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In vitro human‐relevant glioblastoma models as the novel frontier of nanomedicine screening
No full textThe highly heterogeneous tumor microenvironment (TME), the stiff extracellular matrix (ECM) and the Blood Brain Barrier (BBB) hinder treatment efficacy against Glioblastoma (GBM). Hence, the preclinical evaluation of novel drug delivery platforms is key in GBM management. Currently, drug screening relies on animal studies or in vitro models, which do not fully replicate GBM complexity. To fill this gap, this study aims to develop a human-relevant GBM model to investigate the efficacy of nanoparticles (NPs)-based drug delivery systems.
Multicellular tumor spheroids (MTS) were prepared by mixing different cell types at varying ratios (e.g., tumor cells, microglia, and GBM-Stem Cells) to model GBM composition and embedded in polymeric hydrogels resembling the main properties of GBM ECM. MTS infiltration capacity and viability were assessed on the model following treatment with polyurethane NPs for the controlled release of Bortezomib (BTZ), a proteasome inhibitor. The results confirm that BTZ can reduce tumor proliferation and infiltration in ECM-like gels, with the effect depending on the cellular composition.
To verify NPs extravasation across brain capillaries, a vascular network was inducted into the MTS through a commercial microfluidic platform, using brain capillary endothelial cells. Immunostaining and perfusion assays were performed to analyze microvessels properties. CD31-staining showed the homogeneous presence of endothelial cells forming tight junctions (confirmed by ZO-1 staining). Fluorescent NPs injected in the channels were retained without extravasation, confirming previous in vivo observations.
This model represents a prototype for a 3R-compliant replica of GBM microenvironment, combining key cell actors, biomimetic materials, and an in vitro brain microvasculature. The promising results suggest the possibility to increase model complexity, e.g., by including pericytes and astrocytes, to provide a reliable tool for nanomedicine screening
Modelling glioblastoma microenvironment for in vitro assessment of cell-based drug delivery
No full textIntroduction: Glioblastoma (GBM) is the most hard-to-treat brain tumor due to the
heterogeneity of its microenvironment (TME) and to the presence of the Blood Brain Barrier
(BBB). Hence, more efficient drug delivery strategies are needed to improve the outcome of GBM
patients. For instance, cell-mediated transport of nanoparticles (NPs) has the potential to
improve GBM treatment, by exploiting the tumor homing properties of living cells (e.g., microglia).
This study aims to design a reliable in vitro GBM model as an alternative tool for the
investigation of cell-mediated drug delivery.
Methods: A three-dimensional GBM model was realized by encapsulating multicellular spheroids
(MS) in different commercial gels. MSs comprised GBM cells and GBM-associated Stem Cells to
replicate tumor histology, while the gels reproduced the mechanical properties of GBM
extracellular matrix (ECM). MS vascularization was induced through a commercial microfluidic
platform, using brain endothelial cells. Network formation was confirmed by
immunostaining and perfusion assay. Microglia (μG) were incubated with different concentration
of Bortezomib-loaded polyurethane NPs (0.5, 1, 2 mg/mL) for increasing time (2, 24 h). Loading
of NPs in μG was optimized by determining cell viability (CellTiter MTS) and internalization by
flow cytometry. Treatment efficacy after NPs and μG (incubated for 2 h with 1 mg/mL NPs)
administration was assess through viability assay (CellTiter-Glo) on MS. The infiltration in ECM-
like gels and extravasation across vessels were tested on the model.
Results: MS were effectively encapsulated in gels, which preserved integrity and facilitated
invasion. The microfluidic platform successfully induced vascularization, as confirmed by
immunostaining for tight junction proteins (ZO-1). NPs internalization in μG increased with
concentration and over time, with NPs concentrations above 1 mg/mL resulting in more than 90%
internalizatio without significant cytotoxicity (90% viability). BTZ-NPs-μG were
able to infiltrate in different gels, reaching the embedded MS, and successfully reducing MS
proliferation and invasion. The microvasculature was able to replicate the barrier effect against
NPs, while microglia cells penetrated across the vessel towards the MS.
Conclusions: This model represents a prototype for a reliable replica of the heterogeneity of GBM
microenvironment, which combines cells, biomaterials, and microfluidics. The model provided
valuable confirmation of the potential of microglia-mediated drug delivery as a promising
strategy for GBM homing. Future studies should introduce further BBB elements into the model
and compare the system with in vivo observations
Hydrogel-based tumour microenvironments as models of vascularized Glioblastoma for validation of nanomedicines
No full textThe tumour microenvironment (TME) is the main obstacle limiting the efficacy of treatments against
aggressive diseases such as glioblastoma (GBM). The high histological complexity, the infiltration by
tumour-supporting cells, and the presence of biological barriers such as the blood-brain barrier (BBB)
and the rigid extracellular matrix (ECM) hinder the accumulation of molecules and transporters. Reliable
in vitro systems that recapitulate the complexity of GBM TME are needed to support the design of
innovative drugs and carriers able to overcome these barriers.
