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International audience; Fibroblastic reticular cells (FRCs), the T-cell zone stromal cell subtype in the lymph nodes, create a scaffold for adhesion and migration of immune cells, thus allowing them to communicate. Although known to be important for the initiation of immune responses, studies about FRCs and their interactions have been impeded because FRCs are limited in availability and lose their function upon culture expansion. To circumvent these limitations, stromal cell precursors can be mechanotranduced to form mature FRCs. Here, we used a library of designed surface topographies to trigger FRC differentiation from tonsil-derived stromal cells (TSCs). Undifferentiated TSCs were seeded on a TopoChip containing 2176 different topographies in culture medium without differentiation factors, then monitored cell morphology and the levels of ICAM-1, a marker of FRC differentiation. We identified 112 and 72 surfaces that upregulated and downregulated, respectively, ICAM-1 expression. By monitoring cell morphology, and expression of the FRC differentiation marker ICAM-1 via image analysis and machine learning, we discovered correlations between ICAM-1 expression, cell shape and design of surface topographies and confirmed our findings by using flow cytometry. Our findings confirmed that TSCs are mechano-responsive cells and identified particular topographies that can be used to improve FRC differentiation protocols.

Fibrotic tumors, such as pancreatic ductal adenocarcinoma (PDAC), are characterized for high desmoplastic reaction, which results in high intra-tumoral solid stress leading to the compression of blood vessels. These microarchitectural alterations cause loss of blood flow and poor intra-tumoral delivery of therapeutics. Currently, there is a lack of relevant in vitro models capable of replicating these mechanical characteristics and to test anti-desmoplastic compounds. Here, a multi-layered vascularized 3D PDAC model consisting of primary human pancreatic stellate cells (PSCs) embedded in collagen/fibrinogen (Col/Fib), mimicking tumor tissue within adjunct healthy tissue, is presented to study the fibrosis-induced compression of vasculature in PDAC. It is demonstrated how the mechanical and biological stimulation induce PSC activation, extracellular matrix production and eventually vessel compression. The clinical relevance is confirmed by correlating with patient transcriptomic data. Furthermore, the effects of gradual vessel compression on the fluid dynamics occurring within the channel is evaluated in silico. Finally, it is demonstrated how cancer-associated fibroblast (CAF)-modulatory therapeutics can inhibit the cell-mediated compression of blood vessels in PDAC in vitro, in silico and in vivo. It is envisioned that this 3D model is used to improve the understanding of mechanical characteristics in tumors and for evaluating novel anti-desmoplastic therapeutics.

Since tumor stroma poses as a barrier to achieve efficacy of nanomedicines, it is essential to evaluate nano-chemotherapeutics in stroma-mimicking 3D models that reliably predict their behavior regarding these hurdles limiting efficacy. In this study, we evaluated the effect of paclitaxel-loaded polymeric micelles (PTX-PMCs) and polymeric nanoparticles (PTX-PNPs) in a tumor stroma–mimicking 3D in vitro model. PTX-PMCs (77 nm) based on a amphiphilic block copolymer of mPEG-b-p(HPMAm-Bz) and PTX-PNPs (159 nm) based on poly(lactic-co-glycolic acid) were prepared, which had an encapsulation efficiency (EE%) of 81 ± 15% and 45 ± 8%, respectively. 3D homospheroids of mouse 4T1 breast cancer cells and heterospheroids of NIH3T3 fibroblasts and 4T1 (5:1 ratio) were prepared and characterized with high content two-photon microscopy and immunostaining. Data showed an induction of epithelial-mesenchymal transition (α-SMA) in both homo- and heterospheroids, while ECM (collagen) deposition only in heterospheroids. Two-photon imaging revealed that both fluorescently labeled PMCs and PNPs penetrated into the core of homospheroids and only PMCs penetrated into heterospheroids. Furthermore, PTX-PMCs, PTX-PNPs, and free PTX induced cytotoxicity in tumor cells and fibroblasts grown as monolayer, but these effects were substantially reduced in 3D models, in particular in heterospheroids. Gene expression analysis showed that heterospheroids had a significant increase of drug resistance markers (Bcl2, Abgc2) compared to 2D or 3D monocultures. Altogether, this study shows that the efficacy of nanotherapeutics is challenged by stroma-induced poor penetration and development of resistant phenotype. Therefore, this tumor stroma–mimicking 3D model can provide an excellent platform to study penetration and effects of nanotherapeutics before in vivo studies. Graphical Abstract

Intracellular delivery of nanoparticles is crucial in nanomedicine to reach optimal delivery of therapeutics and imaging agents. Single-chain polymer nanoparticles (SCNPs) are an interesting class of nanoparticles due to their unique site range of 5-20 nm. The intracellular delivery of SCNPs can be enhanced by using delivery agents. Here, a positive polymer is used to form polyplexes with SCNPs, similar to the strategy of protein and gene delivery. The size and surface charge of the polyplexes were evaluated. The cellular uptake showed rapid uptake of SCNPs via polyplex formation, and the cytosolic delivery of the SCNPs was presented by confocal microscopy. The ability of SCNPs to act as nanocarriers was further explored by conjugation of doxorubicin.

An increasing number of studies have revealed that the tumor microenvironment conveys signals to tumor cells to acquire resistance against chemotherapy [1-5]. Development of resistance is an intrinsic feature and is induced via multiple mechanisms such as overexpression of membrane drug transporters, altered DNA repair mechanisms and inhibition of pro-apoptotic mechanisms [6-9]. Furthermore, many solid tumors undergo desmoplasia i.e., formation of fibrotic stromal tissue composed of cancer-associated fibroblasts (CAFs) and extracellular matrix (ECM) constituents. ECM constituents are fibrous proteins such as collagen, elastin, and reticulin; link proteins such as fibronectin and laminin; as well as space filling molecules (proteoglycans, glycosaminoglycans). CAFs and ECMs together form a dense network that acts as a barrier for penetration of chemotherapeutic drugs and drug-loaded nanoparticles, thereby negatively affecting therapeutic efficacy [1]. In this thesis, we studied strategies to overcome the above-mentioned barriers that hinder the efficacy of chemotherapeutics either in free form or after encapsulation in nanoparticles. Two crucial barriers studied in this thesis are the following: (i) activation of an anti-apoptotic mechanism in cancer cells; (ii) formation of stroma hindering nanomedicine penetration into tumors. To overcome the resistance towards chemotherapeutics, the anti-apoptosis pathway was inhibited with a peptide delivered intracellularly using nanoparticles and combined with chemotherapeutic agents. Furthermore, a stroma-rich 3D spheroid model was developed and characterized and used to study the penetration and anti-tumor effects of drug-loaded nanoparticles.

Cancer-associated fibroblasts (CAFs) are important drivers in the tumor microenvironment and facilitate the growth and survival of tumor cells, as well as metastasis formation. They may travel together with tumor cells to support their survival and aid in the formation of a metastatic niche. In this study, we aimed to study circulating CAFs (cCAFs) and circulating tumor cells (CTCs) in a preclinical breast tumor model in mice in order to understand the effect of chemotherapy on cCAFs and CTC formation. Tumors with MDA-MB-231 human breast tumor cells with/without primary human mammary fibroblasts (representing CAFs) were coinjected in SCID mice to develop tumors. We found that the tumors with CAFs grew faster than tumors without CAFs. To study the effect of the stroma on CTCs and cCAFs, we isolated cells using microsieve filtration technology and established ITGA5 as a new cCAF biomarker, which showed good agreement with the CAF markers FAP and α-SMA. We found that ITGA5+ cCAFs shed in the blood of mice bearing stroma-rich coinjection-based tumors, while there was no difference in CTC formation. Although treatment with liposomal doxorubicin reduced tumor growth, it increased the numbers of both cCAFs and CTCs in blood. Moreover, cCAFs and CTCs were found to form clusters in the chemotherapy-treated mice. Altogether, these findings indicate that the tumor stroma supports tumor growth and the formation of cCAFs. Furthermore, chemotherapy may exacerbate the formation of cCAFs and CTCs, which may eventually support the formation of a metastasis niche in breast cancer.

