Development of Shell-Crosslinked Knedel-like Nanoparticles as Intracellular Delivery Vehicles and Gene Regulation Agents

This dissertation focuses on the development of polymer-based nanomaterials, termed shell-crosslinked knedel-like: SCK) nanoparticles, as vehicles to carry specific guest molecules or guest structures into the cell. Detailed synthetic procedures for and characterization of well-defined block copolymers, as well as the nanostructures resulted from their self-assembly are reported. The nanoparticles exhibited different but controlled sizes and shapes, depending on the conditions for their preparation. To incorporate functionality into these materials, both pre- and post-particle functionalization methods, as well as their combination, were used. The nanostructures involved in this dissertation include protein transduction domain: PTD)-functionalized SCK, folate-functionalized SCK and cationic SCK: cSCK). Biological evaluation of each type of nanoparticles is described. For the PTD- and the folate-SCKs, a particle shape/size dependence on their ability to undergo cell uptake was found, although their trends were opposite. The cSCKs were designed to bear primary amines, which rendered the nanoparticles a positively charged character in solution that was utilized to condense and protect DNA. The cSCKs were shown to be highly effective in transporting DNA into the cell to allow the DNA to function. Peptide nucleic acids: PNAs) were also effectively transported into the cell by covalent conjugation to or electrostatically complexation with the cSCKs. Finally, the cSCKs were shown to be able to form hierarchical nanoscale structures with anionc, cylindrical SCKs, and transport the cylinders into the cell

Rhodamine-based sensor for real-time imaging of mitochondrial ATP in living fibroblasts

Mitochondria are essential for the production and maintenance of ATP in the eukaryotic cell. To image and monitor intracellular ATP level without cell breakage, biological and chemical sensors were developed in the last years. Here, we have internalized a rhodamine-based sensor RSL+ into living cells and monitored the mitochondrial ATP levels in cultured mouse embryonic fibroblasts. To evaluate the robustness of the sensor we imaged the changes of the mitochondrial ATP levels under non-physiological conditions upon incubation with FCCP, oligomycin, azide, deoxyglucose or phosphoenolpyruvate; all compounds that interfere with ATP homeostasis of the cell. The ATP sensor allowed us to determine the mitochondrial ATP levels in human skin fibroblasts where we observe a similar amount of ATP compared to mouse embryonic fibroblasts. We propose the RSL+ to be a valuable tool for the assessment of mitochondrial dysfunction in human cells derived from mitochondrial OXPHOS patients and for basic studies on bioenergetics metabolism

Homeotic transformation induced by protein transduction

One of the most fundamental features of living organisms is that cells are separated from their external environment by a thin, but highly complex plasma membrane constituted of a lipid bilayer. Although, the lipid bilayer is only a few nanometers in width, it is impermeable to most molecules apart from small hydrophobic ones. The ability of small molecules to diffuse through a lipid bilayer is related to their lipid solubility. Hydrophilic macromolecular Antennapedia homeodomain peptide has been shown to be able to translocate from extracellular space into the cytoplasm of cells in a receptor-independent manner. Its third α-helix domain, designated as “Penetratin”, was proposed to be the functional transduction domain that is responsible for the translocation, and it is widely used for intracellular delivery of various exogenous proteins. Although Penetratin has been regarded to be the only element conferring the capacity of its parent polypeptide to penetrate through the plasma membrane, we found that the complete Antennapedia homeodomain exhibits an appreciably higher level of translocation efficiency as compared to Penetratin. Pharmacological analysis demonstrated that macropinocytic endocytosis plays a significant role underlying the process of the homeodomain internalization, and this is consistent with the observation that internalized polypeptide co-localizes with a fluid phase dye. Our studies identify macropinocytosis as a major mechanism by which Antennapedia homeodomain obtains the access to the interior of cells. In the process of macropinocytosis, signaling from the plasma membrane is required for actin remodeling to generate mechanical deformation forces; the interaction between positively charged Antennapedia homeodomain and negatively charged extracellular heparan sulfate could trigger the signaling cascade for fluid phase endocytosis. This would presumably explain why positively charged peptides, polymers, and liposomes are able to penetrate cells. As a fluid phase macropinocytosis provides cells with a way to non-selectively internalize large quantities of solute, it represents an effective means for drug delivery into cells. Both of “Penetratin” and Antennapedia homeodomain exploit macropinocytosis to a certain extent, the comparison between them may advance our understanding of the mechanisms triggering macropinocytotic endocytosis

