Ladan Parhamifar

Particulate Systems for Targeting of Macrophages: Basic and Therapeutic Concepts

Particulate systems in the form of liposomes, polymeric micelles, polymeric nano- and microparticles, and many others offer a rational approach for selective delivery of therapeutic agents to the macrophage from different physiological portals of entry. Particulate targeting of macrophages and intracellular drug release processes can be optimized through modifications of the drug carrier physicochemical properties, which include hydrodynamic size, shape, composition and surface characteristics. Through such modifications together with understanding of macrophage cell biology, targeting may be aimed at a particular subset of macrophages. Advances in basic and therapeutic concepts of particulate targeting of macrophages and related nanotechnology approaches for immune cell modifications are discussed.

Combined MUC1-specific nanobody-tagged PEG-polyethylenimine polyplex targeting and transcriptional targeting of tBid transgene for directed killing of MUC1 over-expressing tumour cells

We provide evidence for combining a single domain antibody (nanobody)-based targeting approach with transcriptional targeting as a safe way to deliver lethal transgenes to MUC1 over-expressing cancer cells. From a nanobody immune library, we have isolated an anti-DF3/Mucin1 (MUC1) nanobody with high specificity for the MUC1 antigen, which is an aberrantly glycosylated glycoprotein over-expressed in tumours of epithelial origin. The anti-MUC1 nanobody was covalently linked to the distal end of poly(ethylene glycol)(3500) (PEG(3500)) in PEG(3500)-25kDa polyethylenimine (PEI) conjugates and the resultant macromolecular entity successfully condensed plasmids coding a transcriptionally targeted truncated-Bid (tBid) killer gene under the control of the cancer-specific MUC1 promoter. The engineered polyplexes exhibited favourable physicochemical characteristics for transfection and dramatically elevated the level of Bid/tBid expression in both MUC1 over-expressing caspase 3-deficient (MCF7 cells) and caspase 3-positive (T47D and SKBR3) tumour cell lines and, concomitantly, induced considerable cell death. Neither transgene expression nor cell death occurred when the MUC1 promoter was replaced with the CNS-specific synapsin I promoter. Since PEGylated PEI was only responsible for DNA compaction and played no significant role in direct transfection and cell killing, our attempts overcome previously reported PEI-mediated apoptotic and necrotic cell death, which is advantageous for future in vivo transcriptional targeting as this will minimize (or eliminate) non-targeted cell damage.

Polyethylenimine-mediated impairment of mitochondrial membrane potential, respiration and membrane integrity: implications for nucleic acid delivery and gene therapy.

The 25 kDa branched polyethylenimine (PEI) is a highly efficient synthetic polycation used in transfection protocols, but also triggers mitochondrial-mediated apoptotic cell death processes where the mechanistic issues are poorly understood. We now demonstrate that PEI in a concentration- and time-dependent manner can affect functions (membrane potential, swelling and respiration) and ultrastructural integrity of freshly isolated rat liver mitochondria. The threshold concentration for detection of PEI-mediated impairment of rat liver mitochondrial functions is 3 μg/mL, however, lower PEI levels still exert some effects on mitochondrial morphology and respiration, and these may be related to the inherent membrane perturbing properties of this polycation. The PEI-mediated mitochondrial swelling phase is biphasic, with a fast decaying initial period (most prominent from 4 μg/mL PEI) followed by a slower, linear swelling response. The slow phase is presumably the result of a time-dependent transition permeability opening in mitochondria initially resistant to swelling/depolarization, but may further be related to PEI-induced nanoscale structural defects and/or formation of pores in the outer membrane. Respiration assessments further suggested that PEI in the presence of exogenous ADP behaves in a similar fashion to a slow-acting inhibitory compound. PEI further shows an uncoupling property that is detectable at low respiration rates. The relevance of these findings to PEI-mediated initiation of intrinsic apoptotic pathway is discussed.

Poly(3-hydroxybutyrate- co -R-3-hydroxyhexanoate) Nanoparticles with Polyethylenimine Coat as Simple, Safe, and Versatile Vehicles for Cell Targeting: Population Characteristics, Cell Uptake, and Intracellular Trafficking

A simple and highly safe poly(3-hydroxybutyrate-co-R-3-hydroxyhexanoate) nanoparticulate delivery system that targets different cell types is developed. A sub-cytotoxic level of polyethylenimine coat mediates universal cell targeting. Internalized nanoparticles traffic along endolysosomal compartments, endoplasmic reticulum and the Golgi complex. Nanoparticles have no detrimental effects on cell morphology and respiration.

Lactate dehydrogenase assay for assessment of polycation cytotoxicity.

Cellular toxicity and/or cell death entail complex mechanisms that require detailed evaluation for proper characterization. A detailed mechanistic assessment of cytotoxicity is essential for design and construction of more effective polycations for nucleic acid delivery. A single toxicity assay cannot stand alone in determining the type and extent of damage or cell death mechanism. In this chapter we describe a lactate dehydrogenase (LDH) assay for high-throughput screening that can be used as a starting point for further detailed cytotoxicity determination. LDH release is considered an early event in necrosis but a late event in apoptosis. An accurate temporal assessment of the toxic responses is crucial as late apoptosis may convert into necrosis as well as in situations where cell death is initiated without any visible cell morphological changes or responses in assays measuring late events, resulting in early ongoing toxicity being overlooked.

Polyethylenimine architecture-dependent metabolic imprints and perturbation of cellular redox homeostasis.