Nanomedicines promise to deliver useful and valuable research tools and clinical applications. The connection of various nanomedicines to hypersensitivity reactions upon IV administration, called complement activation-related pseudoallergy (CARPA), will be introduced in these studies. Intravenous injection of a variety of nanomedicinal (liposomal, micellar, polymer-conjugated) and protein-based (antibodies, enzymes) drugs can lead to hypersensitivity reactions, also known as infusion or anaphylactoid reactions. The molecular mechanism and severity of these allergy symptoms may alter from case to case and in many cases the major cause, or contributing factor is activation of the complement system. The clinical relevance of CARPA, a non-IgE-mediated allergy syndrome, lies in its unpredictability and occasionally lethal outcome. The research is planned to be centered around PEGylated liposomes and SPIONs (superparamagnetic iron oxide nanoparticles) as successful examples of marketed nanomaterials. These studies will mainly include in vivo pig models, one if not the best animal model for complement related pseudoallergy studies. Several parameters can be monitored during such in vivo experiments, including hemodynamic changes (heart rate, systemic arterial pressure, pulmonary arterial pressure, pCO2, SpO2), blood cell count analysis and thromboxane (TxB2) changes measured with ELISA. Indeed, the role of possible biomarkers as the previously mentioned TxB2, and the promising complement factor H (FH) are planned to be assessed in reviews. Another agent used in therapeutics that can induce C related HSRs similar to nanomedicines are monoclonal antibodies (mABs) with or without PEGylation. In conclusion, these planned experiments and reviews with diverse topics all aim to analyse the mechanics and background of complement related hypersensitivity reactions of polymer coated nanomedicines and biological molecules, trying to find possible preventive options or at least ways to reduce the level of these adverse events.

Bacteriophages are potential alternatives to conventional antibiotics in the treatment of multidrug resistant infections. However, optimal treatment administration protocols are not established. The direct application of bacteriophages in local biomaterials-based carriers offers solutions to many of the limitations of current modalities. In this study, thermo-responsive hyaluronic acid-poly(N-isopropylacrylamide) (HA-pNIPAM) hydrogels and alginate-chitosan microbeads have been developed as bioresorbable local bacteriophage delivery systems designed to enable rapid and delayed phage release, respectively. The long-tailed Staphylococcus aureus phage ISP and the short-tailed Pseudomonas aeruginosa phage LUZ19 were selected as structurally diverse phages to load within both biomaterials. HA-pNIPAM hydrogels were synthesized and their thermo-responsive crosslinking was confirmed after rheological assessment. Alginate microbeads where prepared by ionic-gelation and chitosan surface deposition was confirmed by amine sensitive assays. After embedding phages, their antimicrobial potential was retained when stored at 4 °C. Release of both phages from HA-pNIPAM hydrogels was gradual and consistent over 21 days, whereby, continuous release of the short-tailed LUZ19 was observed for 21 days from the microbeads, while release of the long-tailed ISP portrayed burst-like release during early timepoints, with gradually reducing phage release thereafter. These findings suggest that the release kinetics of phage from biomaterials are impacted by both the biomaterial and the phages themselves. These results highlight the potential of HA-pNIPAM hydrogel and alginate-chitosan microbeads for local delivery of ISP and LUZ19 bacteriophages for both rapid burst release and controlled, extended release.

Alternatively-activated, M2-like tumor-associated macrophages (TAM) strongly contribute to tumor growth, invasiveness and metastasis. Technologies to disable the pro-tumorigenic function of these TAMs are of high interest to immunotherapy research. Here we show that by designing engineered nanoliposomes bio-mimicking peroxidated phospholipids that are recognised and internalised by scavenger receptors, TAMs can be targeted. Incorporation of phospholipids possessing a terminal carboxylate group at the sn-2 position into nanoliposome bilayers drives their uptake by M2 macrophages with high specificity. Molecular dynamics simulation of the lipid bilayer predicts flipping of the sn-2 tail towards the aqueous phase, while molecular docking data indicates interaction of the tail with Scavenger Receptor Class B type 1 (SR-B1). In vivo, the engineered nanoliposomes are distributed specifically to M2-like macrophages and, upon delivery of the STAT6 inhibitor (AS1517499), zoledronic acid or muramyl tripeptide, these cells promote reduction of the premetastatic niche and/or tumor growth. Altogether, we demonstrate the efficiency and versatility of our engineered “tail-flipping” nanoliposomes in a pre-clinical model, which paves the way to their development as cancer immunotherapeutics in humans.

Endotoxins are commonly present in both naturally derived and synthetic biomaterials. This Forum highlights the consequences of endotoxin contamination in biomaterials on different engineered tissues, such as bone, skin, or cartilage, and discusses the potential effects on novel 3D immunocompetent pathophysiological models.

Layer-by-layer (LbL) assembly is the process of building functional multilayered thin films. Owing to its highly modular and versatile nature, it has been used to coat a wide variety of different surfaces, including inorganic substrates, membranes, implants, nanoparticles and even living cells. It provides ways to induce responsiveness through both the chemically engineered macromolecular components, and the way the multilayers are built up. For example, assembly degradability can be adjusted by using degradable polymers or crosslinkers, while physical properties can be altered through the use of additives, or by the assembly method. This chapter is dedicated to LbL fabrication-specific responsiveness, and to recent developments in multilayers composed of specifically tailored polymers. It further focuses on chemically and biologically responsive LbL systems, with main applications in the biomedical field. The introduction covers general aspects of LbL assembly and physicochemical aspects of the assemblies. The second part describes physicochemical aspects in more detail with examples on how variation in deposition conditions, e.g. pH and ionic strength, as well as specific additives, induce responsiveness to the resulting multilayers. It also highlights several reports on compartmentalized multilayered coating fabrication for tunable disassembly or release of incorporated materials. The third part describes multilayers fabricated with chemically tailored biomaterials for different chemical and biological responsiveness. More specifically, multilayer disassembly can be triggered by the inherent responsiveness of one of the multilayer components, through incorporation of labile bonds that respond to specific external triggers, or through disruption of the interlayer interaction between two or more multilayer components.

Micro-extrusion-based 3D printing of complex geometrical and porous calcium phosphate (CaP) can improve treatment of bone defects through the production of personalized bone substitutes. However, achieving printing and post-printing shape stabilities for the efficient fabrication and application of rapid hardening protocol are still challenging. In this work, the coaxial printing of a self-setting CaP cement with water and ethanol mixtures aiming to increase the ink yield stress upon extrusion and the stability of fabricated structures was explored. Printing height of overhang structure was doubled when aqueous solvents were used and a 2 log increase of the stiffness was achieved post-printing. A standard and fast steam sterilization protocol applied as hardening step on the coaxial printed CaP cement (CPC) ink resulted in constructs with 4 to 5 times higher compressive moduli in comparison to extrusion process in the absence of solvent. This improved mechanical performance is likely due to rapid CPC setting, preventing cracks formation during hardening process. Thus, coaxial micro-extrusion-based 3D printing of a CPC ink with aqueous solvent enhances printability and allows the use of the widespread steam sterilization cycle as a standalone post-processing technique for production of 3D printed personalized CaP bone substitutes. Statement of significance Coaxial micro-extrusion-based 3D printing of a self-setting CaP cement with water:ethanol mixtures increased the ink yield stress upon extrusion and the stability of fabricated structures. Printing height of overhang structure was doubled when aqueous solvents were used, and a 2 orders of magnitude log increase of the stiffness was achieved post-printing. A fast hardening step consisting of a standard steam sterilization was applied. Four to 5 times higher compressive moduli was obtained for hardened coaxially printed constructs. This improved mechanical performance is likely due to rapid CPC setting in the coaxial printing, preventing cracks formation during hardening process.

