A xanthene-BODIPY cassette is used as a ratiometric intracellular pH reporter for imaging protein-dye conjugates in living cells. A model was hypothesized to explain the pH-dependent energy transfer efficiencies from the donor to the acceptor based on the electronic chemistry data. Sulfonation conditions were developed for BODIPY dyes to give water-soluble functionalized monosulfonation and disulfonation donors. A water-soluble TBET cassette, which has good photophysical properties, was synthesized using a bissulfonated BODIPY dye as the donor, and their applications for in vitro protein labeling is achieved. Chemoselective cross-coupling reactions were demonstrated for C-S bonds in the BODIPY dye, and similar reactions were applied to make the acceptor of the watersoluble cassette. Chemiluminescent energy transfer cassettes based on fluorescein and Nile Red were synthesized and their spectral properties were studied. Pep-1 (also known as Chariot), R8 (which is not often used as a non-covalent protein carrier), and a new synthesized compound, Azo-R8, was used for the study of non-covalent delivery of four different proteins into mammalian cells. Data from confocal spectroscopy revealed that all three carriers are effective for translocating protein cargos into live cells. At 37 dgrees C, import into endocytic compartments dominates, but at 4 degrees C weak, diffuse fluorescence is observed in the cytosol indicative of a favorable mode of action.

No description available.

Cancer is the second leading cause of death in America. In 2018, there were 9.5 million deaths due to cancer according to the International Agency for Research on Cancer (IARC), and this number is expected to grow to 16.3 million by 2040. Among the type of cancers, neuroblastoma and nerve tissue cancers have a 5-year survival rate of 33%, which is very low. One of the main issues linked to such situations is due to the lack of specificity in removing tumor cells. While clinical therapies work to reduce tumor mass as much as possible, they cannot always target all of them, and once some cancer cells are left behind, they regrow and spread. The work of this thesis seeks to enhance the treatment outcome by utilizing all-trans retinoic acid (ATRA), a metabolite of vitamin A, to induce differentiation of nerve tissue cancer cells and eliminate their ability to self-renew (reemerge). Differentiation therapy is currently utilized in select clinical applications but the utilization of ATRA is limited due to its poor solubility in the blood, low bioavailability, short half-life, and in vivo toxicity. In order to alleviate some of these issues, the ATRA molecule was engineered with a novel cell penetrating peptide and tested for its efficacy. Data and results presented herein report the differentiation induced by the CPP-conjugated ATRA may act as a viable method for neuroblastoma treatment. / Bioengineering

Le peptide Tat est un des "cell penetrating peptides" (CPP) les plus utilisés pour l'internalisation cellulaire de diverses molécules cargos. La molécule chimérique (Tat-cargo) induit une réponse biologique plus efficace comparée au cargo seul. Cependant, en marquant le peptide Tat à l'iode 125, il a été déterminé que seulement moins de 1 % de la quantité initiale de peptide est internalisé. Il y a donc une opportunité d'augmenter l'efficacité de cette internalisation. L'étape cruciale du processus d'internalisation est le passage transmembranaire. Cette thèse présente l'évaluation de l'impact d'un groupement hydrophobe intégré en différentes positions au peptide Tat, afin de favoriser son interaction avec la membrane. Un acide aminé modifié chimiquement comportant un groupement cholestéryle a été développé dans ce sens. Cet aminoacide peut être intégré en toute position du peptide Tat. Différentes positions au sein du peptide Tat ont été cholestérylées et l'effet sur le taux d'internalisation a été étudié par cytométrie en flux et par comptage suite au radiomarquage des peptides à l'iode 125.L'ajout de cholestérol en position centrale du peptide Tat induit une efficacité d'internalisation supérieure d'un facteur 30 alors qu'une augmentation moindre est observée suite à l'ajout du groupement hydrophobe en positions latérales, N- ou C-terminale. / The Tat peptide is one of the most used cell penetrating peptides for internalizing various cargo molecules into cells. The chimaeric molecule thus triggers an efficient cellular biological response when compared with the cargo molecule alone. However, following labeling of the Tat peptide with radiolabeled iodine, less than 1% of the external peptide was internalized. Therefore, there is an opportunity to improve the level of CPP internalization. The ultimate step is the crossing through the plasma membrane. This thesis presents an evaluation of the impact of a hydrophobic group incorporated at different positions to Tat peptide, to promote its interaction with the membrane.A chemically modified amino acid comprising a cholesteryl group was developed in this direction. This amino acid can be inserted at any position within the Tat peptide. Different positions within the Tat peptide were cholesterylated and the effect on the internalization rate of Tat CPP was investigated by flow cytometry and by counting following the radiolabeling of peptides with iodine 125.The addition of cholesterol in the central position of the peptide Tat induces internalization efficiency than a factor of 30 while a smaller increase was observed after the addition of hydrophobic group in lateral positions, N-or C-terminus.