Development of a secreted cell-permeable NF-κB inhibitor to control inflammation

PhDRheumatoid arthritis (RA) is an autoimmune chronic inflammatory disease, of unknown aetiology. Several disease-modulating approaches have been developed in the past years, however these are expensive, usually accompanied by unwanted side-effects and 30% of the patients fail to respond. The transcription factor NF-kB is a key factor in the development and perpetuation of the disease, as it regulates a number of inflammatory genes. The activity of certain signalling pathways can be modulated by delivering into cells inhibitors coupled to Protein Transduction Domains (PTDs). The aim of this study was to develop a secretable PTD-fusion NF-κΒ inhibitor that is produced and secreted by genetically engineered mammalian cells in sufficient amounts to subsequently transduce and regulate NF-κΒ activity in neighbouring cells. Such methodology could be useful for the management of RA by transplantation of engineered cells or directly using gene delivery into the synovial joints. In this study, PTD-fusion proteins were fused to the IL-2 secretion signal and their ability to be secreted from mammalian cells was explored. Secretable forms of TAT-IgG2A and TAT-eGFP were generated as control PTD-fusion proteins, and the TAT-srIκΒα (super repressor IκBα, a NF-κΒ inhibitor) was generated as an NF-κΒ PTD-fusion inhibitor. Western blotting analysis of supernatants from transiently transfected 293T cells revealed that TAT-IgG2A, TAT-eGFP and TAT-srIκΒα are secreted with variable efficiencies. When concentrated, PTD proteins were able to transduce mammalian cells, as demonstrated with Jurkat cells by confocal microscopy and western blotting analysis. The TAT PTD domain was replaced to a more stable, furin cleavage-resistant and less positively charged PTD domain, the TAT3 PTD domain, to ensure that PTDfusion proteins will be secreted more efficiently. This change of the PTD domain did not increase secretion levels of the srΙκBα. Subsequently, the Latent Associated Peptide (LAP) of TGFβ, was fused to the TAT3-srIκΒα inhibitor, via a matrix metalloproteinase (MMP) cleavage linker. This LAP-MMP-PTD-fusion NF-κΒ inhibitor was again poorly secreted. In turn, the srIκΒα inhibitor was replaced with a small synthetic NF-κΒ inhibitor, termed Nemo Binding Domain (NBD), in the form of LAP MMP-TAT3-NBD NF-κΒ inhibitor. Western blotting analysis of supernatants from transiently transfected 293T cells revealed that the LAP-MMP-TAT3-NBD was efficiently secreted. The ability of LAP-MMP-TAT3-NBD to inhibit NF-κΒ was tested in vitro with the use of a cell-assay based on HeLa cells that are permanently transfected with the luciferase gene driven by an NF-κB regulated promoter. In this assay, HeLa cells that were treated with the secreted LAP-MMP-TAT3-NBD, showed reduced levels of luciferase activity after IL-1β stimulation. Subsequently, using a replication-deficient lentiviral vector, genetically engeneered DBA/1 fibroblasts (DTF) able to produce the secreted LAP-MMP-TAT3-NBD were generated. The NF-κB inhibitory properties of the secreted LAP-MMP-TAT3-NBD were tested in vivo in the Carrageenan-induced paw oedema, Antigen Induced Arthritis and Air-Pouch acute inflammation models. Paws of mice that were treated with engineered cells or lentivirus encoding LAPMMP- TAT3-NBD demonstrated milder paw swelling, suggesting that LAP-MMPTAT3- NBD had a protective role in the induction of inflammation. However, the LAPMMP- TAT3-NBD did not demonstrate anti-inflammatory effects in the Air-Pouch model. In this study, I present a method to design PTD-fusion proteins that can be efficiently secreted from mammalian cells and I demonstrate a novel gene therapy approach for the local delivery of a therapeutic agent

Identifying and Characterizing Molecular Parameters that Modulate the Endosomal Escape of Cationic Cell- Penetrating Peptides: A Structure Activity Approach