Clinical kidney journal : CKJ 14(6), 1514-1523 (2021). doi:10.1093/ckj/sfab011, Published by Oxford Univ. Press, Oxford

Fibrosis is a pathological outcome of most chronic inflammatory diseases and characterized with the accumulation of abundant extracellular matrix component produced by myofibroblasts. These prominent cells are found both in fibrosis and fibrotic tumor such as pancreatic cancer. In normal condition, fibroblasts maintain structural integrity of healthy tissue. However, in fibrotic diseases and in cancers, fibroblasts play a vital role in the development of the diseases. During tumor progression, malignant cells recruit surrounding cells including fibroblasts and together create tumor microenvironment (TME). The TME presents a complex network that contribute in the tumor progression, invasion, metastasis, and resistance. With the increasing understanding of the TME and its key players, novel therapeutic strategies have been investigated with focus on the modulation of the TME. This thesis describes the development of specific targeting to achieve better outcome of the treatment. In detail, the first chapter of this thesis describes fibrosis in organs and cancers and their current treatment strategies. In the context of cancer, we specifically targeted human pancreatic stellate cells (hPSCs), the key cells in the pancreatic cancer progression. Relaxin (RLX), a biological that is able to reverse fibroblast activation was investigated in pancreatic cancer (chapter 2). In addition, RLX was also examined in a different fibrotic disease, liver cirrhosis (chapter 3). Another biomolecule, fibroblast growth factor 2 (FGF2) was also investigated in a 3D heterospheroid models of pancreatic cancer in vitro (chapter 4). Next to endogenous biologicals, novel peptides were designed to target integrin α5 (ITGA5), a potential therapeutic target in pancreatic cancer. The properties of peptides, including secondary structures, binding affinity, stability in plasma, and their inhibitory effect against ITGA5 were elucidated (chapter 5). The most promising peptide (cyAV3.3) were then further trailed in more relevant models (chapter 6). In vitro data collectively indicate that cyAV3.3 is able to induce the therapeutic effects of gemcitabine by inhibiting hPSCs. Furthermore, cyAV3.3 reduced the tumor growth in co-injection tumor models in vivo in mice. There is an unmet clinical need for novel therapeutic strategies to treat pancreatic cancer since the common treatments such as radiotherapies and chemotherapies are failing to cure patients. This thesis explores several strategies to actively target and modulate the stroma in pancreatic cancer and shows the benefits of stroma targeting.

Despite promising progress in cancer vaccination, therapeutic effectiveness is often insufficient. Cancer vaccine effectiveness could be enhanced by targeting vaccine antigens to antigen-presenting cells, thereby increasing T-cell activation. CD169-expressing splenic macrophages efficiently capture particulate antigens from the blood and transfer these antigens to dendritic cells for the activation of CD8+ T cells. In this study, we incorporated a physiological ligand for CD169, the ganglioside GM3, into liposomes to enhance liposome uptake by CD169+ macrophages. We assessed how variation in the amount of GM3, surface-attached PEG and liposomal size affected the binding to, and uptake by, CD169+ macrophages in vitro and in vivo. As a proof of concept, we prepared GM3-targeted liposomes containing a long synthetic ovalbumin peptide and tested the capacity of these liposomes to induce CD8+ and CD4+ T-cell responses compared to control liposomes or soluble peptide. The data indicate that the delivery of liposomes to splenic CD169+ macrophages can be optimized by the selection of liposomal constituents and liposomal size. Moreover, optimized GM3-mediated liposomal targeting to CD169+ macrophages induces potent immune responses and therefore presents as an interesting delivery strategy for cancer vaccination.

Background Accurate and up-to-date assessment of demographic metrics is crucial for understanding a wide range of social, economic, and public health issues that affect populations worldwide. The Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) 2019 produced updated and comprehensive demographic assessments of the key indicators of fertility, mortality, migration, and population for 204 countries and territories and selected subnational locations from 1950 to 2019. Methods 8078 country-years of vital registration and sample registration data, 938 surveys, 349 censuses, and 238 other sources were identified and used to estimate age-specific fertility. Spatiotemporal Gaussian process regression (ST-GPR) was used to generate age-specific fertility rates for 5-year age groups between ages 15 and 49 years. With extensions to age groups 10-14 and 50-54 years, the total fertility rate (TFR) was then aggregated using the estimated age-specific fertility between ages 10 and 54 years. 7417 sources were used for under-5 mortality estimation and 7355 for adult mortality. ST-GPR was used to synthesise data sources after correction for known biases. Adult mortality was measured as the probability of death between ages 15 and 60 years based on vital registration, sample registration, and sibling histories, and was also estimated using ST-GPR. HIV-free life tables were then estimated using estimates of under-5 and adult mortality rates using a relational model life table system created for GBD, which closely tracks observed age-specific mortality rates from complete vital registration when available. Independent estimates of HIV-specific mortality generated by an epidemiological analysis of HIV prevalence surveys and antenatal clinic serosurveillance and other sources were incorporated into the estimates in countries with large epidemics. Annual and single-year age estimates of net migration and population for each country and territory were generated using a Bayesian hierarchical cohort component model that analysed estimated age-specific fertility and mortality rates along with 1250 censuses and 747 population registry years. We classified location-years into seven categories on the basis of the natural rate of increase in population (calculated by subtracting the crude death rate from the crude birth rate) and the net migration rate. We computed healthy life expectancy (HALE) using years lived with disability (YLDs) per capita, life tables, and standard demographic methods. Uncertainty was propagated throughout the demographic estimation process, including fertility, mortality, and population, with 1000 draw-level estimates produced for each metric. Findings The global TFR decreased from 2.72 (95% uncertainty interval [UI] 2.66-2.79) in 2000 to 2.31 (2.17-2.46) in 2019. Global annual livebirths increased from 134.5 million (131.5-137.8) in 2000 to a peak of 139.6 million (133.0-146.9) in 2016. Global livebirths then declined to 135.3 million (127.2-144.1) in 2019. Of the 204 countries and territories included in this study, in 2019, 102 had a TFR lower than 2.1, which is considered a good approximation of replacement-level fertility. All countries in sub-Saharan Africa had TFRs above replacement level in 2019 and accounted for 27.1% (95% UI 26.4-27.8) of global livebirths. Global life expectancy at birth increased from 67.2 years (95% UI 66.8-67.6) in 2000 to 73.5 years (72.8-74.3) in 2019. The total number of deaths increased from 50.7 million (49.5-51.9) in 2000 to 56.5 million (53.7-59.2) in 2019. Under-5 deaths declined from 9.6 million (9.1-10.3) in 2000 to 5.0 million (4.3-6.0) in 2019. Global population increased by 25.7%, from 6.2 billion (6.0-6.3) in 2000 to 7.7 billion (7.5-8.0) in 2019. In 2019, 34 countries had negative natural rates of increase; in 17 of these, the population declined because immigration was not sufficient to counteract the negative rate of decline. Globally, HALE increased from 58.6 years (56.1-60.8) in 2000 to 63.5 years (60.8-66.1) in 2019. HALE increased in 202 of 204 countries and territories between 2000 and 2019. Interpretation Over the past 20 years, fertility rates have been dropping steadily and life expectancy has been increasing, with few exceptions. Much of this change follows historical patterns linking social and economic determinants, such as those captured by the GBD Socio-demographic Index, with demographic outcomes. More recently, several countries have experienced a combination of low fertility and stagnating improvement in mortality rates, pushing more populations into the late stages of the demographic transition. Tracking demographic change and the emergence of new patterns will be essential for global health monitoring. Copyright (C) 2020 The Author(s). Published by Elsevier Ltd.

Bone infection is a feared complication for patients with surgically fixed bone fractures and local antibiotic delivery is important in prophylaxis and treatment of these infections. Recent studies indicated that Staphylococcus aureus can penetrate bone tissue through micron-sized canaliculi and evade systemic and currently available local antibiotic treatments. Targeting bacteria within the bone requires highly efficient delivery of antimicrobials to the infected bone tissue. In this work, a biodegradable microsphere carrier loaded with antibiotics and with specific affinity to bone mineral was developed. Two widely used antibiotics, i.e., Gentamicin-dioctyl sulfosuccinate (GM-AOT) and Ciprofloxacin (CF) were embedded in poly(ϵ-caprolactone) (PCL) microspheres fabricated by oil-in-water emulsion techniques with carboxylated poly(vinyl alcohol) (cPVA) as surfactant. The carboxylic acid groups present at the Poly(ϵ-caprolactone)/cPVA (PCL-cPVA) microsphere surface were functionalized with aspartic acid oligomers (ASP) granting bone targeting properties. We report on cPVA synthesis, microsphere formulation, and antibiotic loading of PCL/cPVA-ASP microspheres. Antibiotic loaded PCL/cPVA-ASP microspheres show sustained release of its antibiotic load and can inhibit bacterial growth in vitro for up to 6 days. PCL/cPVA-ASP microspheres show enhanced affinity to mineralized substrates compared to non-functionalized PCL/cPVA microspheres. These findings support further development of these bone targeting antibiotic carriers for potential treatment of persistent bone infections.

