Pancreatic adenocarcinoma (PDAC) is highly resistant to conventional chemotherapeutic interventions, resulting in exceptionally low survival rates. The limited efficacy can in part be attributed to... Show morePancreatic adenocarcinoma (PDAC) is highly resistant to conventional chemotherapeutic interventions, resulting in exceptionally low survival rates. The limited efficacy can in part be attributed to dose limitations and treatment cessation urged by toxicity of currently used chemotherapy. The advent of targeted delivery strategies has kindled hope for circumventing off-target toxicity. We have previously reported a PDAC-specific mesoporous silica nanoparticle (MSN) containing a protease linker responsive to ADAM9, a PDAC-enriched extracellularly deposited protease. Upon loading with paclitaxel these ADAM9-MSNs reduced side effects both in vitro and in vivo, however, disappointing antitumor efficacy was observed in vivo. Here, we propose that an efficient uptake of MSNs by tumor cells might underlie the lack of antitumor efficacy of MSNs functionalized with linker responsive to extracellular proteases. Harnessing this premise to improve antitumor efficacy, we performed an in silico analysis to identify PDAC-enriched intracellular proteases. We report the identification of BACE2, CAPN2 and DPP3 as PDAC enriched intracellular proteases, and report the synthesis of BACE2-, CAPN2- and DPP3- responsive MSNs. Extensive preclinical assessments revealed that paclitaxel-loaded CAPN2- and DPP3-MSNs exhibit high PDAC specificity in vitro as opposed to free paclitaxel. The administration of paclitaxel-loaded CAPN2- and DPP3-MSNs in vivo confirmed the reduction of leukopenia and induced no organ damage. Promisingly, in two mouse models CAPN2-MSNs reduced tumor growth at least as efficiently as free paclitaxel. Taken together, our results pose CAPN2-MSNs as a promising nanocarrier for the targeted delivery of chemotherapeutics in PDAC. Show less
Bi, D.; Hal, A. van; Aschmann, D.; Shen, M.; Zhang, H.; Su, L.; ... ; Bussmann, J. 2024
Improving target versus off-target ratio in nanomedicine remains a major challenge for increasing drug bioavailability and reducing toxicity. Active targeting using ligands on nanoparticle surfaces... Show moreImproving target versus off-target ratio in nanomedicine remains a major challenge for increasing drug bioavailability and reducing toxicity. Active targeting using ligands on nanoparticle surfaces is a key approach but has limited clinical success. A potential issue is the integration of targeting ligands also changes the physicochemical properties of nanoparticles (passive targeting). Direct studies to understand the mechanisms of active targeting and off-targeting in vivo are limited by the lack of suitable tools. Here, the biodistribution of a representative active targeting liposome is analyzed, modified with an apolipoprotein E (ApoE) peptide that binds to the low-density lipoprotein receptor (LDLR), using zebrafish embryos. The ApoE liposomes demonstrated the expected liver targeting effect but also accumulated in the kidney glomerulus. The ldlra−/- zebrafish is developed to explore the LDLR-specificity of ApoE liposomes. Interestingly, liver targeting depends on the LDLR-specific interaction, while glomerular accumulation is independent of LDLR and peptide sequence. It is found that cationic charges of peptides and the size of liposomes govern glomerular targeting. Increasing the size of ApoE liposomes can avoid this off-targeting. Taken together, the study shows the potential of the zebrafish embryo model for understanding active and passive targeting mechanisms, that can be used to optimize the design of nanoparticles. Show less
Strien, J. van; Makurat, D.M.M.; Zeng, Y.; Olsthoorn, R.R.C.L.; Schneider, G.F.; Slutter, B.A.; ... ; Kros, A. 2024