Polyethylenimines (PEIs) are among the most efficient polycationic non-viral transfectants. PEI architecture and size not only modulate transfection efficiency, but also cytotoxicity. However, the underlying mechanisms of PEI-induced multifaceted cell damage and death are largely unknown. Here, we demonstrate that the central mechanisms of PEI architecture- and size-dependent perturbations of integrated cellular metabolomics involve destabilization of plasma membrane and mitochondrial membranes with consequences on mitochondrial oxidative phosphorylation (OXPHOS), glycolytic flux and redox homeostasis that ultimately modulate cell death. In comparison to linear PEI, the branched architectures induced greater plasma membrane destabilization and were more detrimental to glycolytic activity and OXPHOS capacity as well as being a more potent inhibitor of the cytochrome c oxidase. Accordingly, the branched architectures caused a greater lactate dehydrogenase (LDH) and ATP depletion, activated AMP kinase (AMPK) and disturbed redox homeostasis through diminished availability of nicotinamide adenine dinucleotide phosphate (NADPH), reduced antioxidant capacity of glutathione (GSH) and increased burden of reactive oxygen species (ROS). The differences in metabolic and redox imprints were further reflected in the transfection performance of the polycations, but co-treatment with the GSH precursor N-acetyl-cysteine (NAC) counteracted redox dysregulation and increased the number of viable transfected cells. Integrated biomembrane integrity and metabolomic analysis provides a rapid approach for mechanistic understanding of multifactorial polycation-mediated cytotoxicity, and could form the basis for combinatorial throughput platforms for improved design and selection of safer polymeric vectors.

T cells expressing VHH-directed oligoclonal chimeric HER2 antigen receptors: Towards tumor-directed oligoclonal T cell therapy

BACKGROUND Adoptive cell therapy with engineered T cells expressing chimeric antigen receptors (CARs) originated from antibodies is a promising strategy in cancer immunotherapy. Several unsuccessful trials, however, highlight the need for alternative conventional binding domains and the better combination of costimulatory endodomains for CAR construction to improve the effector functions of the engineered T cells. Camelid single-domain antibodies (VHHs), which are the smallest single domain antibodies, can endow great targeting ability to CAR-engineered T cells. METHODS We have developed a method to generate genetically engineered Jurkat T cells armed with a CAR comprising the anti-HER2 VHH as targeting moiety. From an immune camel library, five VHH clones were selected as a set of oligoclonal anti-HER2 VHHs that exhibited diverse binding abilities and joined them to CD28-CD3ζ and CD28-OX40-CD3ζ signaling endodomains. Jurkat T cells expression of VHH-CARs and cell functions were evaluated. RESULTS The oligoclonal engineered T cells showed higher proliferation, cytokine secretion and cytotoxicity than each individual VHH-CAR-engineered Jurkat T cells. CONCLUSIONS The combination of superior targeting ability of oligoclonal VHHs with the third generation CAR can substantially improve the function of engineered T cells. GENERAL SIGNIFICANCE Antigen-specific directed oligoclonal T cells are alternatively promising, but safer systems, to combat tumor cells.

Polycation-Mediated Integrated Cell Death Processes

One of the major challenges in the field of nucleic acid delivery is the design of delivery vehicles with attributes that render them safe as well as efficient in transfection. To this end, polycationic vectors have been intensely investigated with native polyethylenimines (PEIs) being the gold standard. PEIs are highly efficient transfectants, but depending on their architecture and size they induce cytotoxicity through different modes of cell death pathways. Here, we briefly review dynamic and integrated cell death processes and pathways, and discuss considerations in cell death assay design and their interpretation in relation to PEIs and PEI-based engineered vectors, which are also translatable for the design and studying the safety of other transfectants.

Differential Modulation of Cellular Bioenergetics by Poly(l-lysine)s of Different Molecular Weights

Poly(L-lysine)s (PLLs), and related derivatives, have received considerable attention as nonviral vectors. High molecular weight PLLs (H-PLLs) are superior transfectants compared with low Mw PLLs (L-PLLs), but suggested to be more cytotoxic. Through a pan-integrated metabolomic approach using Seahorse XF technology, we studied the impact of PLL size on cellular bioenergetic processes in two human cell lines. In contrast to L-PLLs (1-5 kDa), H-PLLs (15-30 kDa) were more detrimental to both mitochondrial oxidative phosphorylation (OXPHOS) and glycolytic activity resulting in considerable intracellular ATP depletion, thereby initiating necrotic-type cell death. The cellular differences to polycation sensitivity were further related to the mitochondrial state, where the impact was substantial on cells with hyperpolarized mitochondria. These medium-throughput approaches offer better opportunities for understanding inter-related intracellular and cell type-dependent processes instigating a bioenergetics crisis, thus, aiding selection (from available libraries) and improved design of safer biodegradable polycations for nucleic acid compaction and cell type-specific delivery.

AFM visualization of sub-50 nm polyplex disposition to the nuclear pore complex without compromising the integrity of the nuclear envelope

It has been questioned as to whether polyplexes in the cytoplasm can reach the nuclear compartment and if so in what form. By applying atomic force microscopy (AFM) to the nuclear envelope and the nuclear pore complexes, we demonstrate that disposition of polyethylenimine (PEI)/DNA polyplexes that were microinjected into the oocytes of Xenopus laevis, as an example of a non-dividing cell, is exclusive to the nuclear pore complex (NPC). AFM images show NPCs clogged only with sub-50nm polyplexes. This mode of disposition neither altered the morphology/integrity of the nuclear membrane nor the NPC. AFM images further show polyplexes on the nucleoplasmic side of the envelope, presumably indicating species in transit. Transmission electron microscopy studies of ruptured nuclei from transfected human cell lines demonstrate the presence of sub-50nm particles resembling polyplexes in morphology compared with control preparations.