Rakku sisenevate peptiidi/nukleiinhappe komplekside kirjeldamine ja nende rakku sisenemise mehhanismid

Väitekirja elektrooniline versioon ei sisalda publikatsioone.Kõrge spetsiiflisuse ja madala kõrvalmõjude tekkeriski tõttu peetakse nukleiinhappeid väga suure ravipotentsiaaliga molekulideks. Bioloogilise aktiivsuse saavutamiseks on vajalik nukleiinhapete sisenemine rakkudesse ning jõudmine sihtkohta kas rakutuumas või tsütoplasmas. Suure molekulmassi ja negatiivse laengu tõttu pole aga nukleiinhapped võimelised ise rakke ümbritsevat plasmamembraani läbima. Seetõttu on arendatud mitmesuguseid meetodeid, mille abil nukleiinhappeid efektiivsemalt rakkudesse suunata, ning üheks neist on rakku sisenevate peptiidide (RSP; i.k. cell-penetrating peptide, CPP) kasutamine. RSP-d on lühikesed, enamasti katioonsed ja/või amfipaatsed aminohappelised järjestused, mis on võimelised läbima plasmamembraani ning transportima rakkudesse erinevaid bioaktiivseid molekule. Selleks, et RSPd saaksid nukleiinhappeid rakkudesse transportida, on vajalik peptiidse vektori sidumine lastmolekuliga. Lihtsaim võimalus on RSP siduda lastmolekuliga kompleksi moodustamise teel, millega saadakse RSP/nukleiinhappe nanopartiklid, mida hoiavad koos laengulised ja hüdrofoobsed interaktsioonid. RSP/nukleiinhappe nanokompleksid sisenevad rakkudesse efektiivselt ning tagavad kõrge lastmolekuli bioaktiivsuse, samas ei ole antud süsteem veel jõudnud kliiniliste katsete või kasutuseni. Üheks põhjuseks siinjuures on tekkivate nanokomplekside omaduste (nt suurus, kuju, laeng) komplitseeritud kirjeldamine. Teine probleem, mis takistab mittekovalentse strateegia rakendamist, on vähene arusaam nanokomplekside rakku sisenemise mehhanismidest ja rakusisesest suunamisest. Nii RSP/nukleiinhappe komplekside omaduste selgitamine kui nende rakku sisenemise mehhanismide ja rakusisese suunamise mõistmine on aga äärmiselt vajalikud, et parendada transportpeptiidide omadusi ja tagada lastmolekulide võimalikult kõrge aktiivsus nende kasutuselevõtuks biomeditsiinis, vältides sealjuures soovimatuid kõrvalmõjusid. Antud töös kirjeldasime hiljuti väljatöötatud PepFect ja NickFect tüüpi peptiidide ja nukleiinhapete vaheliste nanokomplekside suurust, kuju ja laengut. Lisaks sellele identifitseerisime uuritud RSP/nukleiinhappe komplekside peamised rakku sisenemise mehhanismid ning analüüsisime rakku viidud nukleiinhappe paiknemist ja selle muutumist ajas.Nucleic acids are highly promising candidates for the treatment of various diseases. In order to achieve biological functionality, nucleic acids need to be internalized by cells and reach their action site in cytoplasm or nucleus. However, due to the large size and negative charge, naked nucleic acids are not capable of traversing the plasma membrane of cells. A wide variety of delivery vectors have been designed to facilitate the cellular uptake of nucleic acids. One class of such vectors are cell-penetrating peptides (CPPs), short sequences of 5-40 amino acid residues, which are capable of gaining access to the interior of cells, and importantly, mediate the internalization of coupled cargo molecules. The simplest way to couple CPPs to nucleic acids is to mix the peptide and cargo. The co-incubation of CPPs and nucleic acids leads to the formation of nanocomplexes due to electrostatic and hydrophobic interactions between the peptide and cargo. Co-incubation strategy has been shown to yield high bioactivities of cargos in numerous studies. However, this approach has not reached clinical trials yet. One reason behind this is the complicated physicochemical characterization of the forming nanocomplexes. However, in order to be considered for implementation in biomedicine the properties of CPP/nucleic acid complexes such as size, morphology and charge need to be characterized in detail. Another bottleneck which impedes the implementation of non-covalent strategy for nucleic acid delivery is the poor knowledge of the cellular uptake mechanisms and intracellular trafficking of CPP/nucleic acid nanocomplexes. However, detailed characterization of the cell internalization pathways and cellular trafficking of CPP/cargo complexes are essential for avoiding undesired side effects and refining their properties in order to yield higher activities of delivered cargo. The main objectives of the current thesis were to characterize the physicochemical properties of CPP/nucleic acids nanocomplexes formed by PepFect and NickFect type carrier peptides, and to examine the cellular uptake mechanisms and intracellular trafficking of nanocomplexes and nucleic acids