The plasma membrane functions as a barrier, restricting entry of hydrophilic pharmaceutical agents. Cell-penetrating peptides (CPPs) are capable of transporting bioactive cargos into the cell and have consequently been extensively investigated for their mechanism of entry and capability to deliver various cargos spanning from peptides to plasmids. The main aim of this thesis was to investigate the mechanism and capability of some of these CPPs to deliver mainly oligonucleotides and peptides into the cell. Oligonucleotides in the form of ds DNA decoy for sequestering of transcription factors or PNAs for redirection of splicing. In addition, peptides derived from the interaction interface of a tumor suppressor protein were investigated for their potential to combine a biological effect with internalization. Peptides with or without any cargo were predominantly dependent on some form of endocytic mechanism for internalization, substantiated by using a functional assay, where all tested CPPs were associated with endocytosis for delivery of splice correcting PNAs. A new CPP, M918 proved most efficient in promoting splice correction and internalized mainly via macropinocytosis. In addition, TP10 efficiently delivered dsDNA decoy oligonucleotides for sequestering of the transcription factor Myc with a concomitant biological response, i.e. reduced proliferation. Finally, for the first time, to our knowledge, a novel pro-apoptotic peptide with cell-penetrating properties was designed from the tumor suppressor p14ARF, which decreased proliferation and induced apoptosis in cancer cell-lines, potentially mimicking the full-length protein. Altogether, this thesis highlights the functionality of CPPs and the possibility to develop new CPPs with improved or new properties, having the potential to advance delivery of therapeutic compounds.

High molecular weight biomolecules are becoming important in the development of new therapeutics. However, their size and nature creates a major limitation for their application – poor penetration through biological membranes. A new class of peptides, cell-penetrating peptides (CPPs), has shown the capability to transport various macromolecules inside the cells. However, there are at least two limiting factors for successful application of CPPs: the lack of cell-type specificity and restricted bioavailability resulting from endocytic uptake of CPPs and entrapment in endosomal compartments. This thesis aims at designing delivery vehicles for therapeutic substances. In papers I-III, the CPPs have been rationally modified in order to achieve in vivo selectivity towards cancer cells. The first two papers employ tumor homing peptides as targeting moieties coupled to the N-termini of CPPs. In the third paper, a CPP is C-terminally prolonged with a matrix metalloproteinase 2 (MMP-2) specific cleavage site followed by an inactivating amino acid sequence. In tissues overexpressing MMP-2, i. e. in proximity to cancer, the CPP is activated after proteolytic removal of the inactivating sequence, thus the cargo can be transported inside the cells. In paper IV, several CPPs have been N-terminally modified with a stearyl moiety and applied for the delivery of splice-correcting oligonucleotides. We show that stearyl-TP10 is as effective in oligonucleotide delivery as Lipofectamine™ 2000. Moreover, stearyl-TP10 has preserved efficacy in serum and is not toxic to cells. In conclusion, the rational modifications of CPPs greatly potentiate their application in cargo delivery both in vitro and in vivo.

