Intracellular trafficking of size-tuned nanoparticles for drug delivery

Polymeric nanoparticles (NPs) are widely used as drug delivery systems in nanomedicine. Despite their widespread application, a comprehensive understanding of their intracellular trafficking remains elusive. In the present study, we focused on exploring the impact of a 20 nm difference in size on NP performance, including drug delivery capabilities and intracellular trafficking. For that, poly(ethylene glycol) methyl ether-block-poly(lactide-co-glycolide) (PLGA-PEG) NPs with sizes of 50 and 70 nm were precisely tailored. To assess their prowess in encapsulating and releasing therapeutic agents, we have employed doxorubicin (Dox), a well-established anticancer drug widely utilized in clinical settings, as a model drug. Then, the beneficial effect of the developed nanoformulations was evaluated in breast cancer cells. Finally, we performed a semiquantitative analysis of both NPsâ uptake and intracellular localization by immunostaining lysosomes, early endosomes, and recycling endosomes. The results show that the smaller NPs (50 nm) were able to reduce the metabolic activity of cancer cells more efficiently than NPs of 70 nm, in a time and concentration-dependent manner. These findings are corroborated by intracellular trafficking studies that reveal an earlier and higher uptake of NPs, with 50 nm compared to the 70 nm ones, by the breast cancer cells. Consequently, this study demonstrates that NP size, even in small increments, has an important impact on their therapeutic effect.The authors would like to thank the funders that allowed for carrying out this work, namely the Fundação para a Ciência e a Tecnologia (FCT) for the S. Gimondi fellowship (PD/BD/143140/2019; COVID/BD/153033/2022) and for the Associated Laboratory Project, ICVS/3B’s (UIDP/50026/2020). This work was also supported by HEALTH UNORTE (NORTE-01-0145-FEDER-000039). The authors would also like to thank the contributions to this research from the project “TERM RES Hub—Scientific Infrastructure for Tissue Engineering and Regenerative Medicine”, reference PINFRA/22190/2016 (Norte-01-0145-FEDER-022190), funded by the Portuguese National Science Foundation (FCT) in cooperation with the Northern Portugal Regional Coordination and Development Commission (CCDR-N), for providing relevant lab facilities, state-of-the-art equipment, and highly qualified human resources

Microfluidic-derived docosahexaenoic acid liposomes for targeting glioblastoma and Its inflammatory microenvironment

Glioblastoma (GBM) is the most common malignant primary brain tumor, characterized by limited treatment options and a poor prognosis. Its aggressiveness is attributed not only to the uncontrolled proliferation and invasion of tumor cells but also to the complex interplay between these cells and the surrounding microenvironment. Within the tumor microenvironment, an intricate network of immune cells, stromal cells, and various signaling molecules creates a pro-inflammatory milieu that supports tumor growth and progression. Docosahexaenoic acid (DHA), an essential ω3 polyunsaturated fatty acid for brain function, is associated with anti-inflammatory and anticarcinogenic properties. Therefore, in this work, DHA liposomes were synthesized using a microfluidic platform to target and reduce the inflammatory environment of GBM. The liposomes were rapidly taken up by macrophages in a time-dependent manner without causing cytotoxicity. Moreover, DHA liposomes successfully downregulated the expression of inflammatory-associated genes (IL-6; IL-1β; TNFα; NF-κB, and STAT-1) and the secretion of key cytokines (IL-6 and TNFα) in stimulated macrophages and GBM cells. Conversely, no significant differences were observed in the expression of IL-10, an anti-inflammatory gene expressed in alternatively activated macrophages. Additionally, DHA liposomes were found to be more efficient in regulating the inflammatory profile of these cells compared with a free formulation of DHA. The nanomedicine platform established in this work opens new opportunities for developing liposomes incorporating DHA to target GBM and its inflammatory milieu

Development of polymeric nanoparticles by microfluidic technologies as functional drug delivery devices