This work aims to develop a reliable three-dimensional GBM model to investigate the transport of
polymer nanoparticles (NPs)-based drug platforms. The model combines different cell actors involved in
human GBM, ECM-like biomaterials, and a microfluidic device to reproduce vascularization, to reliably
mimics GBM structure and composition.
GBM spheroids were prepared in low adhesion conditions using a combination of GBM cells and GBMassociated Stem Cells, coupled with other cells of the TME, such as microglia and astrocytes, to replicate tumour histology. The spheroids were encapsulated in natural (collagen-based) or synthetic
(polysaccharide-based) polymer gels with mechanical properties resembling the GBM ECM.
A vascular network was obtained by inserting the GBM spheroids in a commercial microfluidic platform,
containing two lateral perfusion channels coated with human brain endothelial cells, from which
angiogenic sprouting was induced to vascularize the spheroid. Immunostaining confirmed the
homogeneous presence of endothelial cells forming tight junctions. Moreover, the
microvasculature was able to replicate the barrier effect against NPs, resembling our in vivo
observations. The model was used to verify the infiltration capacity and viability following treatment
with polyurethane NPS loaded with a proteasome inhibitor (Bortezomib, BTZ). The results confirm that
the drug can reduce tumour proliferation and infiltration in ECM-like gels, with the effect depending on
the cellular composition. NPs-mediated treatment had lower efficacy than the free drug and was able to
reduce cytotoxicity on non-tumour cells of TME.
This model represents a promising step in developing a reliable replica of human GBM TME by
combining biomaterials and microfluidics. The device could be a valuable tool for the preliminary
validation of drugs, nanomedicines, and alternative transporters (e.g., cell-mediated drug delivery)
Development of green nanoparticles through solvent-free techniques and of alternative in vitro models for their validation
No full textIntroduction:
Recently, nanotechnology has been exploited for the development of drug-delivery systems. Nanoparticle (NP) brought many advantages in cancer therapy, such as targeting, reduction of side, and improved biodistribution.
Traditionally, NPs were synthesized using environmental unsustainable methods, which frequently employed organic solvents.
The aim of this work is the preparation of solvent-free NPs for proteins and RNA delivery to melanoma, which will be tested on a 3D-printed model of metastatic melanoma model (under development).
Methods:
Two solvent-free techniques were investigated to synthetize green NPs: i) Antibody-loaded Chitosan (CS) NPs obtained by ionic gelation and ii) siRNA-loaded phosphate-poly(allylamine-hydrochloride) (PAH) NPs, obtained through electrostatic self-assembly.
NPs were characterized in terms of size (DLS), loading efficiency, cell compatibility, and platelet activation.
Different compositions of a collagen-hyaluronic acid (C/HA) bioink were tested to find the optimal ratio in terms of printability, shape fidelity, and cell viability.
Human fibroblast and human melanoma cells were embedded in the C/HA bioink to obtain a cellularized architecture. Cell viability in the gel (cell titer-blue) was assessed after 1, 4, and 7 days.
Results:
NPs were successfully obtained with both methods, achieving a size of 200 nm for CS NPs and 90 nm for PAH NPS.
SEM and FACS analysis showed that PAH NPs did not trigger platelet activation, at any of the tested concentrations, while CS NPs did not induce activation at concentrations below 200 μg/mL.
Nearly 100% of the siRNA was successfully complexed into PAH NPs, while CS NPs encapsulated nearly 40% of the loaded Ab.
FACS analysis and confocal microscopy showed that NPs were able to significantly enhance siRNA and protein accumulation in cells.
Printability tests revealed that the optimal C/HA ratio is 50:50 and viability tests and confocal microscopy analysis confirmed the hydrogel biocompatibility with the tested cell line.
Conclusions:
CS and PAH NPs of small size and good encapsulation efficiency were successfully obtained without organic solvents. NPs exhibited good cell compatibility and extremely low platelet activation.
3D bioprinted C/HA matrix was realized allowing the cells to grow in a physiological-like environment.
The model will be further characterized and improved by including blood vessels, followed by investigation of NPs ability to penetrate within the tumor and to target metastatic cells.
Acknowledgements:
Carlotta Mattioda acknowledges PON "Ricerca e Innovazione" 2014-2020 Azione IV.R "dottorati su tematiche green" for co-financing her Ph.D scholarship
Development of green approaches based on solvent-free nanoparticles and in vitro models for the management of metastatic melanoma
No full textNanoparticles (NPs) brought many advantages in cancer therapy, but their synthesis process results still
environmentally unsustainable, due to the amount of organic solvent involved.
The aim of this project is the development of green NPs synthesis technique to deliver hydrophobic drugs.
Two green NPs platforms were prepared i) Antibody-loaded chitosan (CS) NPs obtained by ionic gelation and ii) siRNA-loaded phosphate-poly(allylamine-hydrochloride) (PAH) NPs, obtained through electrostatic self-assembly.
Small size NPs with low polydispersity index, and positive Z potential were obtained. No sign of cytotoxicity caused by NPs was observed against melanoma and fibroblasts cell lines. NPs-induced platelet activation was tested to investigate NPs safety after sistemic injection. Platelet activation was evaluated through SEM microscopy and by FACS analysis. PAH NPs did not trigger platelet activation, at any of the tested concentrations, while CS NPs did not induce activation at low concentrations.