Ionizable cationic lipids (ICLs) play an essential role in the effectiveness of lipid nanoparticles (LNPs) for delivery of mRNA therapeutics and vaccines; therefore, critical evaluations of their... Show moreIonizable cationic lipids (ICLs) play an essential role in the effectiveness of lipid nanoparticles (LNPs) for delivery of mRNA therapeutics and vaccines; therefore, critical evaluations of their biological performance would extend the existing knowledge in the field. In the present study, we examined the effects of the three clinically-approved ICLs, Dlin-MC3-DMA, ALC-0315 and SM-102, as well as DODAP, on the in vitro and in vivo performance of LNPs for mRNA delivery and vaccine efficacy. mRNA-LNPs containing these lipids were successfully prepared, which were all found to be very similar in their physicochemical properties and mRNA encapsulation efficiencies. Furthermore, the results of the in vitro studies indicated that these mRNA-LNPs were efficiently taken up by immortalized and primary immune cells with comparable efficiency; however, SM-102-based LNPs were superior in inducing protein expression and antigen-specific T cell proliferation. In contrast, in vivo studies revealed that LNPs containing ALC-0315 and SM-102 yielded almost identical protein expression levels in zebrafish embryos, which were significantly higher than Dlin-MC3-DMA-based LNPs. Additionally, a mouse immunization study demonstrated that a single-dose subcutaneous administration of the mRNA-LNPs resulted in a high production of intracellular cytokines by antigen-specific T cells, but no significant differences among the three clinically approved ICLs were observed, suggesting a weak correlation between in vitro and in vivo outcomes. This study provides strong evidence that ICLs modulate the performance of mRNA-LNPs and that in vitro data does not adequately predict their behavior in vivo. Show less
Small interfering RNA (siRNA) therapeutics have shown tremendous potential for the treatment of a range of diseases, but there is still a need for novel siRNA delivery materials. Here, we introduce... Show moreSmall interfering RNA (siRNA) therapeutics have shown tremendous potential for the treatment of a range of diseases, but there is still a need for novel siRNA delivery materials. Here, we introduce M12L24 cages as siRNA delivery agents. We used four functionalized build-ing blocks to form M12L24 nanocages. Dynamic light scattering showed that well-defined 130 nm nanocage/siRNA assemblies formed with positive zeta potentials. Cell-specific siRNA-mediated green fluorescent protein (GFP) silencing, controlled by the metal used for nanocage formation, was obtained. A Pt12L24 nanocage was highly effective in delivering siRNA to U2OS cells but showed little efficiency for HeLa cells. The less stable Pd12L24 nanocage derived from the same building block displayed effective GFP silencing for HeLa cells but not for U2OS cells. The ease of prepara-tion and the ability to tune the binding strength, together with the specific siRNA delivery efficiency depending on the building blocks and metals, show potential for future siRNA delivery applications. Show less
There is an increasing interest in the application of metal-organic cages (MOCs) in a biomedicinal context, as they can offer non-classical distribution in organisms compared to molecular... Show moreThere is an increasing interest in the application of metal-organic cages (MOCs) in a biomedicinal context, as they can offer non-classical distribution in organisms compared to molecular substrates, while revealing novel cytotoxicity mechanisms. Unfortunately, many MOCs are not sufficiently stable under in vivo conditions, making it difficult to study their structure-activity relationships in living cells. As such, it is currently unclear whether MOC cytotoxicity stems from supramolecular features or their decomposition products. Herein, we describe the toxicity and photophysical properties of highly-stable rhodamine functionalized platinum-based Pt2L4 nanospheres as well as their building blocks under in vitro and in vivo conditions. We show that in both zebrafish and human cancer cell lines, the Pt2L4 nanospheres demonstrate reduced cytotoxicity and altered biodistribution within the body of zebrafish embryos compared to the building blocks. We anticipate that the composition-dependent biodistribution of Pt2L4 spheres together with their cytotoxic and photophysical properties provides the fundament for MOC application in cancer therapy. Show less
Zeng, Y.; Shen, M.; Singhal, A.; Sevink, G.J.A.; Crone, N.S.A.; Boyle, A.L.; Kros, A. 2023
