Tumor-On-A-Chip Models for Predicting In Vivo Nanoparticle Behavior.

Nanosized drug formulations are broadly explored for the improvement of cancer therapy. Prediction of in vivo nanoparticle (NP) behavior, however, is challenging, given the complexity of the tumor and its microenvironment. Microfluidic tumor-on-a-chip models are gaining popularity for the in vitro testing of nanoparticle targeting under conditions that simulate the 3D tumor (microenvironment). In this review, following a description of the tumor microenvironment (TME), the state of the art regarding tumor-on-a-chip models for investigating nanoparticle delivery to solid tumors is summarized. The models are classified based on the degree of compartmentalization (single/multi-compartment) and cell composition (tumor only/tumor microenvironment). The physiological relevance of the models is critically evaluated. Overall, microfluidic tumor-on-a-chip models greatly improve the simulation of the TME in comparison to 2D tissue cultures and static 3D spheroid models and contribute to the understanding of nanoparticle behavior. Interestingly, two interrelated aspects have received little attention so far which are the presence and potential impact of a protein corona as well as nanoparticle uptake through phagocytosing cells. A better understanding of their relevance for the predictive capacity of tumor-on-a-chip systems and development of best practices will be a next step for the further refinement of advanced in vitro tumor models.

Selective Targeting of Tumor Cells in a Microfluidic Tumor Model with Multiple Cell Types.

Organ-on-a-chip technology allows researchers to precisely monitor drug efficacy in 3D tissue culture systems that are physiologically more relevant to humans compared to 2D cultures and that allow better control over experimental conditions as compared to animal models. Specifically, the high control over microenvironmental conditions combined with the broad range of direct measurements that can be performed in these systems makes organ-on-a-chip devices a versatile tool to investigate tumor targeting and drug delivery. Here, we describe a detailed protocol for studying the cell-selective targeting of protein drugs to tumor cells on an organ-on-a-chip system using a co-culture consisting of BT-474 cancer cells and C5120 human fibroblasts as an example.

Potent and selective eradication of tumor cells by an EpCAM-targeted Ras-degrading enzyme.

Despite decades of efforts, an urgent need remains to develop tumor cell-selective rat sarcoma (Ras)-targeting therapies that can treat patients with Ras-driven tumors. Here we report modular engineered proteins that degrade Ras selectively in tumor cells that overexpress the tumor cell marker epithelial cell adhesion molecule (EpCAM) by fusing the Ras degrader Ras-Rap1-specific endopeptidase with the translocation domain of the Pseudomonas aeruginosa exotoxin A (ETA) or diphtheria toxin (DT). Redirection to EpCAM is achieved by a designed ankyrin repeat protein. In two-dimensional tumor cell cultures, complete degradation of Ras proteins after 24 h was observed with EpCAM-targeted Ras degraders fused to ETA or DT in EpCAM-overexpressing MCF7 and HCT116 cells, with median inhibition concentration values at sub-nanomolar levels. The viability of EpCAM-low non-cancerous fibroblasts remained unaffected. In a three-dimensional (3D) tumor-on-a-chip system that mimics the natural tumor microenvironment, effective Ras degradation and selective toxicity toward tumor cells, particularly with the ETA-fused constructs, was determined on-chip. To conclude, we demonstrate the potential of modular engineered proteins to kill tumor cells highly selectively by simultaneously exploiting EpCAM as a tumor-specific cell surface molecule as well as Ras as an intracellular oncotarget in a 3D system mimicking the natural tumor microenvironment.

Generation of Protein-Phosphorodiamidate Morpholino Oligomer Conjugates for Efficient Cellular Delivery via Anthrax Protective Antigen.

Phosphorodiamidate morpholino oligomers (PMOs) offer great promise as therapeutic agents for translation blocking or splice modulation due to their high stability and affinity for target sequences. However, in spite of their neutral charge as compared to natural oligonucleotides or phosphorothioate analogs, they still show little permeability for cellular membranes, highlighting the need for effective cytosolic delivery strategies. In addition, the implementation of strategies for efficient cellular targeting is highly desirable to minimize side effects and maximize the drug dose at its site of action. Anthrax toxin is a three-protein toxin of which the pore-forming protein anthrax protective antigen (PA) can be redirected to a receptor of choice and lethal factor (LF), one of the two substrate proteins, can be coupled to various cargoes for efficient cytosolic cargo delivery. In this protocol, we describe the steps to produce the proteins and protein conjugates required for cytosolic delivery of PMOs through the cation-selective pore generated by anthrax protective antigen. The method relies on the introduction of a unique cysteine at the C-terminal end of a truncated LF (aa 1-254), high-yield expression of the (truncated) toxin proteins in E. coli, functionalization of a PMO with a maleimide group and coupling of the maleimide-functionalized PMO to the unique cysteine on LF by maleimide-thiol conjugation chemistry. Through co-administration of PA with LF-PMO conjugates, an efficient cytosolic delivery of PMOs can be obtained.