NPs showed capacity to load model payloads and to release it in a controlled fashion. FACS analysis and
confocal microscopy showed that PAH NPs were able to significantly enhance siRNA delivery to cells, as
compared to free siRNA administration.
According to 3R principles, a 3D-printed metastatic melanoma model is under development as a NPs testing device, representing an alternative to animal tests.
Skin fibroblasts (Hff-1) were embedded in a collagen/hyaluronic acid-based hydrogel, and allowed to grow
up to four weeks. To recreate the vasculature, a channel was obtained within the hydrogel and seeded with endothelial cells (hUVECs). The model will be inoculated with melanoma cells and used to investigate NPs extravasation towards the primary tumor and their ability to target metastatic melanoma cells present in the channel.
Carlotta Mattioda acknowledges PON "Ricerca e Innovazione" 2014-2020 Azione IV.R "dottorati su tematiche green" for co-financing her Ph.D scholarship
Chitosan Nanoparticles as Therapeutic Protein Nanocarriers: the Effect of pH on Particle Formation and Encapsulation Efficiency
No full textIn the past, tripolyphosphate (TPP)-crosslinked chitosan (CS) nanoparticles (CSNPs) have been widely applied for the delivery of biomacromolecules, because of their mild preparation conditions. However, poor uniformity and burst release limit their application to some extent. In this study, we investigated the effect of pH on the formation and on protein encapsulation efficiency (EE) of CSNPs. Results revealed that smaller particles are formed at lower pH and that the size distribution is conjointly influenced by pH and CS/TPP mass ratio. EE of bovine serum albumin (BSA) increased significantly with pH value. The influence of the pH of the crosslinker (TPP) solution was also studied, showing that CSNPs prepared with basic TPP solution (pH 9.5) had larger size, higher yield, and BSA EE compared with those obtained with acidic TPP solution (pH 5.5). Characterization by Fourier transform infrared-attenuated total Reflectance (FTIR-ATR) spectroscopy and SEM as well as the in vitro BSA release analysis revealed that the pH of the TPP solution might influence CSNPs' properties, by changing the conformation of polymer chains. This study analyzes the formation of CSNPs and protein encapsulation mechanisms at different pH values of both the polymer and the crosslinker solutions, suggesting strategies to overcome some of the major drawbacks of CSNPs as protein nanocarriers for therapeutic applications
Green approaches based on solvent-free methods to prepare nanoparticles and on alternative in vitro models for their validation: application in the treatment of metastatic melanoma
Full text linkNanoparticles (NPs) are advantageous drug-delivery systems due to their ability to maximize drug efficacy and minimize side effects. However, NPs preparation techniques pose environmental issue, as they require large quantities of organic solvents.
This contribution proposes the use of green methods for the preparation of NPs to deliver protein drugs or therapeutic RNAs. Two green NPs platforms were prepared i) Antibody-loaded Chitosan (CS) NPs obtained by ionic gelation and ii) siRNA-loaded phosphate-poly(allylamine-hydrochloride) (PAH) NPs, obtained through electrostatic self-assembly.
NPs of small size (~200 nm), low polydispersity index, and positive Z potential were obtained. NPs toxicity was investigated against melanoma and fibroblasts cell lines, observing no significant reduction in viability, up to 1 mg/mL. Platelet activation after exposure to NPs was tested as a preliminary assessment of their safety after intravenous injection. NPs were incubated with platelets for 30 min, followed by count of platelet adhesion and SEM analysis on adherent platelets, for a qualitative assessment, and by FACS, for a quantitative measure. PAH NPs did not trigger platelet activation, at any of the tested concentrations, while CS NPs did not induce activation at concentrations below 200 μg/mL.
NPs showed capacity to load model payloads (secondary antibody for CS NPs and mock siRNA or BRAF- silencing siRNA for PAH NPs) and to release it in a controlled fashion. FACS analysis and confocal microscopy showed that PAH NPs were able to significantly enhance siRNA delivery to cells, as compared to free siRNA administration.
Cisplatin-loaded CS and BRAF-siRNA loaded PAH NPs were tested against human melanoma spheroids. Since the tumor response to treatment strongly depends on its interactions with the tumor microenvironment (TME), a 3D-printed model of metastatic melanoma is under development as a further NPs testing device, representing an alternative to animal tests. To obtain the model, skin fibroblasts (Hff-1) were embedded in a collagen/hyaluronic acid-based hydrogel, and allowed to grow for one week. To recreate the vasculature, a channel was obtained within the hydrogel and seeded with endothelial cells (hUVECs). The model will be inoculated with melanoma cells and used to investigate NPs extravasation towards the primary tumor and their ability to target metastatic melanoma cells present in the channel.
Carlotta Mattioda acknowledges PON "Ricerca e Innovazione" 2014-2020 Azione IV.R "dottorati su tematiche green" for co-financing her Ph.D scholarship
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