72 research outputs found
Biodegradable Poly(2-Dimethylamino Ethylamino)Phosphazene for In Vivo Gene Delivery to Tumor Cells. Effect of Polymer Molecular Weight
Purpose. Previously, we have shown that complexes of plasmid DNA with the biodegradable polymer poly(2-dimethylamino ethylamino)phosphazene (p(DMAEA)-ppz) mediated tumor selective gene expression after intravenous administration in mice. In this study, we investigated the effect of p(DMAEA)-ppz molecular weight on both in vitro and in vivo tumor transfection, as well as on complex induced toxicity. Materials and Methods. p(DMAEA)-ppz with a broad molar mass distribution was fractionated by preparative size exclusion chromatography. Polyplexes consisting of plasmid DNA and the collected polymer fractions were tested for biophysical properties, (cyto)toxicity and transfection activity. Results. Four p(DMAEA)-ppz fractions were collected with weight average molecular weights ranging from 130 to 950 kDa, and with narrow molecular mass distributions (Mw/Mn from 1.1 to 1.3). At polymer-to-DNA (N/P) ratios above 6, polyplexes based on these polymers were all positively charged (zeta potential 25–29 mV), and had a size of 80–90 nm. The in vitro cytotoxicity of the polyplexes positively correlated with polymer molecular weight. The in vitro transfection activity of the different polyplexes depended on their N/P ratio, and was affected by the degree of cytotoxicity, as well as the colloidal stability of the different polyplexes. Intravenous administration of polyplexes based on the high molecular weight polymers led to apparent toxicity, as a result of polyplex-induced erythrocyte aggregation. On the other hand, administration of polyplexes based on low molecular weight p(DMAEA)-ppz_s (Mw130 kDa) did not show signs of toxicity and resulted in tumor selective gene expression. Conclusion. Polymer molecular weight fractionation enabled us to optimize the transfection efficiency/ toxicity ratio of p(DMAEA)-ppz polyplexes for in vitro and in vivo tumor transfection. KEY WORDS: biodegradable; cationic polymer; DNA; molecular weight; tumor gene delivery
Levodopa-loaded nanoparticles for the treatment of Parkinson's disease
Parkinson's disease (PD) is a neurodegenerative disorder characterized by degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNpc) resulting in dopamine (DA) deficiency, which manifests itself in motor symptoms including tremors, rigidity and bradykinesia. Current PD treatments aim at symptom reduction through oral delivery of levodopa (L-DOPA), a precursor of DA. However, L-DOPA delivery to the brain is inefficient and increased dosages are required as the disease progresses, resulting in serious side effects like dyskinesias. To improve PD treatment efficacy and to reduce side effects, recent research focuses on the encapsulation of L-DOPA into polymeric- and lipid-based nanoparticles (NPs). These formulations can protect L-DOPA from systemic decarboxylation into DA and improve L-DOPA delivery to the central nervous system. Additionally, NPs can be modified with proteins, peptides and antibodies specifically targeting the blood-brain barrier (BBB), thereby reducing required dosages and free systemic DA. Alternative delivery approaches for NP-encapsulated L-DOPA include intravenous (IV) administration, transdermal delivery using adhesive patches and direct intranasal administration, facilitating increased therapeutic DA concentrations in the brain. This review provides an overview of the recent advances for NP-mediated L-DOPA delivery to the brain, and debates challenges and future perspectives on the field
Magnetic beads for the evaluation of drug release from biotinylated polymeric micelles in biological media