Self-assembling molecular drugs combine the easy preparation typical of small-molecule chemotherapy and the tumour-targeting properties of drug-nanoparticle conjugates. However, they require a... Show moreSelf-assembling molecular drugs combine the easy preparation typical of small-molecule chemotherapy and the tumour-targeting properties of drug-nanoparticle conjugates. However, they require a supramolecular interaction that survives the complex environment of a living animal. Here we report that the metallophilic interaction between cyclometalated palladium complexes generates supramolecular nanostructures in living mice that have a long circulation time (over 12 h) and efficient tumour accumulation rate (up to 10.2% of the injected dose per gram) in a skin melanoma tumour model. Green light activation leads to efficient tumour destruction due to the type I photodynamic effect generated by the self-assembled palladium complexes, as demonstrated in vitro by an up to 96-fold cytotoxicity increase upon irradiation. This work demonstrates that metallophilic interactions are well suited to generating stable supramolecular nanotherapeutics in vivo with exceptional tumour-targeting properties. Show less
Membrane fusion is an essential part of the proper functioning of life. As such it is not only important that organisms carefully regulate the process, but also that it is well understood. One way... Show moreMembrane fusion is an essential part of the proper functioning of life. As such it is not only important that organisms carefully regulate the process, but also that it is well understood. One way to facilitate and study membrane fusion is to use artificial, minimalist, fusion peptides. In this study the efficiency and kinetics of two fusion peptides, denoted CPE and CPK, were studied using single-particle TIRF microscopy. CPE and CPK are helical peptides which interact with each other, forming a coiled-coil motif. The peptides can be inserted into a lipid membrane using a lipid anchor, and if these peptides are anchored in opposing lipid membranes, then the coiled-coil interaction can provide the mechanical force necessary to overcome the energy barrier to initiate fusion, much in the same way the SNARE complex does. In this study we find that the fusogenic facilitation of CPE and CPK in liposomes is, at least partially, dependent on the size of the particle. In addition, under certain fusogenic conditions such as when using small liposomes of similar to 60 nm in diameter, CPK alone is enough to facilitate membrane fusion in both bulk and single-particle studies. We show this using bulk lipid mixing assays utilizing FRET and single-particle TIRF, making use of dequenching fluorophores to indicate fusion. This provides us with new insights into the mechanisms of peptide-mediated membrane fusion and illuminates both challenges as well as opportunities when designing drug delivery systems. Show less
The induction of a potent T cell response is essential for successful tumor immunotherapy and protection against many infectious diseases. In the past few years, mRNA vaccines have emerged as... Show moreThe induction of a potent T cell response is essential for successful tumor immunotherapy and protection against many infectious diseases. In the past few years, mRNA vaccines have emerged as potent immune activators and inducers of a robust T cell immune response. The recent approval of the Moderna and the Pfizer/BioNTech vaccines based on lipid nanoparticles (LNP) encapsulating antigen-encoding mRNA has revolutionized the field of vaccines. The advantages of LNPs are their ease of design and formulation resulting in potent, effective, and safe vaccines. However, there is still plenty of room for improvement with respect to LNP efficacy, for instance, by optimizing the lipid composition and tuning LNP for specific purposes. mRNA delivery is known to be strongly dependent on the lipid composition of LNPs and the efficiency is mainly determined by the ionizable lipids. Besides that, cholesterol and helper lipids also play important roles in mRNA transfection potency. Here, a panel of LNP formulations was studied by keeping the ionizable lipids constant, replacing cholesterol with β-sitosterol, and changing the fusogenic helper lipid DOPE content. We studied the ability of this LNP library to induce antigen presentation and T cell proliferation to identify superior LNP candidates eliciting potent T cell immune responses. We hypothesize that using β-sitosterol and increasing DOPE content would boost the mRNA transfection on immune cells and result in enhanced immune responses. Transfection of immortal immune cell lines and bone marrow dendritic cells (BMDCs) with LNPs was studied. Delivery of mRNA coding for the model antigen ovalbumin (OVA-mRNA) to BMDCs with a number of LNP formulations, resulted in a high level of activation, as evidenced by the upregulation of the co-stimulatory receptors (CD40 and CD86) and IL-12 in BMDCs. The enhancement of BMDC activation and T cell proliferation induced by the introduction of β-sitosterol and fusogenic DOPE lipids were cell dependent. Four LNP formulations (C12-200-cho-10%DOPE, C12-200-sito-10%DOPE, cKK-E12-cho-10%DOPE and cKK-E12-sito-30%DOPE) were identified that induced robust T cell proliferation and enhanced IFN-γ, TNF-α, IL-2 expression. These results demonstrate that T cell proliferation is strongly dependent on LNP composition and promising LNP-mRNA vaccine formulations were identified. Show less