Mimicking the Biology of Engineered Protein and mRNA Nanoparticle Delivery Using a Versatile Microfluidic Platform.

To investigate the delivery of next-generation macromolecular drugs, such as engineered proteins and mRNA-containing nanoparticles, there is an increasing push towards the use of physiologically relevant disease models that incorporate human cells and do not face ethical dilemmas associated with animal use. Here, we illustrate the versatility and ease of use of a microfluidic platform for studying drug delivery using high-resolution microscopy in 3D. Using this microfluidic platform, we successfully demonstrate the specific targeting of carbonic anhydrase IX (CAIX) on cells overexpressing the protein in a tumor-mimicking chip system using affibodies, with CAIX-negative cells and non-binding affibodies as controls. Furthermore, we demonstrate this system's feasibility for testing mRNA-containing biomaterials designed to regenerate bone defects. To this end, peptide- and lipid-based mRNA formulations were successfully mixed with colloidal gelatin in microfluidic devices, while translational activity was studied by the expression of a green fluorescent protein. This microfluidic platform enables the testing of mRNA delivery from colloidal biomaterials of relatively high densities, which represents a first important step towards a bone-on-a-chip platform. Collectively, by illustrating the ease of adaptation of our microfluidic platform towards use in distinct applications, we show that our microfluidic chip represents a powerful and flexible way to investigate drug delivery in 3D disease-mimicking culture systems that recapitulate key parameters associated with in vivo drug application.

[Metformin and ageing: can treatment delay age-related diseases?] / Metformine en het verouderingsproces.

Metformin is the most widely used medication for diabetes. In recent years, metformin has attracted attention because it might slow down aging. By direct interference with aging-related processes such as reduced autophagy, the accumulation of DNA damage and inflammation, metformin could reduce the risk of age-related diseases including cardiovascular diseases, cancer and neurodegenerative diseases. This article describes the clinical and translational perspectives of metformin in view of the underlying molecular mechanisms. Similarities and differences between lifestyle interventions such as exercise and therapeutic fasting will be highlighted. Upon a careful evaluation of the known advantages and disadvantages, it can be considered to implement metformin use in specific cases for preventive purposes.

A Hybrid In Silico and Tumor-on-a-Chip Approach to Model Targeted Protein Behavior in 3D Microenvironments.

To rationally improve targeted drug delivery to tumor cells, new methods combining in silico and physiologically relevant in vitro models are needed. This study combines mathematical modeling with 3D in vitro co-culture models to study the delivery of engineered proteins, called designed ankyrin repeat proteins (DARPins), in biomimetic tumor microenvironments containing fibroblasts and tumor cells overexpressing epithelial cell adhesion molecule (EpCAM) or human epithelial growth factor receptor (HER2). In multicellular tumor spheroids, we observed strong binding-site barriers in combination with low apparent diffusion coefficients of 1 µm2·s-1 and 2 µm2 ·s-1 for EpCAM- and HER2-binding DARPin, respectively. Contrasting this, in a tumor-on-a-chip model for investigating delivery in real-time, transport was characterized by hindered diffusion as a consequence of the lower local tumor cell density. Finally, simulations of the diffusion of an EpCAM-targeting DARPin fused to a fragment of Pseudomonas aeruginosa exotoxin A, which specifically kills tumor cells while leaving fibroblasts untouched, correctly predicted the need for concentrations of 10 nM or higher for extensive tumor cell killing on-chip, whereas in 2D models picomolar concentrations were sufficient. These results illustrate the power of combining in vitro models with mathematical modeling to study and predict the protein activity in complex 3D models.

A Computational Investigation of In Vivo Cytosolic Protein Delivery for Cancer Therapy.