The aim of this work was to fabricate microporous poly(trimethylene carbonate) (PTMC) vascular structures by stereolithography (SLA) for applications in tissue engineering and organ models. Leachable CaCO3 particles with an average size of 0.56 &mu, m were used as porogens. Composites of photocrosslinkable PTMC and CaCO3 particles were cast on glass plates, crosslinked by ultraviolet light treatment and leached in watery HCl solutions. In order to obtain interconnected pore structures, the PTMC/CaCO3 composites had to contain at least 30 vol % CaCO3. Leached PTMC films had porosities ranging from 33% to 71% and a pore size of around 0.5 &mu, m. The mechanical properties of the microporous PTMC films matched with those of natural blood vessels. Resins based on PTMC/CaCO3 composites with 45 vol % CaCO3 particles were formulated and successfully used to build vascular structures of various shapes and sizes by SLA. The intrinsic permeabilities of the microporous PTMC films and vascular structures were at least one order of magnitude higher than reported for the extracellular matrix, indicating no mass transfer limitations in the case of cell seeding.

Synthetic immune-stimulatory drugs such as agonists of the Toll-like receptors (TLR) 7/8 are potent activators of antigen-presenting cells (APCs), however, they also induce severe side effects due to leakage from the site of injection into systemic circulation. Here, we report on the design and synthesis of an amphiphilic polymer-prodrug conjugate of an imidazoquinoline TLR7/8 agonist that in aqueous medium forms vesicular structures of 200 nm. The conjugate contains an endosomal enzyme-responsive linker enabling degradation of the vesicles and release of the TLR7/8 agonist in native form after endocytosis, which results in high in vitro TLR agonist activity. In a mouse model, locally administered vesicles provoke significantly more potent and long-lasting immune stimulation in terms of interferon expression at the injection site and in draining lymphoid tissue compared to a nonamphiphilic control and the native TLR agonist. Moreover, the vesicles induce robust activation of dendritic cells in the draining lymph node in vivo.

Fibroblasts are a heterogenic cell-type that play a major role in tissue integrity and wound healing. Under pathological conditions they transform into an aggressive phenotype, being responsible for inducing organ fibrosis as well as supporting tumor progression. Fibroblasts are known to secrete abundant amounts of extracellular matrix and numerous growth factors and cytokines. Cell-specific therapeutic targeting to fibroblasts is of high interest for intervening into several pathologies by developing novel therapeutic modalities. This chapter will shed light on the biology of fibroblasts, their diversity and heterogeneity as well as the targeting strategies using different targeting systems, such as nanoparticles and biologicals.

Objective: Bone infections are challenging to treat because of limited capability of systemic antibiotics to accumulate at the bone site. To enhance therapeutic action, systemic treatments are commonly combined with local antibiotic-loaded materials. Nevertheless, available drug carriers have undesirable properties, including inappropriate antibiotic release profiles and nonbiodegradability. To alleviate such limitations, we aim to develop a drug delivery system (DDS) for local administration that can interact strongly with bone mineral, releasing antibiotics at the infected bone site. Methods: Biodegradable polyesters (poly (ε-caprolactone) or poly (D,l-lactic acid)) were selected to fabricate antibiotic-loaded microspheres by oil in water emulsion. Antibiotic release and antimicrobial effects on Staphylococcus aureus were assessed by zone of inhibition measurements. Microsphere bone affinity was increased by functionalising the bisphosphonate drug alendronate to the microsphere surface using carbodiimide chemistry. Effect of bone targeting microspheres on bone homeostasis was tested by looking at the resorption potential of osteoclasts exposed to the developed microspheres. Results: In vitro, the antibiotic release profile from the microspheres was shown to be dependent on the polymer used and the microsphere preparation method. Mineral binding assays revealed that microsphere surface modification with alendronate significantly enhanced interaction with bone-like materials. Additionally, alendronate functionalised microspheres did not differentially affect osteoclast mineral resorption in vitro, compared with nonfunctionalised microspheres. Conclusion: We report the development and characterisation of a DDS which can release antibiotics in a sustained manner. Surface-grafted alendronate groups enhanced bone affinity of the microsphere construct, resulting in a bone targeting DDS. The Translational Potential of this Article: The DDS presented can be loaded with hydrophobic antibiotics, representing a potential, versatile and biodegradable candidate to locally treat bone infection. Keywords: Alendronate, Bone infection, Bone targeting, Drug delivery, Microparticle, Osteomyelitis

Additive manufacturing or, as also called, three-dimensional (3D) printing is considered as a game-changer in replacing traditional processing methods in numerous applications; yet, it has one intrinsic potential weakness related to bonding of layers formed during the printing process. Prior to finding solutions for improvement, a thorough quantitative understanding of the mechanical properties of the interface is needed. Here, a quantitative analysis of the nanomechanical properties in 3D printed photopolymers formed by digital light processing (DLP) stereolithography (SLA) is shown. Mapping of the contact Young's modulus across the layered structure is performed by atomic force microscopy (AFM) with a submicrometer resolution. The peakforce quantitative nanomechanical mapping (PF-QNM) mode was employed in the AFM experiments. The layered specimens were obtained from an acrylate-based resin (PR48, Autodesk), containing also a light-absorbing dye. We observed local depressions with values up to 30% of the maximum stiffness at the interface between the consecutively deposited layers, indicating local depletion of molecular cross-link density. The thickness values of the interfacial layers were approximately 11 μm, which corresponds to ∼ 22% of the total layer thickness (50 μm). We attribute this to heterogeneities of the photopolymerization reaction, related to (1) atmospheric oxygen inhibition and (2) molecular diffusion across the interface. Additionally, a pronounced stiffness decay was observed across each individual layer with a skewed profile. This behavior was rationalized by a spatial variation of the polymer cross-link density related to the variations of light absorption within the layers. This is caused by the presence of light absorbers in the printed material, resulting in a spatial decay of light intensity during photopolymerization.

Widespread application of islet transplantation as a cure for type 1 Diabetes Mellitus patients is limited mainly due to the need for life-long immunosuppression associated with serious side effects. Immunoisolation by encapsulation of islets in semi-permeable membranes may overcome this issue as it allows for transplantation potentially obviating the need for immunosuppressive drug therapy. This work focuses on the development and validation of a model of the mass transfer and secretion kinetics of insulin – the major regulator of glucose homeostasis – in a new microwell membrane based encapsulation device for pancreatic islets. Due to the high number and complexity of parameters that determine the effectiveness of the device, the implementation of a reliable numerical model is of critical importance for optimization of the micro-confined bioengineered environment. The simultaneous solution of mass transfer and uptake/production reactions occurring through the polymeric membrane system and within the islets allows detailed description of the spatial distribution profiles of insulin, glucose, and oxygen with the aim to assure an adequate insulin dynamics. The simulation tool, experimentally validated, is used to improve the scaffold geometry with the aim to assure adequate responsiveness and function over time for future clinical implementation of the device.