Characteristics of Cell-Penetrating Peptide/Nucleic Acid Nanoparticles

Nucleic acids are highly promising candidates for the treatment of various genetic diseases. However, due to the large size and negative charge, nucleic acids are not efficiently taken up by cells, and thus, their clinical potential remains limited so far. Therefore, various delivery vehicles have been designed to assist the cellular uptake of nucleic acids. Among these, cell-penetrating peptides (CPPs) have gained increasing popularity as efficient and nontoxic delivery vectors. CPPs can be coupled to nucleic acids either by covalent or noncovalent association. Noncovalent coupling, which is based on the formation of nanoparticle-like nanocomplexes (NP), has received much attention in recent years, and the number of studies employing the strategy is explosively increasing due to the high therapeutic potential. However, the properties of CPP/nucleic acid NPs have not been characterized in sufficient detail yet. We performed a comprehensive analysis of the size and morphology of nucleic acid nanoparticles with novel transfection peptides, PepFects (PFs) and NickFects (NFs), using negative staining transmission electron microscopy (TEM). In addition, we examined whether the attachment of fluorescence or (nano)gold label to nucleic acid affects the nanocomplex formation or its morphology. We demonstrated that transportan-10-based new generation CPPs from PF and NF families condense nucleic acids to NPs of homogeneous size and shape. The size and shape of assembled nanoparticles depend on the type of the complexed nucleic acid and the sequence of the used peptide, whereas the label on the nucleic acid does not influence the gross characteristics of formed NPs

Saturated Fatty Acid Analogues of Cell-Penetrating Peptide PepFect14: Role of Fatty Acid Modification in Complexation and Delivery of Splice-Correcting Oligonucleotides

Modifying cell-penetrating peptides (CPPs) with fatty acids has long been used to improve peptide-mediated nucleic acid delivery. In this study we have revisited this phenomenon with a systematic approach where we developed a structure–activity relationship to describe the role of the acyl chain length in the transfection process. For that we took a well-studied CPP, PepFect14, as the basis and varied its N-terminal acyl chain length from 2 to 22 carbons. To evaluate the delivery efficiency, the peptides were noncovalently complexed with a splice-correcting oligonucleotide (SCO) and tested in HeLa pLuc705 reporter cell line. Our results demonstrate that biological splice-correction activity emerges from acyl chain of 12 carbons and increases linearly with each additional carbon. To assess the underlying factors regarding how the transfection efficacy of these complexes is dependent on hydrophobicity, we used an array of different methods. For the functionally active peptides (C12–22) there was no apparent difference in their physicochemical properties, including complex formation efficiency, hydrodynamic size, and zeta potential. Moreover, membrane activity studies with peptides and their complexes with SCOs confirmed that the toxicity of the complexes at higher molar ratios is mainly caused by the free fraction of the peptide which is not incorporated into the peptide/oligonucleotide complexes. Finally, we show that the increase in splice-correcting activity correlates with the ability of the complexes to associate with the cells. Collectively these studies lay the ground work for how to design highly efficient CPPs and how to optimize their oligonucleotide complexes for lowest toxicity without losing efficiency