The ability to specifically block or degrade cytosolic targets using therapeutic proteins would bring tremendous therapeutic opportunities in cancer therapy. Over the last few years, significant progress has been made with respect to tissue targeting, cytosolic delivery, and catalytic inactivation of targets, placing this aim within reach. Here, we developed a mathematical model specifically built for the evaluation of approaches towards cytosolic protein delivery, involving all steps from systemic administration to translocation into the cytosol and target engagement. Focusing on solid cancer tissues, we utilized the model to investigate the effects of microvascular permeability, receptor affinity, the cellular density of targeted receptors, as well as the mode of activity (blocking/degradation) on therapeutic potential. Our analyses provide guidance for the rational optimization of protein design for enhanced activity and highlight the importance of tuning the receptor affinity as a function of receptor density as well as the receptor internalization rate. Furthermore, we provide quantitative insights into how enzymatic cargoes can enhance the distribution, extent, and duration of therapeutic activity, already at very low catalytic rates. Our results illustrate that with current protein engineering approaches, the goal of delivery of cytosolic delivery of proteins for therapeutic effects is well within reach.

Assessing the use of tumor-specific DARPin-toxin fusion proteins for ex vivo purging of cancer metastases from human ovarian cortex before autotransplantation.

OBJECTIVE: To assess the use of tumor-specific designed ankyrin repeat proteins (DARPins) fused to a domain of Pseudomonas aeruginosa exotoxin A for purging of cancer metastases from the ovarian cortex. DESIGN: Experimental study. SETTING: University medical center. PATIENT(S): Human ovarian cortex. INTERVENTION(S): Ovarian cortex harboring artificially induced breast cancer metastases was treated with DARPins targeted to epithelial cell adhesion molecule (EpCAM) and human epidermal growth factor receptor 2 (HER2). MAIN OUTCOME MEASURE(S): The presence of any remaining cancer cells after purging was analyzed by (immuno)histochemistry and reverse transcriptase polymerase chain reaction. Effects on the viability of the ovarian cortex were determined by (immuno)histology, a follicular viability assay, and an assay to determine the in vitro growth capacity of small follicles. RESULT(S): After purging with EpCAM-targeted DARPin, all EpCAM-positive breast cancer cells were eradicated from the ovarian cortex. Although treatment had no effect on the morphology or viability of small follicles, a sharp decrease in oocyte viability during in vitro growth was observed, presumably due to low-level expression of EpCAM on oocytes. The HER2-targeted DARPins had no detrimental effects on the morphology, viability, or in vitro growth of small follicles. HER2-positive breast cancer foci were fully eliminated from the ovarian cortex, and the reverse transcriptase polymerase chain reaction showed a decrease to basal levels of HER2 mRNA after purging. CONCLUSION(S): Purging cancer metastases from ovarian cortex without impairing ovarian tissue integrity is possible by targeting tumor cell surface proteins with exotoxin A-fused DARPins. By adapting the target specificity of the cytotoxic DARPin fusions, it should be possible to eradicate metastases from all types of malignancies.

EpCAM-Binding DARPins for Targeted Photodynamic Therapy of Ovarian Cancer.

Ovarian cancer is the most lethal gynecological malignancy due to late detection associated with dissemination throughout the abdominal cavity. Targeted photodynamic therapy (tPDT) aimed at epithelial cell adhesion molecule (EpCAM), overexpressed in over 90% of ovarian cancer metastatic lesions, is a promising novel therapeutic modality. Here, we tested the specificity and activity of conjugates of EpCAM-directed designed ankyrin repeat proteins (DARPins) with the photosensitizer IRDye 700DX in in vitro and in vivo ovarian cancer models. EpCAM-binding DARPins (Ec1: Kd = 68 pM; Ac2: Kd = 130 nM) and a control DARPin were site-specifically functionalized with fluorophores or IRDye 700DX. Conjugation of anti-EpCAM DARPins with fluorophores maintained EpCAM-specific binding in cell lines and patient-derived ovarian cancer explants. Penetration of DARPin Ec1 into tumor spheroids was slower than that of Ac2, indicative of a binding site barrier effect for Ec1. DARPin-IRDye 700DX conjugates killed EpCAM-expressing cells in a highly specific and illumination-dependent fashion in 2D and 3D cultures. Furthermore, they effectively homed to EpCAM-expressing subcutaneous OV90 xenografts in mice. In conclusion, the high activity and specificity observed in preclinical ovarian cancer models, combined with a high specificity in patient material, warrant a further investigation of EpCAM-targeted PDT for ovarian cancer.

Metabolic Switching of Tumor Cells under Hypoxic Conditions in a Tumor-on-a-chip Model.