To improve the reliability of in vitro release studies of drug delivery systems, we developed a novel in vitro method for the evaluation of drug release from polymeric micelles in complex biological media. Polymeric micelles based on poly(N-2-hydroxypropyl methacrylamide)-block-poly(N-2-benzoyloxypropyl methacrylamide) (p(HPMAm)-b-p(HPMAm-Bz)) of which 10% of the chains was functionalized with biotin at the p(HPMAm) terminus were prepared using a solvent extraction method. The size of the micelles when loaded with a hydrophobic agent, namely paclitaxel (a clinically used cytostatic drug) or curcumin (a compound with multiple pharmacological activities), was around 65 nm. The biotin decoration allowed the binding of the micelles to streptavidin-coated magnetic beads which occurred within 10 min and reached a binding efficiency of 90 ± 6%. Drug release in different media was studied after the magnetic separation of micelles bound to the streptavidin-coated beads, by determination of the released drug in the media as well as the retained drug in the micellar fraction bound to the beads. The in vitro release of paclitaxel and curcumin at 37 °C in PBS, PBS containing 2% v/v Tween 80, PBS containing 4.5% w/v bovine serum albumin, mouse plasma, and whole mouse blood was highly medium-dependent. In all media studied, paclitaxel showed superior micellar retention compared to curcumin. Importantly, the presence of serum proteins accelerated the release of both paclitaxel and curcumin. The results presented in this study show great potential for predicting drug release from nanomedicines in biological media which in turn is crucial for their further pharmaceutical development
The Impact of Nanobody Density on the Targeting Efficiency of PEGylated Liposomes
Nanoparticles (NPs) are commonly modified with tumor-targeting moieties that recognize proteins overexpressed on the extracellular membrane to increase their specific interaction with target cells. Nanobodies (Nbs), the variable domain of heavy chain-only antibodies, are a robust targeting ligand due to their small size, superior stability, and strong binding affinity. For the clinical translation of targeted Nb-NPs, it is essential to understand how the number of Nbs per NP impacts the receptor recognition on cells. To study this, Nbs targeting the hepatocyte growth factor receptor (MET-Nbs) were conjugated to PEGylated liposomes at a density from 20 to 800 per liposome and their targeting efficiency was evaluated in vitro. MET-targeted liposomes (MET-TLs) associated more profoundly with MET-expressing cells than non-targeted liposomes (NTLs). MET-TLs with approximately 150-300 Nbs per liposome exhibited the highest association and specificity towards MET-expressing cells and retained their targeting capacity when pre-incubated with proteins from different sources. Furthermore, a MET-Nb density above 300 Nbs per liposome increased the interaction of MET-TLs with phagocytic cells by 2-fold in ex vivo human blood compared to NTLs. Overall, this study demonstrates that adjusting the MET-Nb density can increase the specificity of NPs towards their intended cellular target and reduce NP interaction with phagocytic cells
Negative feedback regulation of MAPK signaling is an important driver of chronic lymphocytic leukemia progression
Despite available targeted treatments for the disease, drug-resistant chronic lymphocytic leukemia (CLL) poses a clinical challenge. The objective of this study is to examine whether the dual-specific phosphatases DUSP1 and DUSP6 are required to negatively regulate mitogen-activated protein kinases (MAPKs) and thus counterbalance excessive MAPK activity. We show that high expression of DUSP6 in CLL correlates with poor clinical prognosis. Importantly, genetic deletion of the inhibitory phosphatase DUSP1 or DUSP6 and blocking DUSP1/6 function using a small-molecule inhibitor reduces CLL cell survival in vitro and in vivo. Using global phospho-proteome approaches, we observe acute activation of MAPK signaling by DUSP1/6 inhibition. This promotes accumulation of mitochondrial reactive oxygen species and, thereby, DNA damage and apoptotic cell death in CLL cells. Finally, we observe that DUSP1/6 inhibition is particularly effective against treatment-resistant CLL and therefore suggest transient DUSP1/6 inhibition as a promising treatment concept to eliminate drug-resistant CLL cells
Anti-PEG antibodies compromise the integrity of PEGylated lipid-based nanoparticles via complement