A hybrid gene delivery platform, micro Cell Vesicle Technology (mCVT), produced from the fusion of plasma membranes and cationic lipids, is presently used to improve the transfection efficiency of hard-to-transfect (HTT) cells. The plasma membrane components of mCVTs impart specificity in cellular uptake and reduce cytotoxicity in the transfection process, while the cationic lipids complex with the genetic material and provide structural integrity to mCVTs. This journal is

Pancreatic ductal adenocarcinoma (PDAC), characterized with abundant tumor stroma, is a highly malignant tumor with poor prognosis. The tumor stroma largely consists of cancer-associated fibroblasts (CAFs) and extracellular matrix (ECM), and is known to promote tumor growth and progression as well as acts as a barrier to chemotherapy. Inhibition of tumor stroma is highly crucial to induce the effect of chemotherapy. In this study, we delivered fibroblast growth factor 2 (FGF2) to human pancreatic stellate cells (hPSCs), the precursors of CAFs, using superparamagnetic iron oxide nanoparticles (SPIONs). FGF2 was covalently conjugated to functionalized PEGylated dextran-coated SPIONs. FGF2-SPIONs significantly reduced TGF-β induced hPSCs differentiation (α-SMA and collagen-1 expression) by inhibiting pSmad2/3 signaling and inducing ERK1/2 activity, as shown with western blot analysis. Then, we established a stroma-rich self-assembling 3D heterospheroid model by co-culturing PANC-1 and hPSCs in 3D environment. We found that FGF2-SPIONs treatment alone inhibited the tumor stroma-induced spheroid growth. In addition, they also potentiated the effect of gemcitabine, as shown by measuring the spheroid size and ATP content. These effects were attributed to the reduced expression of the hPSC activation and differentiation marker, α-SMA. Furthermore, to demonstrate an application of SPIONs, we applied an external magnetic field to spheroids while incubated with FGF2-SPIONs. This resulted in an enhanced effect of gemcitabine in our 3D model. In conclusion, this study presents a novel approach to target FGF2 to tumor stroma using SPIONs and thereby enhancing the effect of gemcitabine as demonstrated in the complex 3D tumor spheroid model.

International audience; Microfluidic droplet generators excel in generating monodisperse micrometer-sized droplets and particles. However, the low throughput of conventional droplet generators hinders their clinical and industrial translation. Current approaches to parallelize microdevices are challenged by the two-dimensional nature of the standard fabrication methods. Here, we report the facile production of three-dimensionally (3D) parallelized microfluidic droplet generators consisting of stacked and radially multiplexed channel designs. Computational fluid dynamics simulations form the design basis for a microflow distributor that ensures similar flow rates through all droplet generators. Stereolithography is the selected technique to fabricate microdevices, which enables the manufacturing of hollow channels with dimensions as small as 50 μm. The microdevices could be operated up to 4 bars without structural damage, including deformation of channels, or leakage of the on-chip printed Luer-Lok type connectors. The printed microdevices readily enable the production of water-in-oil emulsions, as well as polymer containing droplets that act as templates for both solid and core-shell hydrogel microparticles. The cytocompatibility of the 3D printed device is demonstrated by encapsulating mesenchymal stem cells in hydrogel microcapsules, which results in the controllable formation of stem cell spheroids that remain viable and metabolically active for at least 21 days. Thus, the unique features of stereolithography fabricated microfluidic devices allow for the parallelization of droplet generators in a simple yet effective manner by enabling the realization of (complex) 3D designs.

Non-alcoholic steatohepatitis (NASH) and alcohol-associated steatohepatitis (ASH) are the major cause of liver-related mortality with limited therapeutic options available. In this study, we investigated the role of Src tyrosine kinase in the pathogenesis of (N)ASH. We examined Src kinase expression in livers from patients with NASH, ASH, cirrhosis and biliary atresia, and from preclinical mouse models: methionine choline deficient (MCD)-diet-induced NASH, Lieber–DeCarli ethanol (EtOH)-diet-induced ASH, carbon tetrachloride (CCl4)- and bile duct ligation (BDL)-induced liver fibrosis. Functional studies, using Src kinase inhibitor KX2-391, were performed in NASH and ASH mouse models, and in relevant in vitro models. Transcriptomic analysis revealed increased Src kinase expression in human and mouse diseased livers that positively correlated with disease progression. Src kinase inhibition ameliorated lipid biosynthesis (steatosis); monocytes/macrophages infiltration and inflammatory cytokines expression (inflammation) and hepatic stellate cells (HSCs) activation and collagen expression (fibrosis) in MCD-diet and EtOH-diet induced liver disease mouse models. In vitro Src inhibition attenuated FFA-induced hepatocytic lipid accumulation, LPS/IFNγ-induced nitric oxide release and expression of several inflammatory signatures in macrophages and TGFβ-induced HSCs activation, contractility and collagen expression. Moreover, Src inhibition diminished gene expression of inflammatory markers in LPS/IFNγ-stimulated BMDMs and LPS-stimulated PCLS, and reduced FA accumulation, macrophage activation and collagen deposition in 3D human liver spheroids. Mechanistic studies further revealed that Src kinase mediated the effects through FAK/PI3K/AKT pathway. Our results demonstrate a multicellular and functional role of Src kinase highlighting Src kinase as a promising therapeutic target in (non)alcoholic steatohepatitis.

Liver fibrosis and its progression to liver cirrhosis and hepatocellular carcinoma (HCC) is a growing health problem affecting millions of people worldwide. This growing global health burden is attributed to hepatitis viral infections (viral hepatitis), metabolic disorders such as obesity and diabetes (non-alcoholic fatty liver disease, NAFLD) and alcohol abuse (alcohol-associated liver disease, ALD), among others. Unfortunately, no clinically approved drugs are available for the treatment of liver diseases. Liver transplantation is the only option available in case of end-stage liver failure (liver cirrhosis). Macrophages and hepatic stellate cells (HSCs) are known to play a crucial role in the development of liver diseases, both during acute and chronic liver injury. Therefore, in this thesis, we developed and investigated novel therapeutic approaches targeting macrophages and HSCs. We explored the effects of a small molecule SYK inhibitor, R406 (without and with nanoparticles) in vitro and in vivo. We assessed the implication of the SYK pathway in macrophages and found that inhibition of the SYK pathway using R406 resulted in the inhibition of inflammation markers i.e., nitric oxide (NO) release, and enhanced gene expression of IL-1β, FcγR1, iNOS, CCL2, IL-6, and CCR2, in a dose-dependent manner. We found that both R406-PLGA nanoparticles as well as R406 itself could significantly inhibit the gene expression of several inflammatory markers in vitro. In a subsequent in vivo study, using the MCD-diet NASH mouse model, we found that R406-PLGA nanoparticles significantly ameliorated liver inflammation, fibrosis, and steatosis as compared to free R406. The selective small molecule SRC kinase inhibitor, KX2-391 inhibited the phosphorylation of SRC and significantly reduced the NO release in RAW macrophages. Gene expression analysis in RAW macrophages and BMDMs evidenced that SRC pathway inhibition mediated by KX2-391 attenuated the expression of different inflammatory markers including iNOS, CCL2, and FcγR1. Additionally, in a precision-cut liver slices (PCLS) model, KX2-391 decreased the gene expression of SRC, iNOS and CCL2 without affecting the cell/tissue viability. Moreover, KX2-391 attenuated hepatocytic lipid accumulation; TGFβ-induced HSCs activation, contractility and collagen expression. KX2-391 attenuated inflammation, fibrosis, and steatosis in both NASH and ASH mouse models. Mechanistic studies further revealed that SRC mediated the effects through the FAK/PI3K/AKT pathway. We observed that FGF2 was able to inhibit TGFβ-induced HSCs activation (confirmed by reduced collagen-I and α-SMA expression), migration of HSCs (confirmed by wound healing assays) and contraction of HSCs (confirmed by 3D-gel contraction assays). These results laid the foundation for our further studies in which we conjugated FGF2 to superparamagnetic iron oxide nanoparticles (FGF2-SPIONs) to improve the stability and half-life of FGF2 and thereby to improve the therapeutic efficacy of FGF2 for the treatment of liver fibrosis. After performing several in vitro and in vivo experiments, the results revealed that the potency of FGF2-SPIONs conjugate is significantly improved as compared to free FGF2. In conclusion, the approaches described in this thesis are promising to be explored further for the treatment of liver diseases like NASH or ALD-based liver fibrosis.

In this chapter, the authors provide a detailed overview of the physical properties and barrier within the tumor stroma and describe different treatment strategies to overcome the physical barrier and eventually reduce tumor progression. In the recent years, however, many therapeutic strategies have been developed to inhibit this physical barrier presented by the tumor stroma including the inhibition of cancer-associated fibroblasts, degradation of the extracellular matrix (ECM), and others. The tumor stroma has shown many distinct physical properties when compared to healthy tissues, such as an elevated stiffness, a solid stress within the tissue, high interstitial pressure, hypoxia, and compressed and/or leaky blood vasculature. The desmoplastic reaction in tumors is mainly driven by cellular components within the tumor stroma, which display an enhanced production of Extracellular Matrix proteins such as collagen type I or fibronectin. Decorin is an omnipresent component of the ECM and is a small leucine-rich proteoglycan consisting of a single glycosaminoglycans chain and a core protein.