Hypoxia switches the metabolism of tumor cells and induces drug resistance. Currently, no therapeutic exists that effectively and specifically targets hypoxic cells in tumors. Development of such therapeutics critically depends on the availability of in vitro models that accurately recapitulate hypoxia as found in the tumor microenvironment. Here, we report on the design and validation of an easy-to-fabricate tumor-on-a-chip microfluidic platform that robustly emulates the hypoxic tumor microenvironment. The tumor-on-a-chip model consists of a central chamber for 3D tumor cell culture and two side channels for medium perfusion. The microfluidic device is fabricated from polydimethylsiloxane (PDMS), and oxygen diffusion in the device is blocked by an embedded sheet of polymethyl methacrylate (PMMA). Hypoxia was confirmed using oxygen-sensitive probes and the effect on the 3D tumor cell culture investigated by a pH-sensitive dual-labeled fluorescent dextran and a fluorescently labeled glucose analogue. In contrast to control devices without PMMA, PMMA-containing devices gave rise to decreases in oxygen and pH levels as well as an increased consumption of glucose after two days of culture, indicating a rapid metabolic switch of the tumor cells under hypoxic conditions towards increased glycolysis. This platform will open new avenues for testing anti-cancer therapies targeting hypoxic areas.

ENABLE 2017, the First European PhD and Post-Doc Symposium. Session 1: Building the Foundations of Biology: Synthetic and Cellular Research.

The European Academy for Biomedical Science (ENABLE) is an initiative funded by the European Union Horizon 2020 program involving four renowned European Research Institutes (Institute for Research in Biomedicine—IRB Barcelona, Spain; Radboud Institute for Molecular Life Sciences—RIMLS, the Netherlands; Novo Nordisk Foundation Center for Protein Research—NNF CPR, Denmark; European School of Molecular Medicine—SEMM, Italy) and an innovative science communication agency (Scienseed). With the aim of promoting biomedical science of excellence in Europe, ENABLE organizes an annual three-day international event. This gathering includes a top-level scientific symposium bringing together leading scientists, PhD students, and post-doctoral fellows; career development activities supporting the progression of young researchers and fostering discussion about opportunities beyond the bench; and outreach activities stimulating the interaction between science and society. The first European PhD and Postdoc Symposium, entitled “Breaking Down Complexity: Innovative Models and Techniques in Biomedicine”, was hosted by the vibrant city of Barcelona. The scientific program of the conference was focused on the most recent advances and applications of modern techniques and models in biomedical research and covered a wide range of topics, from synthetic biology to translational medicine. Overall, the event was a great success, with more than 200 attendees from all over Europe actively participating in the symposium by presenting their research and exchanging ideas with their peers and world-renowned scientists.

ENABLE 2017, the First EUROPEAN PhD and Post-Doc Symposium. Session 4: From Discovery to Cure: The Future of Therapeutics.

The EUROPEAN ACADEMY FOR BIOMEDICAL SCIENCE (ENABLE) is an initiative funded by the European Union Horizon 2020 program involving four renowned European Research Institutes (Institute for Research in Biomedicine-IRB Barcelona, Spain; Radboud Institute for Molecular Life Sciences-RIMLS, the Netherlands; Novo Nordisk Foundation Center for Protein Research-NNF CPR, Denmark; European School of Molecular Medicine-SEMM, Italy) and an innovative science communication agency (Scienseed). With the aim of promoting biomedical science of excellence in Europe, ENABLE organizes an annual three-day international event. This gathering includes a top-level scientific symposium bringing together leading scientists, PhD students, and post-doctoral fellows; career development activities supporting the progression of young researchers and fostering discussion about opportunities beyond the bench; and outreach activities stimulating the interaction between science and society. The first European PhD and Postdoc Symposium, entitled "Breaking Down Complexity: Innovative Models and Techniques in Biomedicine", was hosted by the vibrant city of Barcelona. The scientific program of the conference was focused on the most recent advances and applications of modern techniques and models in biomedical research and covered a wide range of topics, from synthetic biology to translational medicine. Overall, the event was a great success, with more than 200 attendees from all over Europe actively participating in the symposium by presenting their research and exchanging ideas with their peers and world-renowned scientists.

ENABLE 2017, the First EUROPEAN PhD and Post-Doc Symposium. Session 3: In Vitro to In Vivo: Modeling Life in 3D.