Tese de doutoramento em Engenharia de Tecidos, Medicina Regenerativa e Células EstaminaisNanoparticles (NPs) are entities with dimensions ranging from 1 and 100 nanometers (nm) in size. As a result of their narrow dimension, NPs exhibit a large surface area-to-volume ratio compared to bulk material, which lead to improved properties. Indeed, NPs have been widely explored in different research fields, demonstrating a strong potential in nanomedicine as drug delivery systems. However, their performance is strictly related to their physicochemical characteristics. Among the different parameters that can be tuned in the resulting NPs, the size stands out for its key role in determining NPs fate, biodistribution, cell interaction and resulting biological effects. However, the ability to produce NPs with precise and defined sizes remains a challenge for the most common and traditional synthesis techniques. Polymeric NPs are mainly synthesized through the nanoprecipitation process. This reaction leads to the production of NPs through the nucleation of the individual polymer chains until their growth into the final entities. During this process, one of the main factors that affects the resulting product size is the mixing of the organic and the aqueous phase. Indeed, the mixing guides the diffusion process between the two miscible phases and, consequently, the formation of NPs. To achieve high mixing performances, the work herein presented leverages microfluidic technology to obtain NPs with a defined size. Indeed, microfluidics allow to investigate the behavior of fluids flowing through microscale channels and can be efficiently applied to NPs production. At first, we assessed the impact of several experimental parameters (polymer concentration, flow rates, and flow rate ratio between the aqueous and organic solutions) on the resulting NPs features, in particular their size. Then, three sizes of interest were selected (30, 50 and 70 nm) to investigate the role of the NPs size on drug delivery and biological effects. Additionally, it was explored the NPs size influence on their ability for biological barriers crossing, as well as cellular internalization and trafficking. The employed micromixer also revealed being a powerful platform for the handling of high molecular weight polymers. Indeed, the synthesis of chitosan-hyaluronic acid NPs was successfully carried out by the microfluidic chip, leading to smaller NPs compared to the conventional method (dropwise). The data herein reported indicates that the NPs size can have a significant impact in their pharmacokinetics and cells response, suggesting that the precise control of NPs features can tailor the delivery of bioactive agents and enhance their biological efficacy.As nanopartículas (NPs) são estruturas com dimensões entre 1 e 100 nanómetros (nm). Devido à sua dimensão, as NPs exibem uma grande relação área de superfície-volume, o que melhora as suas propriedades para diferentes aplicações. As NPs têm sido exploradas em diferentes áreas de investigação, demonstrando um forte potencial na nanomedicina como sistemas de libertação de fármacos. O desempenho das NPs está estritamente relacionado com as suas características físico químicas. Entre as diferentes características das NPs, o tamanho tem importantes implicações na biodistribuição, interação celular e consequentes efeitos biológicos. Porém, a capacidade de produzir NPs com tamanhos precisos e definidos continua a ser um desafio para as técnicas de síntese atuais. As NPs poliméricas são sintetizadas principalmente através do processo de nanoprecipitação. Neste processo, as NPs começam a formar-se através da nucleação das cadeias poliméricas individuais, até atingir o seu tamanho final. Durante este processo, um dos principais fatores que afetam o tamanho das NPs é a eficiência da mistura da fase orgânica com a aquosa. De facto, a mistura guia o processo de difusão entre as duas fases miscíveis e, consequentemente, a formação das NPs. Para alcançar altas performances de mistura, neste trabalho aproveitou-se a tecnologia de microfluídica para obter NPs com um tamanho definido. A tecnologia de microfluídica permite manipular o comportamento de fluidos em canais com geometria à microescala, e pode ser aplicada de forma eficiente na produção de NPs. Primeiramente, avaliou-se o impacto de vários parâmetros experimentais (concentração de polímero, fluxos e rácio de fluxos entre a solução aquosa e orgânica) nas características das NPs, nomeadamente no seu tamanho. Seguidamente, foram selecionados três tamanhos (30, 50 e 70 nm) para estudar diferentes questões, nomeadamente, o papel do tamanho das NPs na entrega de fármacos e consequentemente os seus efeitos biológicos. A influência do tamanho das NPs na capacidade destas atravessarem barreiras biológicas, direcionar vias de internalização, e localização intracelular, foi também estudada. O micro-misturador utilizado revelou ser uma poderosa plataforma para a manipulação de polímeros de elevado peso molecular. A síntese de NPs de quitosano-ácido hialurónico foi alcançada com sucesso pelo chip, levando a NPs menores em comparação com o método tradicional (adição gota a gota). Os resultados obtidos indicam que o tamanho das NPs pode ter um impacto significativo na farmacocinética e na resposta celular, sugerindo que o controle preciso das características das NPs pode permitir ajustar a entrega de agentes ativos e aumentar sua eficácia biológica.I would like to acknowledge the Portuguese Foundation for Science and Technology (FCT) for my PhD scholarship (PD/BD/143140/2019)