PEGylation of lipid-based nanoparticles and other nanocarriers is widely used to increase their stability and plasma half-life. However, either pre-existing or de novo formed anti-PEG antibodies can induce hypersensitivity reactions and accelerated blood clearance through binding to the nanoparticle surfaces, leading to activation of the complement system. In this study, we investigated the consequences and mechanisms of complement activation by anti-PEG antibodies interacting with different types of PEGylated lipid-based nanoparticles. By using both liposomes loaded with different (model) drugs and LNPs loaded with mRNA, we demonstrate that complement activation triggered by anti-PEG antibodies can compromise the bilayer/surface integrity, leading to premature drug release or exposure of their mRNA contents to serum proteins. Anti-PEG antibodies also can induce deposition of complement fragments onto the surface of PEGylated lipid-based nanoparticles and induce the release of fluid phase complement activation products. The role of the different complement pathways activated by lipid-based nanoparticles was studied using deficient sera and/or inhibitory antibodies. We identified a major role for the classical complement pathway in the early activation events leading to the activation of C3. Our data also confirm the essential role of amplification of C3 activation by alternative pathway components in the lysis of liposomes. Finally, the levels of pre-existing anti-PEG IgM antibodies in plasma of healthy donors correlated with the degree of complement activation (fixation and lysis) induced upon exposure to PEGylated liposomes and mRNA-LNPs. Taken together, anti-PEG antibodies trigger complement activation by PEGylated lipid-based nanoparticles, which can potentially compromise their integrity, leading to premature drug release or cargo exposure to serum proteins
Metallated phthalocyanines and their hydrophilic derivatives for multi-targeted oncological photodynamic therapy
Background and aim: A photosensitizer (PS) delivery and comprehensive tumor targeting platform was developed that is centered on the photosensitization of key pharmacological targets in solid tumors (cancer cells, tumor vascular endothelium, and cellular and non-cellular components of the tumor microenvironment) before photodynamic therapy (PDT). Interstitially targeted liposomes (ITLs) encapsulating zinc phthalocyanine (ZnPC) and aluminum phthalocyanine (AlPC) were formulated for passive targeting of the tumor microenvironment. In previous work it was established that the PEGylated ITLs were taken up by cultured cholangiocarcinoma cells. The aim of this study was to verify previous results in cancer cells and to determine whether the ITLs can also be used to photosensitize cells in the tumor microenvironment and vasculature. Following positive results, rudimentary in vitro and in vivo experiments were performed with ZnPC-ITLs and AlPC-ITLs as well as their water-soluble tetrasulfonated derivatives (ZnPCS4 and AlPCS4) to assemble a research dossier and bring this platform closer to clinical transition. Methods: Flow cytometry and confocal microscopy were employed to determine ITL uptake and PS distribution in cholangiocarcinoma (SK-ChA-1) cells, endothelial cells (HUVECs), fibroblasts (NIH-3T3), and macrophages (RAW 264.7). Uptake of ITLs by endothelial cells was verified under flow conditions in a flow chamber. Dark toxicity and PDT efficacy were determined by cell viability assays, while the mode of cell death and cell cycle arrest were assayed by flow cytometry. In vivo systemic toxicity was assessed in zebrafish and chicken embryos, whereas skin phototoxicity was determined in BALB/c nude mice. A PDT efficacy pilot was conducted in BALB/c nude mice bearing human triple-negative breast cancer (MDA-MB-231) xenografts. Results: The key findings were that (1) photodynamically active PSs (i.e., all except ZnPCS4) were able to effectively photosensitize cancer cells and non-cancerous cells; (2) following PDT, photodynamically active PSs were highly toxic-to-potent as per anti-cancer compound classification; (3) the photodynamically active PSs did not elicit notable systemic toxicity in zebrafish and chicken embryos; (4) ITL-delivered ZnPC and ZnPCS4 were associated with skin phototoxicity, while the aluminum-containing PSs did not exert detectable skin phototoxicity; and (5) ITL-delivered ZnPC and AlPC were equally effective in their tumor-killing capacity in human tumor breast cancer xenografts and superior to other non-phthalocyanine PSs when appraised on a per mole administered dose basis. Conclusions: AlPC(S4) are the safest and most effective PSs to integrate into the comprehensive tumor targeting and PS delivery platform. Pending further in vivo validation, these third-generation PSs may be used for multi-compartmental tumor photosensitization