The EUROPEAN ACADEMY FOR BIOMEDICAL SCIENCE (ENABLE) is an initiative funded by the European Union Horizon 2020 program involving four renowned European research institutes (Institute for Research in Biomedicine-IRB Barcelona, Spain; Radboud Institute for Molecular Life Sciences-RIMLS, the Netherlands; Novo Nordisk Foundation Center for Protein Research-NNF CPR, Denmark; European School of Molecular Medicine-SEMM, Italy) and an innovative science communication agency (Scienseed). With the aim to promote biomedical science of excellence in Europe, ENABLE organizes an annual three-day international event. This gathering includes a top-level scientific symposium bringing together leading scientists, PhD students, and post-doctoral fellows; career development activities supporting the progression of young researchers and fostering discussion about opportunities beyond the bench; outreach activities stimulating the interaction between science and society. The first European PhD and Postdoc Symposium, entitled "Breaking Down Complexity: Innovative models and techniques in biomedicine", was hosted by the vibrant city of Barcelona. The scientific program of the conference was focused on the most recent advances and applications of modern techniques and models in biomedical research and covered a wide range of topics, from synthetic biology to translational medicine. Overall, the event was a great success, with more than 200 attendees from all over Europe actively participating in the symposium by presenting their research and exchanging ideas with their peers and world-renowned scientists.

Mimicking Tumors: Toward More Predictive In Vitro Models for Peptide- and Protein-Conjugated Drugs.

Macromolecular drug candidates and nanoparticles are typically tested in 2D cancer cell culture models, which are often directly followed by in vivo animal studies. The majority of these drug candidates, however, fail in vivo. In contrast to classical small-molecule drugs, multiple barriers exist for these larger molecules that two-dimensional approaches do not recapitulate. In order to provide better mechanistic insights into the parameters controlling success and failure and due to changing ethical perspectives on animal studies, there is a growing need for in vitro models with higher physiological relevance. This need is reflected by an increased interest in 3D tumor models, which during the past decade have evolved from relatively simple tumor cell aggregates to more complex models that incorporate additional tumor characteristics as well as patient-derived material. This review will address tissue culture models that implement critical features of the physiological tumor context such as 3D structure, extracellular matrix, interstitial flow, vascular extravasation, and the use of patient material. We will focus on specific examples, relating to peptide-and protein-conjugated drugs and other nanoparticles, and discuss the added value and limitations of the respective approaches.

Intermolecular biparatopic trapping of ErbB2 prevents compensatory activation of PI3K/AKT via RAS-p110 crosstalk.

Compensatory mechanisms, such as relief of AKT-ErbB3-negative feedback, are known to desensitize ErbB2-dependent tumours to targeted therapy. Here we describe an adaptation mechanism leading to reactivation of the PI3K/AKT pathway during trastuzumab treatment, which occurs independently of ErbB3 re-phosphorylation. This signalling bypass of phospho-ErbB3 operates in ErbB2-overexpressing cells via RAS-PI3K crosstalk and is attributable to active ErbB2 homodimers. As demonstrated by dual blockade of ErbB2/RAS and ErbB3 by means of pharmacological inhibition, RNA interference or by specific protein binders obstructing the RAS-p110α interaction, both routes must be blocked to prevent reactivation of the PI3K/AKT pathway. Applying these general principles, we developed biparatopic designed ankyrin repeat proteins (DARPins) trapping ErbB2 in a dimerization-incompetent state, which entail pan-ErbB inhibition and a permanent OFF state in the oncogenic signalling, thereby triggering extensive apoptosis in ErbB2-addicted tumours. Thus, these novel insights into mechanisms underlying network robustness provide a guide for overcoming adaptation response to ErbB2/ErbB3-targeted therapy.

Efficient cell-specific uptake of binding proteins into the cytoplasm through engineered modular transport systems.

Through advances in protein scaffold engineering and selection technologies, highly specific binding proteins, which fold under reducing conditions, can be generated against virtually all targets. Despite tremendous therapeutic opportunities, intracellular applications are hindered by difficulties associated with achieving cytosolic delivery, compounded by even correctly measuring it. Here, we addressed cytosolic delivery systematically through the development of a biotin ligase-based assay that objectively quantifies cytosolic delivery in a generic fashion. We developed modular transport systems that consist of a designed ankyrin repeat protein (DARPin) for receptor targeting and a different DARPin for intracellular recognition and a bacterial toxin-derived component for cytosolic translocation. We show that both anthrax pores and the translocation domain of Pseudomonas exotoxin A (ETA) efficiently deliver DARPins into the cytosol. We found that the cargo must not exceed a threshold thermodynamic stability for anthrax pores, which can be addressed by engineering, while the ETA pathway does not appear to have this restriction.

Enhanced cellular uptake of albumin-based lyophilisomes when functionalized with cell-penetrating peptide TAT in HeLa cells.