Crone, N.S. A.; Hilten, N. van; Ham, A. van der; Risselada. H.J.; Kros, A.; Boyle, A.L. 2023
Plasma lipid transport and metabolism are essential to ensure correct cellular function throughout the body. Dynamically regulated in time and space, the well-characterized mechanisms underpinning... Show morePlasma lipid transport and metabolism are essential to ensure correct cellular function throughout the body. Dynamically regulated in time and space, the well-characterized mechanisms underpinning plasma lipid transport and metabolism offers an enticing, but as yet underexplored, rationale to design synthetic lipid nanoparticles with inherent cell/tissue selectivity. Herein, a systemically administered liposome formulation, composed of just two lipids, that is capable of hijacking a triglyceride lipase-mediated lipid transport pathway resulting in liposome recognition and uptake within specific endothelial cell subsets is described. In the absence of targeting ligands, liposome-lipase interactions are mediated by a unique, phase-separated ("parachute") liposome morphology. Within the embryonic zebrafish, selective liposome accumulation is observed at the developing blood-brain barrier. In mice, extensive liposome accumulation within the liver and spleen - which is reduced, but not eliminated, following small molecule lipase inhibition - supports a role for endothelial lipase but highlights these liposomes are also subject to significant "off-target" by reticuloendothelial system organs. Overall, these compositionally simplistic liposomes offer new insights into the discovery and design of lipid-based nanoparticles that can exploit endogenous lipid transport and metabolism pathways to achieve cell selective targeting in vivo. Show less
Warmenhoven, H.; Leboux, R.J.T.; Bethanis, A.; Strien, J. van; Logiantara, A.; Schijndel, H. van; ... ; Ree, R. van 2023
Although aluminum hydroxide (alum) is widely accepted and used as safe vaccine adjuvant, there is some concern about possible toxicity upon long-lasting repeated exposure during subcutaneous... Show moreAlthough aluminum hydroxide (alum) is widely accepted and used as safe vaccine adjuvant, there is some concern about possible toxicity upon long-lasting repeated exposure during subcutaneous allergen immunotherapy (SCIT). Our objective was to evaluate allergen-bearing liposomes as possible alternative for alum-adsorption in SCIT. A self-assembling, coiled-coil forming peptide pair was used to anchor the major birch pollen allergen Bet v 1 to the surface of cationic liposomes. The resulting nanoparticulate liposomes were characterized with respect to their physicochemical, allergenic and immunological properties. Allergenicity was studied by ImmunoCAP inhibition and rat basophil leukemia (RBL) cell assays. Immunogenicity (immunoglobulin responses) and immune skewing (cytokine responses) were evaluated upon immunization of naïve mice, and compared to alum-adsorbed Bet v 1. Bet v 1-bearing cationic liposomes with a diameter of ∼200 nm showed a positive zeta potential. The coiled-coil conjugation of Bet v 1 to the surface of liposomes resulted in about a 15-fold lower allergenicity than soluble Bet v 1 as judged by RBL assays. Moreover, the nanoparticles induced Bet v 1-specific IgG1/IgG2a responses in mice that were several orders of magnitude higher than those induced by alum-adsorbed Bet v 1. This strong humoral response was accompanied by a relatively strong IL-10 induction upon PBMC stimulation with Bet v 1. In conclusion, their hypo-allergenic properties, combined with their capacity to induce a strong humoral immune response and a relatively strong IL-10 production, makes these allergen-covered cationic liposomes a promising alternative for aluminum salt-adsorption of allergen currently used in SCIT. Show less