A completely different approach to create a biologically relevant in vitro model is the culture of biological samples themselves in the form of tumor tissues or slices. In the recent years, two approaches have emerged more or less in parallel to create 3D in vitro models that are easy to control and reproduce while maintaining a high biological relevance. The first approach is a cell-based methodology that uses the cells own specific properties to create 3D culture systems involving spheroids, tumoroids, and organoids. The second approach uses a (biomedical) engineering methodology, where the design of the model is mainly driven by the researcher to investigate certain cellular functions and interactions. In the recent years, the term “organoid” has been focus of countless studies demonstrating the potential of the novel culture technique that is able to develop 3D organ-like in vitro models resembling key biological morphology, development, and characteristics.

Inflammatory cells recruit to the site of the injury due to secretion of chemo-attractants during the normal wound healing process. These inflammatory cells, once recruited, produce an abundant amount of growth factors and cytokines which in turn activate the local fibroblasts. The terms “tumor microenvironment” and “tumor stroma” are commonly used by researchers in the field. To clarify, the tumor stroma is the non-neoplastic part of the tumor microenvironment composed of various cell types such as cancer-associated fibroblasts, vascular cells, and different immune cells. The tumor stroma has been recognized as a key therapeutic target for the development of anti-cancer treatments, as it affects many hallmarks of cancer. In the last decade, the tumor stroma has been proposed to play a crucial role in many tumor-associated mechanisms such as tumorigenesis, neoplastic cell proliferation, metabolism, angiogenesis, metastasis, and immune cell infiltration.

Tumor cells while growing and progressing similarly affect their surrounding microenvironment. This microenvironment co-evolves into an activated state, activating, and altering the release of a number of cytokines or chemokines. These alterations in signaling promote cancer initiation and progression. One of hallmarks of cancer progression is the formation of a so-called tumor stroma surrounding the tumor cells, which includes endothelial cells, fibroblasts, macrophages, and other immune cells as well as extracellular matrix. Cancer-Associated Fibroblasts (CAFs) are one of main contributors to tumor stroma and desmoplastic reaction within the TME. CAFs are very diverse in origin and phenotype in a tumor microenvironment. Integrins in tumor stroma is one of greatly researched topics with focuses on functions of integrins in cancer, activation of signaling pathways, and molecular mechanisms used in cancer progression, migration, and metastasis. Thus, targeting integrins expressed in the tumor stroma has become an important field of exploration for scientists leading to many drugs targeting specific integrins or integrin subunits.

This review on hybrid hydrogels prepared from methacrylated gelatin (GelMA) and synthetic polymers presents their advantageous properties and potential applications in biomedical engineering. It describes the preparation of the different types of frequently used hybrid hydrogel networks: co-networks, interpenetrating networks (IPNs), and semi-interpenetrating networks (semi-IPNs) and gives an overview of the different methods of preparing biomedical devices from these materials.

In this chapter, the author highlight dysregulated MicroRNAs (miRNA), as potential therapeutic targets in different tumor stromal cells describe their functions in the regulation of the tumor microenvironment. They also discusses the role of tumor-related miRNAs as biomarkers for cancer diagnosis and prognosis. TAMs show the ability to promote the tumor development and progression by enhancing angiogenesis, immune suppression, tumor cell invasion, and metastatic programming of tumor tissue, and thus reducing patient survival. MiRNAs can also be delivered by inorganic nanoparticle–based vectors, such as gold nanoparticles, iron-nanoparticles and silica-based nanoparticles. Tumor stroma plays an essential role during tumor growth, thus targeting tumor stromal cells provides possibilities to inhibit the promotion activities in tumor progression and metastasis. The authors summarizes various miRNA delivery approaches that have been or can potentially be applied to deliver miRNA as therapeutics into the stromal cells.

An efficient, scalable, and good manufacturing practice (GMP) compatible process was developed for the production of docetaxel-loaded poly(ethylene glycol)-b-poly(N-2-benzoyloxypropyl methacrylamide) (mPEG-b-p(HPMA-Bz)) micelles. First, the synthesis of the mPEG-b-p(HPMA-Bz) block copolymer was optimized through step-by-step investigation of the batch synthesis procedures. This resulted in the production of 1 kg of mPEG-b-p(HPMA-Bz) block copolymer with a 5 kDa PEG block and an overall molecular weight of 22.5 kDa. Second, the reproducibility and scalability of micelle formation was investigated for both batch and continuous flow setups by assessing critical process parameters. This resulted in the development of a new and highly efficient continuous flow process, which led to the production of 100 mL of unloaded micelles with a size of 55 nm. Finally, the loading of the micelles with the anticancer drug docetaxel was successfully fine-tuned to obtain precise control on the loaded micelle characteristics. As a result, 100 mL of docetaxel-loaded micelles (20 mg/mL polymer and 5 mg/mL docetaxel in the feed) with a size of 55 nm, an encapsulation efficiency of 65%, a loading capacity of 14%, and stable for at least 2 months in water at room temperature were produced with the newly developed continuous flow process. In conclusion, this study paves the way for efficient and robust large-scale production of docetaxel-loaded micelles with high encapsulation efficiencies and stability, which is crucial for their applicability as a clinically relevant drug delivery platform.

Ultrasound-triggered drug-loaded microbubbles have great potential for drug delivery due to their ability to locally release drugs and simultaneously enhance their delivery into the target tissue. We have recently shown that upon applying ultrasound, nanoparticle-loaded microbubbles can deposit nanoparticles onto cells grown in 2D monolayers, through a process that we termed "sonoprinting". However, the rigid surfaces on which cell monolayers are typically growing might be a source of acoustic reflections and aspherical microbubble oscillations, which can influence microbubble-cell interactions. In the present study, we aim to reveal whether sonoprinting can also occur in more complex and physiologically relevant tissues, by using free-floating 3D tumor spheroids as a tissue model. We show that both monospheroids (consisting of tumor cells alone) and cospheroids (consisting of tumor cells and fibroblasts, which produce an extracellular matrix) can be sonoprinted. Using doxorubicin-liposome-loaded microbubbles, we show that sonoprinting allows to deposit large amounts of doxorubicin-containing liposomes to the outer cell layers of the spheroids, followed by doxorubicin release into the deeper layers of the spheroids, resulting in a significant reduction in cell viability. Sonoprinting may become an attractive approach to deposit drug patches at the surface of tissues, thereby promoting the delivery of drugs into target tissues. ispartof: Journal Of Controlled Release vol:316 pages:79-92 ispartof: location:Netherlands status: published

Urea removal is one of the biggest challenges in dialysate regeneration in Wearable Artificial Kidney (WAK) devices. In this work, a new Mixed Matrix Membrane (MMM) is developed for urea removal in WAK applications. The MMM consists of polystyrene-based ninhydrin particles within a polyethersulfone/polyvinylpyrrolidone polymer blend matrix. The MMM is prepared via dry-wet spinning technique and characterized in terms of its morphology via electron microscopy and clean water permeance. Urea removal is studied both in static and in dynamic conditions. Thanks to the good dispersion of small size ninhydrin particles (size < 63 µm), the MMM removed under static conditions, at 70 °C, 2.1 ± 0.1 mmol of urea per grams of particles at 24 h, while urea removal by the particles in suspension reached 1.7 ± 0.1 mmol/g under the same conditions. Importantly, in continuous recirculation experiments, performed at 70 °C using a laboratory scale module, the MMM removed 3.4 ± 0.3 mmol of urea per grams of particles, in 4 h, due to the high particle accessibility by urea within the membrane. Based on these results it is estimated that only 215 g of MMM are needed for removing the daily produced urea from spent dialysate (400 mmol) making MMM suitable for application to WAK, where miniaturization and lightweight are required.