Lyophilisomes are a novel class of biodegradable proteinaceous nano/micrometer capsules with potential use as drug delivery carrier. Cell-penetrating peptides (CPPs) including the TAT peptide have been successfully implemented for intracellular delivery of a broad variety of cargos including various nanoparticulate pharmaceutical carriers. In the present study, lyophilisomes were modified using CPPs in order to achieve enhanced cellular uptake. Lyophilisomes were prepared by a freezing, annealing, and lyophilization method and a cystein-elongated TAT peptide was conjugated to the lyophilisomes using a heterobifunctional linker. Fluorescent-activated cell sorting (FACS) was utilized to acquire a lyophilisome population with a particle diameter smaller than 1000 nm. Cultured HeLa, OVCAR-3, Caco-2 and SKOV-3 cells were exposed to unmodified lyophilisomes and TAT-conjugated lyophilisomes and examined with FACS. HeLa cells were investigated in more detail using a trypan blue quenching assay, confocal microscopy, and transmission electron microscopy. TAT-conjugation strongly increased binding and cellular uptake of lyophilisomes in a time-dependent manner in vitro, as assessed by FACS. These results were confirmed by confocal microscopy. Transmission electron microscopy indicated rapid cellular uptake of TAT-conjugated lyophilisomes via phagocytosis and/or macropinocytosis. In conclusion, TAT-peptides conjugated to albumin-based lyophilisomes are able to enhance cellular uptake of lyophilisomes in HeLa cells.

Molecular parameters of siRNA--cell penetrating peptide nanocomplexes for efficient cellular delivery.

Cell-penetrating peptides (CPPs) are versatile tools for the intracellular delivery of various biomolecules, including siRNA. Recently, CPPs were introduced that showed greatly enhanced delivery efficiency. However, the molecular basis of this increased activity is poorly understood. Here, we performed a detailed analysis of the molecular and physicochemical properties of seven different siRNA-CPP nanoparticles. In addition, we determined which complexes are internalized most efficiently into the leukemia cell-line SKNO-1, and subsequently inhibited the expression of a luciferase reporter gene. We demonstrated effective complexation of siRNA for all tested CPPs, and optimal encapsulation of the siRNA was achieved at very similar molar ratios independent of peptide charge. However, CPPs with an extreme high or low overall charge proved to be exceptions, suggesting an optimal range of charge for CPP-siRNA nanoparticle formation based on opposite charge. The most active CPP (PepFect6) displayed high serum resistance but also high sensitivity to decomplexation by polyanionic macromolecules, indicating the necessity for partial decomplexation for efficient uptake. Surprisingly, CPP-siRNA complexes acquired a negative ζ-potential in the presence of serum. These novel insights shed light on the observation that cell association is necessary but not sufficient for activity and motivate new research into the nature of the nanoparticle-cell interaction. Overall, our results provide a comprehensive molecular basis for the further development of peptide-based oligonucleotide transfection agents.

Lyophilisomes as a new generation of drug delivery capsules.

Nanoparticulate drug delivery systems are currently explored to overcome critical challenges associated with classical administration forms. In this study, we present a drug delivery system based on a novel class of proteinaceous biodegradable nano/micro capsules, lyophilisomes. Lyophilisomes can be prepared from biomolecules without the need for amphiphilicity. Albumin-based lyophilisomes were prepared by freezing, annealing and lyophilizing, resulting in capsules ranging from 100 to 3000 nm. Lyophilisomes were loaded with the anti-tumor drugs doxorubicin and curcumin using different concentrations and time/temperature regimes. Incubation in 0.1 mg/ml doxorubicin or 1.0 mg/ml curcumin resulted in an entrapment efficiency of 95±1% and 4±1%, respectively. This corresponds to a drug loading of 0.24 mg doxorubicin per milligram albumin and 0.10 mg curcumin per milligram albumin. Drug release profiles from doxorubicin and curcumin-loaded lyophilisomes were studied in culture medium and showed slow release for doxorubicin (2.7% after 72 h), and rapid release for curcumin (55% after 72 h). When applied to cells, non-loaded lyophilisomes did not influence cell viability, even at high concentrations (1 mg/ml). Lyophilisomes were internalized by cells. When loaded with doxorubicin and curcumin, lyophilisomes strongly reduced cell proliferation and viability of SKOV-3 and HeLa cells, respectively, to a level similar or better compared to an equal amount of free drugs. In conclusion, albumin lyophilisomes show potential as (nano)carriers of drugs for tumor cell elimination.