, such as a variable surface area to volume ratio, which is very useful for many biomedical and technological applications. Within this scenario, small nanoparticle aggregates can have their properties reduced due to the increased size and alterations in their shape/morphology. In this work, silver nanoparticles aggregation was studied through chemical reduction of silver nitrate in the presence of sodium borohydride (reducing agent) and sodium citrate (capping agent). By changing the amount of reducing agent along the reaction, unaggregated and partially aggregated samples were obtained and characterized by UV–vis, zeta potential, and SAXS techniques. pH was measured in every step of the reaction in order to correlate these results with those obtained from structural techniques. Addition of the reducing agent first causes the reduction of Ag⁺ to silver nanoparticles. For higher concentrations of sodium borohydrate, the average AgNPs size is increased and NPs aggregation is observed. It was found that zeta potential and pH values have a strong influence on AgNPs formation, since reducing agent addition can induce partial removal of citrate weakly associated on the AgNPs surface and increase the ionic strength of the solution, promoting partial aggregation of the particles. This aggregation state was duly identified by coupling SAXS, zeta potential and pH measurements. In addition, the SAXS technique showed that aggregates formed along the process are elongated-like particles due to the exponential decay evidenced through SAXS curves

Are antibiotic-functionalized nanoparticles a promising tool in antimicrobial therapies?

Sem informação122325872590FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP2013/22429-9; 2014/22322-2; 2015/25406-5; 2017/01167-