Brain metastases are a most disturbing situation for breast cancer patients as there is basically no adequate treatment available. Any potential drug formulation has to be able to cross the blood-brain barrier (BBB) and specific to metastatic brain tumors without causing unacceptable adverse effects. Here, we developed transferrin-functionalized chimeric polymersomes carrying siRNA against polo-like kinase 1 (Tf@TBP-CPs-siPLK1) for treating brain metastatic MDA-MB 231 triple negative breast cancer (TNBC) xenografts in mice. To facilitate the loading of siPLK1, chimaeric polymersomes (CPs) were designed with spermine in the watery core and transferrin-binding peptide (TBP) at the surface, enabling attachment of transferrin after the siRNA loading step and thereby circumventing interference of transferrin with siRNA loading. Tf@TBP-CPs-siPLK1 encapsulating 3.8 wt% siRNA had a mean size of about 50 nm and a neutral zeta potential in phosphate buffer (PB). By virtue of the presence of transferrin, Tf@TBP-CPs demonstrated greatly (ca. 5-fold) enhanced internalization in MDA-MB 231 cells and transcytosis in the endothelial (bEnd.3) monolayer model in vitro as well as markedly improved accumulation in the orthotopically xenografted MDA-MB 231 tumor in the brain in vivo compared with control CPs lacking transferrin, supporting that transferrin mediates efficient BBB penetration and high specificity towards MDA-MB 231 cells. As a result, Tf@TBP-CPs-siPLK1 effectively inhibited tumor progression and prolonged the lifespan of the mice significantly. Selective transferrin coating appears to be a particularly facile strategy to fabricate BBB-permeable and targeted vesicles for potent RNAi therapy of brain metastatic breast cancer.

Simple Summary Pancreatic ductal adenocarcinoma (PDAC) is the most prevalent and aggressive type of pancreatic cancer with a low 5-year survival rate of only 8%. The cellular arrangement plays a crucial role in PDAC, which is characterized by a highly fibrotic environment around the tumor cells, preventing treatments from reaching their target. For the development of novel drug candidates, it is crucial to mimic this cellular arrangement in a laboratory environment. We successfully developed a reproducible three-dimensional cell culture model that demonstrates the PDAC characteristic arrangement and showed a PDAC relevant gene profile when comparing with the genetic profile of PDAC patients. We finally demonstrated the use of the model for the evaluation of novel anti-fibrotic therapy against PDAC by studying drug-induced reduction of fibrosis in PDAC enabling nanoparticles to penetrate and reach the tumor cells. This model is useful for the evaluation of novel treatments against PDAC in a biologically relevant manner. Abstract Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive tumor type with low patient survival due to the low efficacy of current treatment options. Cancer-associated fibroblasts (CAFs) in the tumor microenvironment (TME) create a dense fibrotic environment around the tumor cells, preventing therapies from reaching their target. Novel 3D in vitro models are needed that mimic this fibrotic barrier for the development of therapies in a biologically relevant environment. Here, novel PDAC microtissues (µtissues) consisting of pancreatic cancer cell core surrounded by a CAF-laden collagen gel are presented, that is based on the cells own contractility to form a hard-to-penetrate barrier. The contraction of CAFs is demonstrated facilitating the embedding of tumor cells in the center of the µtissue as observed in patients. The µtissues displayed a PDAC-relevant gene expression by comparing their gene profile with transcriptomic patient data. Furthermore, the CAF-dependent proliferation of cancer cells is presented, as well as the suitability of the µtissues to serve as a platform for the screening of CAF-modulating therapies in combination with other (nano)therapies. It is envisioned that these PDAC µtissues can serve as a high-throughput platform for studying cellular interactions in PDAC and for evaluating different treatment strategies in the future.

Lung diseases are one of the most common causes of death. Moreover, the rise of lung-associated viruses, such as SARS-CoV-2, emphasise the relevance of and need for lung models that generate reliable data of potential drugs and treatments. Thus, lungs-on-chips (LOCs) have been developed, which implement more physiological features, e.g. microfluidics and mechanical stretch of cells, than traditional lung models to better mimic the tissue, and thus yield more reliable results. However, the properties of the porous membranes on which the cells in LOCs and lung models in general are cultured, are often overlooked, while these properties are known to influence cell behaviour. For example, many membranes are made from stiff materials, which do not reflect the stiffness of the lungs and do not allow mechanical stretch. In this thesis, we used poly(trimethylene carbonate) (PTMC), a cytocompatible, flexible material. We developed porous and form-stable PTMC membranes using temperature-induced, liquid-induced and evaporation-induced phase separation (i.e. TIPS, LIPS and EIPS, respectively) together with photo-crosslinking. Different parameters were changed to influence membrane properties. For TIPS, we focused on lowering the temperature during crosslinking. With LIPS, we looked at the effects of pore formers and crosslinking on membrane properties. Using EIPS, we investigated e.g. the effects of the molecular weight of PTMC, the non-solvent type and concentration, and the temperature and humidity during EIPS. We also combined EIPS with micromoulding to produce membranes with alveoli-like microwells. The different membranes were characterised, including their morphology, gel content and water permeance. We developed a lung epithelial-endothelial model of Calu-3 lung epithelial cells and human lung microvascular endothelial cells (LMVECs) on the flat and microstructured EIPS-based membranes. The models were characterised through Zona Occludens-1 (ZO-1)/CD31 and live/dead staining, as well as electrical resistance and FITC-dextran permeability assays. Stainings for epithelial- and endothelial-to-mesenchymal transitions (EMT and EndoMT, respectively) were also performed. Lastly, the co-culture model on the microstructured membranes was integrated in a commercial organ-on-chip platform and ZO-1/CD31 and live/dead staining were performed on this model. The presented novel fabrication methods and PTMC membranes could lead to the use and development of more biomimetic membranes and LOCs.

Monocytes are antigen-presenting cells (APCs) that play diverse roles in promoting or regulating inflammatory responses, but their role in T cell stimulation is not well defined. In inflammatory conditions, monocytes frequently show increased expression of CD169/Siglec-1, a type-I interferon (IFN-I)-regulated protein. However, little is known about the phenotype and function of these CD169+ monocytes. Here, we have investigated the phenotype of human CD169+ monocytes in different diseases, their capacity to activate CD8+ T cells, and the potential for a targeted-vaccination approach. Using spectral flow cytometry, we detected CD169 expression by CD14+ CD16- classical and CD14+ CD16+ intermediate monocytes and unbiased analysis showed that they were distinct from dendritic cells, including the recently described CD14-expressing DC3. CD169+ monocytes expressed higher levels of co-stimulatory and HLA molecules, suggesting an increased activation state. IFNα treatment highly upregulated CD169 expression on CD14+ monocytes and boosted their capacity to cross-present antigen to CD8+ T cells. Furthermore, we observed CD169+ monocytes in virally-infected patients, including in the blood and bronchoalveolar lavage fluid of COVID-19 patients, as well as in the blood of patients with different types of cancers. Finally, we evaluated two CD169-targeting nanovaccine platforms, antibody-based and liposome-based, and we showed that CD169+ monocytes efficiently presented tumor-associated peptides gp100 and WT1 to antigen-specific CD8+ T cells. In conclusion, our data indicate that CD169+ monocytes are activated monocytes with enhanced CD8+ T cell stimulatory capacity and that they emerge as an interesting target in nanovaccine strategies, because of their presence in health and different diseases.

To provide an integrin binding peptide that is particularly ITGA5, ITGA11, α5β1 integrin and an α11β1 binding peptide.SOLUTION: An integrin binding peptide, a pharmaceutical composition containing a peptide thereof, and use thereof as a therapeutic agent, a diagnostic agent, an imaging agent, and a targeting agent.SELECTED DRAWING: None

Non

Pancreatic ductal adenocarcinoma (PDAC) is an extremely aggressive type of cancer with an overall survival rate of less than 7-8%, emphasizing the need for novel effective therapeutics against PDAC. However only a fraction of therapeutics which seemed promising in the laboratory environment will eventually reach the clinic. One of the main reasons behind this low success rate is the complex tumor microenvironment (TME) of PDAC, a highly fibrotic and dense stroma surrounding tumor cells, which supports tumor progression as well as increases the resistance against the treatment. In particular, the growing understanding of the PDAC TME points out a different challenge in the development of efficient therapeutics - a lack of biologically relevant in vitro and in vivo models that resemble the complexity and heterogeneity of PDAC observed in patients. The purpose and scope of this review is to provide an overview of the recent developments in different in vitro and in vivo models, which aim to recapitulate the complexity of PDAC in a laboratory environment, as well to describe how 3D in vitro models can be integrated into drug development pipelines that are already including sophisticated in vivo models. Hereby a special focus will be given on the complexity of in vivo models and the challenges in vitro models face to reach the same levels of complexity in a controllable manner. First, a brief introduction of novel developments in two dimensional (2D) models and ex vivo models is provided. Next, recent developments in three dimensional (3D) in vitro models are described ranging from spheroids, organoids, scaffold models, bioprinted models to organ-on-chip models including a discussion on advantages and limitations for each model. Furthermore, we will provide a detailed overview on the current PDAC in vivo models including chemically-induced models, syngeneic and xenogeneic models, highlighting hetero- and orthotopic, patient-derived tissues (PDX) models, and genetically engineered mouse models. Finally, we will provide a discussion on overall limitations of both, in vitro and in vivo models, and discuss necessary steps to overcome these limitations to reach an efficient drug development pipeline, as well as discuss possibilities to include novel in silico models in the process.