For over 20 years, cell-penetrating peptides (CPPs) have been used as delivery vectors transporting macromolecules (cargos) into live cells for cell biology manipulations and therapeutic applications. While the exact mechanism of cell penetration remains controversial, it is thought that CPPs are first internalized inside endocytic vesicles and have been reported to escape to the cytosolic space of cells. However, while the internalization of CPPs inside vesicles has proven to be highly efficient, their endosomal escape activity remains suboptimal and poorly understood. This is a severe limitation of the CPP-mediated delivery approach because the macromolecules’ escape is essential if the cargo is to interact with its cytosolic/nuclear targets. Multiple approaches to enhance CPP’s endosomolytic activity have been explored (summarized in Chapter 1). Multivalency has been proposed as a possible new approach, in which multiple copies of a molecule are attached as one functional unit. It has been hypothesized that the use of multivalent CPPs will result in an increase in their local concentration at the membrane; and as a consequence enhance their membrane disruption property. In Chapter 2, I report on the generation of a multivalent CPP: dfTAT, a dimeric version of the most studied CPP, TAT. Similar to TAT, dfTAT enters cells via macropinocytosis. Interestingly, while monomeric TAT is extremely inefficient at escaping the endocytic pathway, I demonstrate that dfTAT escapes endosomes with an unprecedented level of efficiency. Cytosolic penetration of dfTAT can be achieved in multiple cell lines and primary cells, without impacting the cell’s physiology. Most importantly, dfTAT can efficiently deliver a wide variety of macromolecules and cell-impermeable small molecules efficiently into the cytosolic space of live cells. In Chapter 3, I report on the role that chirality and protease-resistance have on the endosomal escape behavior of dfTAT. By inverting dfTAT’s chirality from L to D stereochemistry, I generated a protease-resistant dfTAT (D-dfTAT). Interestingly, I show that while the mechanism of cellular entry remains the same, D-dfTAT’s cellular internalization is substantially lower than dfTAT. Nonetheless, once in the endosome, DdfTAT escapes with a higher efficiency than its L-counterpart. Finally, while dfTAT treatment is relatively innocuous to cells, D-dfTAT unexpectedly exerted a prolonged anti-proliferative activity. The literature underscores the important role that arginine residues play in CPP cellular internalization. However, due to the low endosomolytic activity of most CPPs, residues vital for endosomal escape have not yet been established. In Chapter 4, I determined that the arginine residues are both necessary and sufficient for dfTAT endosomolytic activity. Interestingly, a minimum number of 12 arginine residues is required for sufficient endosomal escape. Overall, this work identifies a novel endosomolytic agent and identifies some of the molecular features important for cellular permeation. In turn, my results lay the foundation for new and optimized versions of the dfTAT prototype that may find applications in a variety of delivery approaches

Homeotic transformation induced by protein transduction

One of the most fundamental features of living organisms is that cells are separated from their external environment by a thin, but highly complex plasma membrane constituted of a lipid bilayer. Although, the lipid bilayer is only a few nanometers in width, it is impermeable to most molecules apart from small hydrophobic ones. The ability of small molecules to diffuse through a lipid bilayer is related to their lipid solubility. Hydrophilic macromolecular Antennapedia homeodomain peptide has been shown to be able to translocate from extracellular space into the cytoplasm of cells in a receptor-independent manner. Its third α-helix domain, designated as “Penetratin”, was proposed to be the functional transduction domain that is responsible for the translocation, and it is widely used for intracellular delivery of various exogenous proteins. Although Penetratin has been regarded to be the only element conferring the capacity of its parent polypeptide to penetrate through the plasma membrane, we found that the complete Antennapedia homeodomain exhibits an appreciably higher level of translocation efficiency as compared to Penetratin. Pharmacological analysis demonstrated that macropinocytic endocytosis plays a significant role underlying the process of the homeodomain internalization, and this is consistent with the observation that internalized polypeptide co-localizes with a fluid phase dye. Our studies identify macropinocytosis as a major mechanism by which Antennapedia homeodomain obtains the access to the interior of cells. In the process of macropinocytosis, signaling from the plasma membrane is required for actin remodeling to generate mechanical deformation forces; the interaction between positively charged Antennapedia homeodomain and negatively charged extracellular heparan sulfate could trigger the signaling cascade for fluid phase endocytosis. This would presumably explain why positively charged peptides, polymers, and liposomes are able to penetrate cells. As a fluid phase macropinocytosis provides cells with a way to non-selectively internalize large quantities of solute, it represents an effective means for drug delivery into cells. Both of “Penetratin” and Antennapedia homeodomain exploit macropinocytosis to a certain extent, the comparison between them may advance our understanding of the mechanisms triggering macropinocytotic endocytosis

Membrane translocating peptides for the delivery of proteins

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Folate Receptor-Targeted Delivery of Small Interfering RNA to Cancer Cells