Microfluidic-driven mixing of high molecular weight polymeric complexes for precise nanoparticle downsizing

Chitosan (CHIT) and hyaluronic acid (HA) are two polysaccharides (PSs) with high value in several biomedical applications. In this study, we present a microfluidic method to synthetize CHIT-HA NPs to overcome the disadvantages of the dropwise approach generally used for nanoprecipitation of polyelectrolyte complexes. The proposed microfluidic approach enables to generate monodisperse suspensions of NPs with ≈100 nm of size compared to the dropwise method that generated ≈2 times bigger NPs. Finally, we evaluated the potential of obtained NPs in an inflammatory scenario. The treatment with NPs led to the reduction of the main inflammatory molecules produced by macrophages (PGE2, IL-6, IL-8, MCAF and TNF-α) and fibroblasts (IL-1 α, PGE2, TNF-α) stimulated with lipopolysaccharide or conditioned medium, respectively. This study demonstrates that our approach can be used to enhance the synthesis of nanocarriers based on bioactive macromoleculesThe authors would like to thank funding that allowed to carry out this work, namely the Fundação para a Ciência e a Tecnologia (FCT) for the S. Gimondi fellowship (PD/BD/143140/2019) and PATH program (PD/00169/2013). This work was also supported by Cells4_IDs (PTDC/BTM-SAL/28882/2017) and the NORTE 2020 Structured Project, co-funded by Norte2020 (NORTE-01-0145-FEDER-000021)

Intracellular Trafficking of Size-Tuned Nanoparticles for Drug Delivery

Polymeric nanoparticles (NPs) are widely used as drug delivery systems in nanomedicine. Despite their widespread application, a comprehensive understanding of their intracellular trafficking remains elusive. In the present study, we focused on exploring the impact of a 20 nm difference in size on NP performance, including drug delivery capabilities and intracellular trafficking. For that, poly(ethylene glycol) methyl ether-block-poly(lactide-co-glycolide) (PLGA-PEG) NPs with sizes of 50 and 70 nm were precisely tailored. To assess their prowess in encapsulating and releasing therapeutic agents, we have employed doxorubicin (Dox), a well-established anticancer drug widely utilized in clinical settings, as a model drug. Then, the beneficial effect of the developed nanoformulations was evaluated in breast cancer cells. Finally, we performed a semiquantitative analysis of both NPs’ uptake and intracellular localization by immunostaining lysosomes, early endosomes, and recycling endosomes. The results show that the smaller NPs (50 nm) were able to reduce the metabolic activity of cancer cells more efficiently than NPs of 70 nm, in a time and concentration-dependent manner. These findings are corroborated by intracellular trafficking studies that reveal an earlier and higher uptake of NPs, with 50 nm compared to the 70 nm ones, by the breast cancer cells. Consequently, this study demonstrates that NP size, even in small increments, has an important impact on their therapeutic effect

Abstract 1693: The combination of romidepsin and bendamustin is synergistically cytotoxic and reverses the malignant phenotype in preclinical models of T-cell lymphoma