Differential Endosomal Pathways for Radically Modified Peptide Vectors

In the current work we characterize the uptake mechanism of two NickFect family members, NF51 and NF1, related to the biological activity of transfected plasmid DNA (pDNA). Both vectors condense pDNA into small negatively charged nanoparticles that transfect HeLa cells with equally high efficacy and the delivery is mediated by SCARA3 and SCARA5 receptors. NF1 condenses DNA into less homogeneous and less stable nanoparticles than NF51. NF51/pDNA nanoparticles enter the cells via macropinocytosis, while NF1/pDNA complexes use clathrin- or caveolae-mediated endocytosis and macropinocytosis. Analysis of separated endosomal compartments uncovered lysomotropic properties of NF51 that was also proven by cotransfection with chloroquine. In summary we characterize how radical modifications in peptides, such as introducing a kink in the structure of NF51 or including extra negative charge by phospho-tyrosine substitution in NF1, resulted in equally high efficacy for gene delivery, although this efficacy is achieved by using differential transfection pathways

PepFect14 Peptide Vector for Efficient Gene Delivery in Cell Cultures

The successful applicability of gene therapy approaches will heavily rely on the development of efficient and safe nonviral gene delivery vectors, for example, cell-penetrating peptides (CPPs). CPPs can condense oligonucleotides and plasmid DNA (pDNA) into nanoparticles, thus allowing the transfection of genetic material into cells. However, despite few promising attempts, CPP-mediated pDNA delivery has been relatively inefficient due to the unfavorable nanoparticle characteristics or the nanoparticle entrapment to endocytic compartments. In many cases, both of these drawbacks could be alleviated by modifying CPPs with a stearic acid residue, as demonstrated in the delivery of both the pDNA and the short oligonucleotides. In this study, PepFect14 (PF14) peptide, previously used for the transport of shorter oligonucleotides, is demonstrated to be suited also for the delivery of pDNA. It is shown that PF14 forms stable nanoparticles with pDNA with a negative surface charge and size of around 130–170 nm. These nanoparticles facilitate efficient gene delivery and expression in a variety of regular adherent cell lines and also in difficult-to-transfect primary cells. Uptake studies indicate that PF14/pDNA nanoparticles are utilizing class A scavenger receptors (SCARA) and caveolae-mediated endocytosis as the main route for cellular internalization. Conclusively, PF14 is an efficient nonviral vector for gene delivery

Lipid-based Transfection Reagents Exhibit Cryo-induced Increase in Transfection Efficiency

The advantages of lipid-based transfection reagents have permitted their widespread use in molecular biology and gene therapy. This study outlines the effect of cryo-manipulation of a cationic lipid-based formulation, Lipofectamine 2000, which, after being frozen and thawed, showed orders of magnitude higher plasmid delivery efficiency throughout eight different cell lines, without compromising cell viability. Increased transfection efficiency with the freeze-thawed reagent was also seen with 2'-O-methyl phosphorothioate oligonucleotide delivery and in a splice-correction assay. Most importantly, a log-scale improvement in gene delivery using the freeze-thawed reagent was seen in vivo. Using three different methods, we detected considerable differences in the polydispersity of the different nucleic acid complexes as well as observed a clear difference in their surface spreading and sedimentation, with the freeze-thawed ones displaying substantially higher rate of dispersion and deposition on the glass surface. This hitherto overlooked elevated potency of the freeze-thawed reagent facilitates the targeting of hard-to-transfect cells, accomplishes higher transfection rates, and decreases the overall amount of reagent needed for delivery. Additionally, as we also saw a slight increase in plasmid delivery using other freeze-thawed transfection reagents, we postulate that freeze-thawing might prove to be useful for an even wider variety of transfection reagents