Metallated phthalocyanines and their hydrophilic derivatives for multi-targeted oncological photodynamic therapy
Background and aim: A photosensitizer (PS) delivery and comprehensive tumor targeting platform was developed that is centered on the photosensitization of key pharmacological targets in solid tumors (cancer cells, tumor vascular endothelium, and cellular and non-cellular components of the tumor microenvironment) before photodynamic therapy (PDT). Interstitially targeted liposomes (ITLs) encapsulating zinc phthalocyanine (ZnPC) and aluminum phthalocyanine (AlPC) were formulated for passive targeting of the tumor microenvironment. In previous work it was established that the PEGylated ITLs were taken up by cultured cholangiocarcinoma cells. The aim of this study was to verify previous results in cancer cells and to determine whether the ITLs can also be used to photosensitize cells in the tumor microenvironment and vasculature. Following positive results, rudimentary in vitro and in vivo experiments were performed with ZnPC-ITLs and AlPC-ITLs as well as their water-soluble tetrasulfonated derivatives (ZnPCS4 and AlPCS4) to assemble a research dossier and bring this platform closer to clinical transition. Methods: Flow cytometry and confocal microscopy were employed to determine ITL uptake and PS distribution in cholangiocarcinoma (SK-ChA-1) cells, endothelial cells (HUVECs), fibroblasts (NIH-3T3), and macrophages (RAW 264.7). Uptake of ITLs by endothelial cells was verified under flow conditions in a flow chamber. Dark toxicity and PDT efficacy were determined by cell viability assays, while the mode of cell death and cell cycle arrest were assayed by flow cytometry. In vivo systemic toxicity was assessed in zebrafish and chicken embryos, whereas skin phototoxicity was determined in BALB/c nude mice. A PDT efficacy pilot was conducted in BALB/c nude mice bearing human triple-negative breast cancer (MDA-MB-231) xenografts. Results: The key findings were that (1) photodynamically active PSs (i.e., all except ZnPCS4) were able to effectively photosensitize cancer cells and non-cancerous cells; (2) following PDT, photodynamically active PSs were highly toxic-to-potent as per anti-cancer compound classification; (3) the photodynamically active PSs did not elicit notable systemic toxicity in zebrafish and chicken embryos; (4) ITL-delivered ZnPC and ZnPCS4 were associated with skin phototoxicity, while the aluminum-containing PSs did not exert detectable skin phototoxicity; and (5) ITL-delivered ZnPC and AlPC were equally effective in their tumor-killing capacity in human tumor breast cancer xenografts and superior to other non-phthalocyanine PSs when appraised on a per mole administered dose basis. Conclusions: AlPC(S4) are the safest and most effective PSs to integrate into the comprehensive tumor targeting and PS delivery platform. Pending further in vivo validation, these third-generation PSs may be used for multi-compartmental tumor photosensitization
Red Blood Cells : Chasing Interactions
Human red blood cells (RBC) are highly differentiated cells that have lost all organelles and most intracellular machineries during their maturation process. RBC are fundamental for the nearly all basic physiologic dynamics and they are key cells in the body's respiratory system by being responsible for the oxygen transport to all cells and tissues, and delivery of carbon dioxide to the lungs. With their flexible structure RBC are capable to deform in order to travel through all blood vessels including very small capillaries. Throughout their in average 120 days lifespan, human RBC travel in the bloodstream and come in contact with a broad range of different cell types. In fact, RBC are able to interact and communicate with endothelial cells (ECs), platelets, macrophages, and bacteria. Additionally, they are involved in the maintenance of thrombosis and hemostasis and play an important role in the immune response against pathogens. To clarify the mechanisms of interaction of RBC and these other cells both in health and disease as well as to highlight the role of important key players, we focused our interest on RBC membrane components such as ion channels, proteins, and phospholipids
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