Abstract Background: Peripheral T-cell lymphomas (PTCLs) represent approximately 10-15% of all non-Hodgkin lymphomas (NHL) in the Western world, and their incidence is increasing. Cases of PTCL tend to have an aggressive clinical course, with poor patient responses to conventional chemotherapy and poor long-term survival. So far, treatment approaches have mirrored diffuse large B-cell lymphoma (DLBCL) and CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone) and CHOP-like chemotherapy are commonly used despite suboptimal results. Histone deacetylase inhibitors (HDACs) have been approved for the treatment of relapsed or refractory PTCLs given their single-agent activity in these diseases. To continue to improve responses in patients with PTCL, it is important to assess the utility of romidepsin as frontline therapy and as a component of combination therapies. Aim: Interactions between romidepsin (R) and bendamustine (B), a potent cytotoxic alkylating drug active against a panel of other lymphoproliferative disorders, was investigated in in preclinical models of T-cell Lymphoma. Experimental Design: Assays for cytotoxicity on 5 different T-cell lymphoma and leukemia cell lines (Jurkat, HD-MAR2, Karpas, Sup-T1, HH), mathematical analysis for synergism (Chou-Talalay equation), flow cytometry, and the Itk-Syk transgenic mouse model (K. Pechloff, 2010) were used to explore the in vitro and in vivo activities of R and B alone and in combination in T-cell lymphoid malignancies. Results: In vitro, romidepsin and bendamustine exhibited concentration- and time-dependent cytotoxicity against all the 5 different T-cell lymphoma and leukemia cell lines. Romidepsin showed synergism when combined with bendamustine in all cell lines studied. Romidepsin also induced potent apoptosis and caspase activation when combined with bendamustine across the panel. The impact of schedule on the activity of the combination was determined by assessing cell viability after treatment with B and R as follows: (1) simultaneous exposure; (2) B pretreatment followed by exposure to R; and (3) R pretreatment followed by exposure to B. In a new mouse model of PTCL in which a status of permanent T cell activation mediated by the Itk-Syk transcript induces highly malignant PTCLs with 100% penetrance that resemble the human disease, the combination of romidepsin and bendamustine enhanced efficacy compared with either drug alone. Conclusions: Collectively, these data strongly support the potential therapeutic role of romidepsin in combination with bendamustine for PTCLs and might constitute the basis for future phase I-II clinical trials. Citation Format: Cristiana Carniti, Silvia Gimondi, Antonio Vendramin, Sara Rizzitano, Paolo Corradini. The combination of romidepsin and bendamustin is synergistically cytotoxic and reverses the malignant phenotype in preclinical models of T-cell lymphoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1693. doi:10.1158/1538-7445.AM2014-1693</jats:p

Graft Monocytic Myeloid-Derived Suppressor Cell Content Predicts the Risk of Acute Graft-versus-Host Disease after Allogeneic Transplantation of Granulocyte Colony-Stimulating Factor–Mobilized Peripheral Blood Stem Cells

AbstractMyeloid-derived suppressor cells (MDSCs) are powerful immunomodulatory cells that in mice play a role in infectious and inflammatory disorders, including acute graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation. Their relevance in clinical acute GVHD is poorly known. We analyzed whether granulocyte colony-stimulating factor (G-CSF) administration, used to mobilize hematopoietic stem cells, affected the frequency of MDSCs in the peripheral blood stem cell grafts of 60 unrelated donors. In addition, we evaluated whether the MDSC content in the peripheral blood stem cell grafts affected the occurrence of acute GVHD in patients undergoing unrelated donor allogeneic stem cell transplantation. Systemic treatment with G-CSF induces an expansion of myeloid cells displaying the phenotype of monocytic MDSCs (Linlow/negHLA-DR−CD11b+CD33+CD14+) with the ability to suppress alloreactive T cells in vitro, therefore meeting the definition of MDSCs. Monocytic MDSC dose was the only graft parameter to predict acute GVHD. The cumulative incidence of acute GVHD at 180 days after transplantation for recipients receiving monocytic MDSC doses below and above the median was 63% and 22%, respectively (P = .02). The number of monocytic MDSCs infused did not impact the relapse rate or the transplant-related mortality rate (P > .05). Although further prospective studies involving larger sample size are needed to validate the exact monocytic MDSC graft dose that protects from acute GVHD, our results strongly suggest the modulation of G-CSF might be used to affect monocytic MDSCs graft cell doses for prevention of acute GVHD

Synergistic Anti-Tumor Efficacy of BET Inhibitors JQ1 and Otx-015 in Combination with Dasatinib in Preclinical Models of T-Cell Lymphomas

