Non-viral delivery systems

Non-viral delivery systems encompass a broad range, from organic to inorganic nanoparticles. Some inorganic nanoparticles that need to be mentioned are carbon nanotubes, which are biocompatible and formed by covalent carbon linkages through different C-C hybridizations, silica-based delivery systems, especially mesoporous which exhibit attractive characteristics of high cargo loading, gold nanoparticles that show low toxic and immunogenic nature, high permeability and retention effect that facilitates penetration and accumulation of drugs at the tumor sites, calcium phosphate nanoparticles that enable incorporation of nucleic acids therapeutics on their surface, and metal−organic frameworks.^(23–27)

Lipid nanoparticles, the leading non-viral delivery systems for nucleic acid therapeutics, represent a large group of organic nanoparticles. The most useful are a large unilamellar sub-class of liposomes in a size range of 100 nm. They possess a highly efficient reproducible encapsulation process, scalable, robust manufacturing, and stability of the product for at least one year at 4 °C. Because of their neutral surface, they can avoid rapid accumulation with

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serum proteins and macrophages. A step forward was made with introduction of cationic lipids, which can be easily assembled with negatively charged RNA and DNA molecules.

However, they were unstable, too large, too positively charged and expressed toxic side effect. These drawbacks were reduced by ionizable cationic lipids such as 1,2-dioleoyl-3-dimethylammonium propane (DODAP) and the gold standard for delivery into the liver dilinoleylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA DLin-MC3-DMA is three times more potent than DODAP, despite only a slight difference in chemical structure. They form small nanoparticles in the size range of 100 nm or less, with low surface charge (ideal pKa between 6.2–6.8) at a physiological pH value that protect them from clearance by the immune system, minimalize nonspecific interaction of LNPs, and prevent aggregation with proteins.^(28,29) Their uptake is also facilitated through ApoE which associates preferably with neutral particles and mediates uptake via receptors containing ApoE binding ligands in the liver and brain tissue. They enter the cell through endocytosis where ionizable nanoparticles get protonated because of a higher endosomal pH (pH 5.5) and consequently combine with the phospholipids that are negatively charged, forming a cone shape that disrupts the endosomal bilayer and allows the DNA molecule to escape into the cytosol.⁽²⁹⁾ Unsaturated acyl chains are also supposed to have beneficial effects regarding the endosomal-escape theory. Circulation in the blood can be prolonged with PEG-lipids. However, potency of gene silencing is reduced but with introduction of dissociating PEG lipids, silencing can be improved. The most promising is their potential for delivery of mRNA to the liver and utilizing them as a factory to produce proteins, neutralizing antibodies, and as vaccines. They also show low toxicity. Yet, their uptake mediated by endocytosis is less than 5 %, as up to 60 % is recycled in the extracellular medium and the rest is degraded in lysosomes.⁽²⁸⁾ The second largest group are particles based on polymers such as polymeric nanoparticles, polymer micelles, polymersomes, polyelectrolyte polyplexes, polymer-lipid hybrid systems, and polymer-drug/protein conjugates. They mainly consist of self-assembled multivalent cationic polymers and polyanionic nucleic acids, which are formed by entropy-driven ionic interactions. Due to versatile polymer chemistry, a variety of polymers can be integrated into new carriers. Some polymeric systems are already approved for clinical use, still, macromolecular drug formulations have to correspond to the pharmaceutical standards and many challenges need to be surmounted regarding precision chemistry, purification, and high-end analytics. Furthermore, tumor heterogeneity represents another challenge.^(19,30)

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There are also some more exotic materials that can be used as non-viral vectors, such as black phosphorus-based, reactive oxygen and nitrogen species (RONS) scavenger-based systems, perfluorocarbon nanoparticles, sulfide-based systems, and oxide-based systems.

Properties of these systems are mostly related to the characteristics of the material.⁽³¹⁾ 1.4 Polyplexes as delivery systems

1.4.1 Delivery pathways

Polyplexes can be administrated only intravenously, their size ranging from 6 nm up to 400 nm which mostly depends on the number and type of nucleic acids. Polyplexes containing one molecule of pDNA can have a size of 25 nm, while those containing siRNA, only 6 nm.⁽¹⁹⁾ Nanoparticles, whose hydrodynamic parameter is smaller than 5-10 nm, are rapidly cleared by the kidneys, whereas nanoparticles above 200 nm are recognized by reticuloendothelial system and consequently degraded by macrophages. Particles of size from 10 to 400 nm, can passively accumulate in tumors by enhanced permeability and retention effect (EPR), which is a consequence of the tumor’s leaky blood vessels that enable penetration into the tumorous tissue. Nevertheless, different types of cancers are not equally permeable and that is why polyplexes of different sizes should be applied when targeting specific tumors.^(19,32) Due to their positive charge, their stability in blood circulation can be limited. Although a highly positive charge of nanoparticles can prevent aggregation due to electrostatic repulsion, it may cause non-specific interactions with negatively charged groups of plasma proteins, vessel endothelium and blood cells that can lead to partial or complete dissociation. They can form large self-aggregates or aggregates with blood components (mostly accumulated in pulmonary capillaries), such as erythrocytes or other blood cells, which can lead to serious conditions on larger scale. Moreover, they can also be recognized by innate immune system. This can be avoided by appropriate shielding with hydrophilic polymers as PEG, hydroxyethyl starch or pHPMA. They enable prolonged blood circulation, reduced unspecific cell uptake, cytotoxicity and aggregation, and increase accumulation of polyplexes in tumorous tissue. However, shielding polymers may affect the efficiency of endosomal escape, which can be solved with introduction of a pH-labile bond that can be cleaved in slightly acidic environment such as endocytic vesicles and tumors, thus restoring the endosomolytic capability.⁽³³⁾ The main way polyplexes enter the cytosol is by engulfment into the endosomal compartment and subsequent endosomal escape. There are three proposed mechanisms of endosomal escape for polyplexes: endosomal escape using osmotic