RNA interference (RNAi) is a conservative post-transcriptional gene silencing mechanism that can be mediated by small interfering RNAs (siRNAs). Given the effectiveness and specificity of RNAi, the administration of siRNA molecules is a promising approach to cure diseases caused by abnormal gene expression. However, as siRNA is susceptible to degradation by nucleases and it can hardly penetrate cell membranes due to its polyanionic nature, a successful translation of the RNAi mechanism for therapeutic purposes is contingent on the development of safe and efficient delivery systems. This dissertation described the development of novel siRNA delivery systems on the basis of polymeric and dendrimeric materials and also demonstrated the application of one optimized delivery system to deliver therapeutic siRNAs in a cardiovascular disease model in vivo. We studied a linear peptide polymer made from cell penetrating peptide monomers and investigated the contribution of the polymeric structure, degradability, and ligand conjugation to the siRNA loading capacity, biocompatibility, and transfection efficiency of polymeric materials. With the obtained knowledge and experience, we invented a neutral crosslinked delivery system aiming to solve the inherent drawbacks of traditional cationic delivery systems that are based on electrostatic interactions. The new concept utilized buffering amines to temporarily bind siRNA and a crosslinking reaction to immobilize the formed particles, and targeting ligands modified on the neutral dendrimer surface further enhanced the interactions between the delivery vehicles and target cells. The obtained delivery system allowed stability, safety, controllability, and targeting ability for siRNA delivery, and the method developed here could be transformed to other polymeric or dendrimeric cationic materials to make them safer and more efficient. To exploit the therapeutic potential of siRNA delivery, we developed a tadpole-shaped dendrimeric material to deliver siRNA against an Angiotensin II receptor in a rat ischemia-reperfusion model. Our results showed that the nonaarginine-conjugated tadpole dendrimer was capable of delivering siRNA effectively to cardiac cells both in vitro and in vivo, and the successful down-regulation of the Angiotensin II receptor preserved the cardiac functions and reduced the infarct size post-myocardial infarction. This dissertation paves a way for transforming multifunctional non-viral siRNA delivery systems into potent therapeutic strategies for the management of cardiovascular diseases.

Liver transplantation is the only life-saving treatment for patients with end-stage liver disease; however, organ availability is insufficient to meet demands. Steatotic livers are extended criteria donor (ECD) organs that could be used for transplantation if not for an increased susceptibility ischemia reperfusion injury (IRI). Heme oxygenase-1 is a gene, that when upregulated has be shown to reduce IRI in animal models of transplantation. Increasing HO-1 activity in steatotic livers by delivery of a functional cell-penetrating HO-1 protein (through the use of cell-penetrating peptides) may provide protection against IRI, making these organs useful for transplantation. The purpose of this thesis was the generation and testing of a cell-penetrating HO-1 protein. HO-1 and EGFP gene sequences were cloned into the pET-28B(+) vector in frame with a CPP or TAT sequence. Resulting plasmids were cloned into E. coli, and protein expression was induced using IPTG. Proteins were purified using Ni-NTA affinity chromatography under denaturing and non-denaturing conditions. Non-denatured proteins were tested for HO-1 activity and the ability of both denatured and non-denatured proteins to transduce cells in vitro was tested by fluorescence microscopy. The cell-penetrating ability of nondenatured proteins was further tested in J774, HepG2 and HUVEC cells using immunofluorescence. Five HO-1 and two EGFP cell-penetrating proteins were generated expressed and purified successfully. Purified non-denatured HO-1 retains its enzymatic activity. Non-denatured CPP-EGFP and CPP-HO1 penetrated cells more effectively than their denatured counterparts. CPP-EGFP and CPP-HO1 proteins are able to penetrate multiple cell types in vitro. Successful generation and testing of a cell-penetrating HO-1 protein, for use in an animal model of steatotic liver transplantation. This protein demonstrates promise for use as a potential therapeutic agent in the field of liver transplantation.