Nanomedicine, the application of nanotechnology for medical purpose, has attracted a great deal of attention over the past decades, particularly in the field of cancer therapy where more effective and safer diagnosis and treatment modalities are highly needed. Nanomedicines promise and have shown to offer significant advantages over traditional therapeutic and diagnostic approaches in cancer therapy including enhanced efficacy and/or mitigated toxicity benefits owing to ameliorated pharmacokinetics and favorably altered tissue distribution profile (including the distribution to pathological areas) of the associated drug molecules. Particulate nanomedicines have received a great deal of interest as nanomaterials for achieving above mentioned advantages. Polymersomes (Ps) are structures self-assembled from various amphiphilic block copolymers and exhibit a nano-vesicular architecture that enables encapsulation of hydrophilic or hydrophobic molecules in the internal aqueous compartment or the surrounding polymeric bilayer, respectively. In comparison to the safety concerns of inorganic nanoparticulates for clinical translation, the biocompatibility and/or biodegradability of Ps promise to result in lower or no toxicity in vivo. Actively targeted reduction-responsive Ps were developed exhibiting the following features: high stability in the blood circulation, significant tumor accumulation, enhanced tumor cell uptake and fast intracellular drug release. Besides, Ps also enable the possibility of image-guided drug delivery for optimized and personalized treatment (theranostics). In this thesis, we designed and evaluated Ps systems based on either PEG-disulfide polycarbonate and/or PEG-iodinated polycarbonate for targeted tumor imaging and therapy.

The three main cancer treatment modalities are surgery, radiotherapy, and chemotherapy Unfortunately, they are too often associated with high risks of injury, or toxicity to healthy tissues. Therefore, more selective treatment paradigms and adjuvant combination methods that can kill tumor cells with the promise of limited or no side-effects are being investigated including immunotherapy, drug delivery systems (DDS), and drug delivery devices (DDDs). Ideally, combining two or more of these strategies will realize even more selective treatments with better safety and efficacy. In this thesis work two ‘different scale’ strategies for the treatment of cancer have been explored:1) NANO-scale: thermosensitive liposomes that target tumor vasculature and imageable thermosensitive liposomes for MR-HIFU therapy and 2) MICRO-scale: imageable microparticles as a drug delivery device. Investigation of such strategies has not only therapeutic clinical implications but also can shed light on therapeutic mechanisms and improve our understanding required for the optimization of future drug delivery approaches. The image-able thermosensitive liposome in combination with heat sources such as high intensity focused ultrasound (HIFU), radiofrequency (RF), microwave (MW), or laser ablation used for enhanced drug deposition and monitoring of the fate of delivered drug within the tumor. The cNGR-LTSLs demonstrate improved avidity of a cNGR-targeted LTSL for CD13-positive HT- 1080 cells, as a result of multiple cNGR coupling to the liposome surface. The improved avidity of cNGR-targeted LTSL to vascular receptors achieved by surface attachment of the ligand may translate into increased drug delivery to tumor cells in vivo, as the target vascular receptors are well accessible from the bloodstream. The micro-sized particles reported are radiopaque drug-eluting microparticles containing iodine or bismuth radiopacifiers which were successfully synthesized by direct chemical conjugation of a radio-dense triiodo phenyl species or bismuth chelated macrocycle to microparticles, and which are also able to carry cationic drugs like doxorubicin. These image-able embolic microparticles are visible under fluoroscopy and CBCT which enables real- time feedback of bead location (and thus localization of drug and therapeutic ischemic effect) during and after DEB-TACE. This thesis addresses the successful formulation of image-guided, targeted and locoregional drug delivery systems based on nanometer- and micrometer-sized particles, respectively.

Electrospun scaffolds prepared from combinations of synthetic and natural macromers (macromolecular monomers) are interesting materials for tissue engineering applications. In this study, hybrid fibrous mats composed of 10/0, 8/2, 6/4, 3/7, 1/9 and 0/10 (wt/wt) three-armed methacrylated poly(trimethylene carbonate) (PTMC-tMA) and methacrylated gelatin (GelMA) were prepared by electrospinning and photo-crosslinking. The elastomeric PTMC fibers only maintained a stable structure when electrospun in combination with GelMA. The hybrid materials showed two glass transition temperatures, which indicated the occurrence of phase separation. By varying the ratios of PTMC-tMA/GelMA from 8/2_1/9 (wt/wt), electrospun mats with a fiber diameter ranging from 1037 to 419 nm, water uptake from 106 to 315%, porosity from 88.3 to 94.3%, tensile modulus from 0.643 to 0.216 MPa, elongation at break from 445 to 96%, maximum strength from 4.127 to 0.381 MPa and toughness from 1023 to 15 N/mm2 were prepared. Culturing of human smooth muscle cells on hybrid fibrous mats made from 6/4, 3/7 and 1/9 PTMC-tMA/GelMA showed similar cell attachment and proliferation as compared to 100% GelMA fibrous mats. The toughness of the latter hybrid fibrous mats (539, 33 and 15 N/mm2, respectively) was significantly higher than that of the 100% GelMA fibrous mats (3 N/mm2). In conclusion, tough hybrid fibrous mats with good cell adhesive properties were prepared by electrospinning and photo-crosslinking of mixtures of PTMC-tMA and GelMA.

Background CriPec technology enables the generation of drug-entrapped biodegradable core-crosslinked polymeric micelles (CCPM) with high drug loading capacity, tailorable size, and drug release kinetics. Docetaxel (DTX)-entrapped CCPM, also referred to as CPC634, have demonstrated favorable pharmacokinetics, tolerability, and enhanced tumor uptake in patients. Clinical efficacy evaluation is ongoing. CPC634 is currently stored (shelf life > 5 years) and shipped as a frozen aqueous dispersion at temperatures below -60°C, in order to prevent premature release of DTX and hydrolysis of the core-crosslinks. Consequently, like other aqueous nanomedicine formulations, CPC634 relies on cold chain supply, which is unfavorable for commercialization. Lyophilization can help to bypass this issue. Methods and results Freeze-drying methodology for CCPM was developed by employing CPC634 as a model formulation, and sucrose and trehalose as cryoprotectants. We studied the residual moisture content and reconstitution behavior of the CPC634 freeze-dried cake, as well as the size, polydispersity index, morphology, drug retention, and release kinetics of reconstituted CPC634. Subsequently, the freeze-drying methodology was validated in an industrial setting, yielding a CPC634 freeze-dried cake with a moisture content of less than 0.1 wt%. It was found that trehalose-cryoprotected CPC634 could be rapidly reconstituted in less than 5 min at room temperature. Critical quality attributes such as size, morphology, drug retention, and release kinetics of trehalose-cryoprotected freeze-dried CPC634 upon reconstitution were identical to those of non-freeze-dried CPC634. Conclusion Our findings provide proof-of-concept for the lyophilization of drug-containing CCPM and our methodology is readily translatable to large-scale manufacturing for future commercialization.

Nano-formulating dexamethasone, and administering it via intravenous injection or inhalation, may help to improve anti-COVID-19 treatment efficacy by targeting the potent corticosteroid drug to hyper-activated immune cells, by potentiating its anti-oedema activity and by exploiting its anti-fibrotic effects.