Abstract Background: Approximately 50% of patients with peripheral T-cell lymphoma (PTCL) enter long-term remission after standard chemotherapy and stem cell transplantation. Patients who do not respond to chemotherapy have few treatment options highlighting the critical need for new effective and targeted therapeutics. Aberrant T cell receptor (TCR) and tyrosine kinase (TK) signaling have been described in PTCL (Agostinelli 2014;Netchiporouka 2014). Single-agent TK inhibitors (TKIs) have significantly improved patient outcomes across multiple tumor subtypes. However, TKI therapy is rarely curative. The recent discovery of a subgroup of PTCL characterized by high levels of GATA3 and c-Myc expression and poor prognosis (Iqbal 2014; Manso 2016), establishes the rationale of targeting c-Myc in PTCLs. Based on the demonstration that pharmacologic inhibition of c-Myc is achievable through targeting bromodomain and extra terminal (BET) family of chromatin adapters, the therapeutic potential of BET inhibition was assessed in a panel of T cell lymphoma and leukemia cell lines. Since expression of c-Myc is regulated by the TCR, we also hypothesized that simultaneous targeting of c-Myc and TCR would significantly enhance the antiproliferative effects of BET inhibitors (BETis) and TKI alone in preclinical models of PTCL. Methods: Five T-cell lymphoma and leukemia cell lines (Jurkat, HD-MAR-2, Karpas 299, Sup-T1, HH) were incubated with escalating doses of JQ1 (a small-molecule BETi with the highest affinity for BRD4) and OTX-015 (a BETi with a broader affinity for BRD2, BRD3, BRD4) and the tyrosine-kinase-inhibitor Dasatinib. Analysis of cell viability, cell cycle distribution, apoptosis and mitochondrial depolarization was performed using flow cytometry. Effects of treatments were assessed using gene expression profiling (GEP) and western blotting (WB). Combinations were evaluated using the Chou-Talalay Combination Index (CI), calculated with CompuSyn software (CompuSyn Inc, Paramus, NJ). Results: JQ1 and OTX-015 show antiproliferative activity with IC50 at nanomolar concentrations in all cell lines. As assessed determining viable cells by PI exclusion and flow cytometry, JQ1 and OTX-015 are similarly active in a dose-dependent manner in all cell lines. To understand the activity of JQ1 and OTX-015, we analyzed cell-cycle distribution using flow cytometry. JQ1 and OTX-015 induce a cell cycle arrest with G1-phase accumulation and decrease S-phase with the exception of SUPT1 cells that are characterized by a cell cycle arrest in G2-phase. Minimal increase in the sub-G1 population is observed in all cell lines, suggesting that JQ1 and OTX-015 mainly exert a cytostatic effect. We then examined GATA3 and c-Myc protein levels in all cell lines: varying amounts of GATA3 and c-Myc proteins were observed but a strong correlation between GATA3 and c-Myc expression was detected. After JQ1 and OTX-015 exposure, c-Myc protein level decrease in all cell lines apart from SUP-T1 cell line. Here c-Myc level do not change significantly upon BETis exposure, suggesting that BETis target other pathways relevant for SUP-T1 survival. Dasatinib efficiently inhibits the proliferation in all cell lines at micromolar concentrations in a dose-dependent manner. Dasatinib induces G0/G1-phase arrest and an increase in sub-G1 population indicating a modest induction of apoptosis confirmed by caspase-9 activation and mitochondrial depolarization. Compared to all single agents, combined treatments with sub-optimal concentrations of Dasatinib and JQ1 or OTX-015 exert synergistic lethal activity against all tested cell lines (C.I.&lt;1). To uncover the main biological processes behind the synergistic interactions of BETis and Dasatinib, cell cycle analysis was assessed indicating that both combinations induce a significant increase of sub-G1 population associated with massive mitochondrial depolarization and cleavage of Caspase-9 and PARP. Conclusions: The experiments presented here support the combination of BET inhibitors with the TK inhibitor Dasatinib for PTCLs. Our data suggest a synergistic interaction for the combination of both BETis and Dasatinib in vitro. Mechanistically, combined treatments exert synergistic anti-tumor effects in all cell lines through growth inhibitory effects, direct induction of cell death by promotion of caspase-dependent apoptosis and mitochondrial depolarization. Disclosures No relevant conflicts of interest to declare. </jats:sec