The vitamin folic acid (folate, FA) has been extensively explored as a targeting ligand to deliver a variety of diagnostic/prognostic/therapeutic agents into various tumors through the assistance of its receptor – the folate receptor (FR). FR is over-expressed in many types of human cancer and can mediate internalization of FA-conjugates through an endocytic pathway. The discovery of small interfering RNA (siRNA), which is cable of inducing potent gene silencing in a sequence-specific manner, provides an excellent molecular tool to suppress aberrant gene expression in malignancies, and therefore siRNA has the potential to revolutionize cancer therapeutics. Towards the goal of developing an efficient and cancer-specific siRNA strategy, three types of FA-conjugated molecules have been synthesized to investigate targeted siRNA delivery to cancer cells in vitro. In the first section, FA-linked siRNA was synthesized through our one-step in vitro transcription using FA-HAD-AMP as a transcriptional initiator. FR-dependent cellular uptake and moderate specific gene down-regulation (50%) were observed in a stable cell line (Gluc-KB), which was established in this work by integrating Gaussia luciferase (Gluc) gene to the genome of KB cells (human nasopharyngeal carcinoma). Gluc-KB provides a platform to better evaluate gene expression changes upon siRNA treatment. In the second section, a FA-functionalized, multivalent copolymer (FAPol13) was introduced to: (i) complex with siRNA to form a FAPol13/siRNA complex, (ii) protect siRNAs from enzymatic degradation, (iii) enhance cellular uptake by conjugating several FA molecules to copolymer, and (iv) increase RNAi efficacy eventually. A typical FAPol13 compound has three functional groups: cationic, hydrophilic, and FA moieties for providing siRNA packaging site, water solubility, and cell-selectivity, respectively. This nontoxic polymer successfully delivered siRNAs against Gluc, survivin (Sur), Caspase 8 associated protein 2 (Casp8ap2) genes in KB cells and efficiently reduced their expression (i.e., 62% and 68% downregulation from siSv and siGLuc treatments, respectively). Strikingly, treatment of KB cells with FAPol13/siCasp8ap2 significantly repressed cell growth and induced cells into apoptosis (24.5%, p = 0.01). Furthermore, a real-time imaging system, employing fluorescence approaches (time-lapse, z-stacking, and colocalization analysis), has been developed to evaluate siRNA binding, cellular uptake, and escape from compartments, respectively. In particular, Pearson’s correlation coefficient (PCC) was employed to quantify siRNA endosomal escape, one of the crucial steps of siRNA intracellular trafficking. This method was further applied to the analysis of siRNA delivery in HeLa (human cervical carcinoma) and SKOV3 (human ovary carcinoma). In the third section, a gold nanoparticle (AuNP) capable of packaging and protecting siRNAs was utilized to transport siRNA to cancer cells. Cytotoxicity assay, cellular uptake, and gene down-regulation studies of the AuNP-siRNA system indicate that AuNP can be an efficient siRNA platform. In the fourth section, a correlation between FR expression level and the delivering effectiveness of FA-conjugates was established by comparing cellular absorption, cellular uptake, and RNAi efficiency among KB, HeLa, SKOV3 and A549 (human lung carcinoma) cells. Collectively, we have demonstrated effective FR-mediated and cancer cell-specific siRNA delivery by several approaches. Further studies may lead to the development of therapeutic siRNA delivery systems

Cell Division, Cytotoxicity, and the Assays Used in the Detection of Cytotoxicity

Cell division is a phenomenon that is encountered in all cells in nature. While normal cell division results in proliferation in single-celled organisms, and development and repair in multicellular organisms, aberrant and untimely cell division results in tumor formation. Therefore, the understanding of the cell division is hidden in identifying the details of the molecular mechanisms that govern cellular division at the exact time and under right conditions. Sometimes these molecular mechanisms are distorted by both intrinsic and extracellular factors, and the division process halts or deviates to an abnormal pathway. At this point, it is essential that the abnormal cells are removed from the tissue by an appropriate mechanism. In this context, in this review, general and specific information about cell division and its molecular control mechanisms were discussed, and different types of cell death mechanisms were mentioned accordingly. In addition, chemical, biological, and physical cytotoxic agents that negatively affect cell division and their mechanisms of action are explained. Finally, a brief review of the principles of different cytotoxicity (cell viability and proliferation) test systems has been performed to provide a source of information for investigators who study cell viability, proliferation, or different types of cellular death pathways