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

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.

Abstract Myeloid-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 (Lin low/neg HLA-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

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)

On the size-dependent internalization of sub-hundred polymeric nanoparticles

The understanding of the interaction between nanoparticles (NPs) and cells is crucial to design nanocarriers with high therapeutic relevance. In this study, we exploited a microfluidics device to synthesize homogeneous suspensions of NPs with ≈ 30, 50, and 70 nm of size. Afterward, we investigated their level and mechanism of internalization when exposed to different types of cells (endothelial cells, macrophages, and fibroblasts). Our results show that all NPs were cytocompatible and internalized by the different cell types. However, NPs uptake was size-dependent, being the maximum uptake efficiency observed for the 30 nm NPs. Moreover, we demonstrate that size can lead to distinct interactions with different cells. For instance, 30 nm NPs were internalized with an increasing trend over time by endothelial cells, while a steady and a decreasing trend were observed when incubated with LPS-stimulated macrophages and fibroblasts, respectively. Finally, the use of different chemical inhibitors (chlorpromazine, cytochalasin-D, and nystatin), and low temperature (4 ◦C) indicated that phagocytosis/micropinocytosis are the main internalization mechanism for all NPs sizes. However, different endocytic pathways were initiated in the presence of particular NP sizes. In endothelial cells, for example, caveolin-mediated endocytosis occurs primarily in the presence of 50 nm NPs, whereas clathrin-mediated endocytosis substantially promotes the internalization of 70 nm NPs. This evidence demonstrates the importance of size in the NPs design for mediating interaction with specific cell types.The authors would like to thank funding that allowed to carry out this work, namely the 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/BTMSAL/28882/2017) and the NORTE 2020 Structured Project, co-funded by Norte2020 (NORTE-01-0145-FEDER-000021)

Macrophage cell membrane infused biomimetic liposomes for glioblastoma targeted therapy

Glioblastoma (GBM) is a highly aggressive malignant brain tumor currently without an effective treatment. Inspired by the recent advances in cell membrane biomimetic nanocarriers and by the key role of macrophages in GBM pathology, we developed macrophage membrane liposomes (MML) for GBM targeting. For the first time, it was assessed the role of macrophage polarization states in the effectiveness of these drug delivery systems. Interestingly, we observed that MML derived from M2 macrophages (M2 MML) presents higher uptake and increased delivery of the anticarcinogenic drug doxorubicin compared to M1 macrophage-derived nanocarriers (M1 MML) and control liposomes (CL). Moreover, the lowest uptake by macrophages of MML reveals promising immune escaping properties. Notably, M2 macrophages unveiled a higher expression of integrin CD49d, a crucial protein involved in the bilateral communication of macrophages with tumor cells. Therefore, our findings suggest the potential of using M2 macrophage membranes to develop novel nanocarriers targeting GBM.This work is supported by the Portuguese Foundation for Science and Technology under the doctoral program in Tissue Engineering, Regenerative Medicine, and Stem Cells (PD/00169/2013), by DM scholarship (PD/BD/143038/2018) and bythe project HEALTH-UNORTE (NORTE-01-0145-FEDER-000039

Decellularized kidney extracellular matrix-based hydrogels for renal tissue engineering

Kidney regeneration is hindered by the limited pool of intrinsic reparative cells. Advanced therapies targeting renal regeneration have the potential to alleviate the clinical and financial burdens associated with kidney disease. Delivery systems for cells, extracellular vesicles, or growth factors aimed at enhancing regeneration can benefit from vehicles enabling targeted delivery and controlled release. Hydrogels, optimized to carry biological cargo while promoting regeneration, have emerged as promising candidates for this purpose. This study aims to develop a hydrogel from decellularized kidney extracellular matrix (DKECM) and explore its biocompatibility as a biomaterial for renal regeneration. The resulting hydrogel crosslinks with temperature and exhibits a high concentration of extracellular matrix. The decellularization process efficiently removes detergent residues, yielding a pathogen-free biomaterial that is non-hemolytic and devoid of α-gal epitope. Upon interaction with macrophages, the hydrogel induces differentiation into both pro-inflammatory and anti-inflammatory phenotypes, suggesting an adequate balance to promote biomaterial functionality in vivo. Renal progenitor cells encapsulated in the DKECM hydrogel demonstrate higher viability and proliferation than in commercial collagen-I hydrogels, while also expressing tubular cells and podocyte markers in long-term culture. Overall, the injectable biomaterial derived from porcine DKECM is anticipated to elicit minimal host reaction while fostering progenitor cell bioactivity, offering a potential avenue for enhancing renal regeneration in clinical settings

Microfluidic mixing system for precise PLGA-PEG nanoparticles size control

In this study, a microfluidic device was employed to produce polymeric nanoparticles (NPs) with well-controlled sizes. The influence of several parameters in the synthesis process, namely, polymer concentration, flow rate and flow rate ratio between the aqueous and organic solutions was investigated. To evaluate the NPs size effect, three diameters were selected (30, 50 and 70 nm). Their cytocompatibility was demonstrated on endothelial cells and macrophages. Additionally, their efficacy to act as drug carriers was assessed in an in vitro inflammatory scenario. NPs loaded and released diclofenac (DCF) in a size-dependent profile (smaller sizes presented lower DCF content and higher release rate). Moreover, 30 nm NPs were the most effective in reducing prostaglandin E2 concentration. Therefore, this study demonstrates that microfluidics can generate stable NPs with controlled sizes, high monodispersity and enhanced batch-to-batch reproducibility. Indeed, NPs size is a crucial parameter for drug encapsulation, release and overall biological efficacy.FCT -Fundação para a Ciência e a Tecnologia(NORTE-01-0145-FEDER-000021