Regulation of biological processes through the use of genetic elements is a central part of biological research and also holds great promise for future therapeutic applications. Oligonucleotides comprise a class of versatile biomolecules capable of modulating gene regulation. Gene therapy, the concept of introducing genetic elements in order to treat disease, presents a promising therapeutic strategy based on such macromolecular agents. Applications involving charged macromolecules such as nucleic acids require the development of the active pharmaceutical ingredient as well as efficient means of intracellular delivery. Cell-penetrating peptides are a promising class of drug delivery vehicles, capable of translocation across the cell membrane together with molecules otherwise unable to permeate cells, which has gained significant attention. In order to increase the effectiveness of cell-penetrating peptide-mediated delivery, further understanding of the mechanisms of uptake is needed in addition to improved design to make the cell-penetrating peptides more stable and, in some cases, targeted. This thesis encompasses four scientific studies aimed at investigating cell-penetrating peptide and oligonucleotide designs amenable to therapeutic applications as well as elucidating the mechanisms underlying uptake of cell-penetrating peptide:oligonucleotide nanoparticles. It also includes an example of a therapeutic application of cell-penetrating peptide-mediated delivery of oligonucleotides. Paper I presents a study evaluating a range of chemically modified anti-miRNAs for use in the design of therapeutic oligonucleotides. All varieties of oligonucleotides used in the study target miRNA-21 and are evaluated using a dual luciferase reporter system. Paper II introduces a novel cell-penetrating peptide, PepFect15, aiming at combining the desirable properties of improved peptide stability and efficient cellular uptake with a propensity for endosomal escape, to produce a delivery vector well suited for delivery of oligonucleotides. The performance of this new cell-penetrating peptide was evaluated based on its delivery capabilities pertaining to splice-correcting oligonucleotides and anti-miRNAs. Paper III investigates the involvement of scavenger receptor class A in the uptake of various cell-penetrating peptides together with their oligonucleotide cargo. Finally, paper IV aims at using cell-penetrating peptide-mediated delivery to improve the efficiency of telomerase inhibition by antisense oligonucleotides targeting the telomerase enzyme ribonucleotide component. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.</p>

Atypical gene expression has a major influence on the disease profile of several severe human disorders. Oligonucleotide (ON) based therapeutics has opened an avenue for compensating deviant protein expression by acting on biologically important nucleic acids, mainly RNAs. Antisense ONs (asONs) can be designed to target complementary specific RNA sequences and thereby to influence the corresponding protein synthesis. However, cellular uptake of ONs is poor and is, together with the target specificity of the asONs, the major limiting factor for the development of ON based therapeutics. In this thesis, the mechanisms of well-characterized cell-penetrating peptides (CPPs) are evaluated and CPPs are adapted for cellular ON-delivery. The functionality of ON derivatives in cells is investigated and by optimization of asONs, targeting pre-messenger RNA, high efficiency and specificity is achieved. The optimization of the asONs is based on sequence design and through the choice of nucleic acid analogue composition. It is concluded that asONs, partly composed of locked nucleic acids are attractive for splice-switching applications but these mixmers must be designed with limited number of locked nucleic acid monomers to avoid risk for off-target activity. A protocol allowing for convenient characterization of internalization routes for CPPs is established and utilized. A mechanistic study on cellular CPP uptake and translocation of associated ON cargo reveals the importance of the optimal combination of for example charge and hydrophobicity of CPPs for efficient cellular uptake. Formation of non-covalent CPP:ON complexes and successful cellular delivery is achieved with a stearylated version of the well-recognized CPP, transportan 10. The results illustrate that CPPs and ON derivatives have the potential to become winning allies in the competition to develop therapeutics regulating specific protein expression patterns involved in the disease profile of severe human disorders. / At the time of doctoral defense, the following papers were unpublished and had s status as follows: Paper 4: Accepted.Peper 5: In press. / VINNOVA-SAMBIO Multidisciplinary BIO