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pressure, escape induced by nanoparticle swelling and membrane destabilization.⁽³⁴⁾ Osmotic pressure induced escape, or the so-called “proton sponge effect”, exploits low pH in the endosome. After the uptake, pH in the endosome decreases from physiological pH down to about 6.3 in the early endosome, to 5.5 in the late endosome, to 6.5 in the recycling endosome and to 4.7 in the lysosome.⁽³⁵⁾ Polymers with a buffering capacity inhibit the drop in pH and thus stimulate proton pumps to deliver H⁺ into the endosome to achieve the desired pH. This leads to an influx of chloride counterions and water molecules that increases the pressure and eventually lyses the endosome. Some nanoparticles may swell in the endosome

Figure 1: Illustration of three proposed hypotheses of endosomal escape for polyplexes: 1.

Endosomal escape using osmotic pressure, 2. Escape induced by nanoparticle swelling and 3. Membrane destabilization.⁽³⁴⁾

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or lysosome, increasing to as much as two times their normal size because of endosomal buffering and subsequent drop in pH. What follows is increased mechanical strain, triggered by swelling, causing the rapture of the endosome membrane and the escape of the nanoparticle.⁽³⁴⁾ Another way is by destabilization and disruption of endosomal membrane through charge−charge and hydrophobic interactions.⁽³⁵⁾ Still, endosomal escape is extremely scarce. In one study, scientists quantitatively determined endosomal escape using nanoscopy techniques and discovered that less than 2 % of siRNA-gold escaped the endosome.⁽³⁶⁾ Nonetheless, other delivery strategies that avoid lysosomal degradation can be applied, such as retrograde transport into Golgi organelles or endoplasmic reticulum, instead, to optimize endosomal escape.⁽¹⁹⁾ Transfection can be also improved with lysosomotropic agents such as chloroquine, polyvinylpyrrolidone (PVP) or sucrose. These endosomotropic agents accumulate in endosomes, induce buffering and osmotic swelling of endosome which causes more efficient endosomal escape into cytosol.^(37,38) Chloroquine is one of the most used lysosomotropic agents and can improve the transfection efficiency up to 50-fold.⁽¹⁹⁾ However, despite its relatively high in vitro efficacy, this approach has high in vivo limitations due to systemic toxicity.^(37,38)

Cytosol is the final destination for molecular therapeutics such as mRNA, antisense, siRNA and miRNA, whereas pDNA and splice-switching oligonucleotides have to go further into the nucleus.⁽¹⁹⁾ Some DNA fragments, shorter than 200–300 bp are capable of penetrating into the nucleus by passive diffusion via the nuclear pore complex (NPC). There are also some approaches how to facilitate uptake into the nucleus, such as: covalent conjugation of DNA with a peptidic nuclear localization, use of trans-cyclohexane1,2-diol (TCHD) that temporary perturbs the barrier function of the NPCs and thus facilitates nuclear entry, but leads to high toxicity, all-trans-retinoic acid (ATRA) that interacts with the retinoic acid receptor⁽³⁷⁾, and the adenoviral peptide conjugated with pDNA that enables attachment to the microtubular motor protein dynein and triggers transport along microtubules, to name just a few.⁽¹⁹⁾ Transfection can be very successful during mitosis. 85–90 % of transfected cells acquired the pDNA after mitosis. ⁽³⁷⁾

1.4.2 Cationic polymers

Cationic polymers are regarded as the best polymers for delivering nucleic therapeutics.

They are able to bind with the negatively charged nucleic acid phosphate backbone through electrostatic interactions and so protect and condense nucleic acids into rods and toroids,

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releasing it into cytosol through endosomal membrane destabilization.⁽³⁹⁾ First generation polymers were DEAE-dextran, PLL (poly-L-lysine), PLR (poly-L-arginin) to name a few.

PLR and PLL are able to produce stable complexes with nucleic acids at a physiological pH.

However, due to lack of buffering capacity, they are not able to deliver the payload efficiently. On the contrary, polyhistidine has high buffering capacity but is not sufficiently protonated at a physiological pH to enable efficient nucleic acid complexation. Introduction of polyethylenimine (PEI), which consists of many repeating diaminoethane units and is only partly protonated at a physiological pH, which is still enough to successfully bind nucleic acid and trigger the proton-sponge effect in acidifying endosomes in the pH range from 7.4 and down to > 5. Linear PEI is renowned as a gold-standard for pDNA delivery because of its above mentioned characteristics.⁽⁴⁰⁾ Interestingly, branched PEIs are generally less effective than the more flexible linear PEIs – a very important fact showing how such seemingly insignificant structural changes can influence the effectiveness of carrier.⁽⁴¹⁾ However, direct PEI membrane destabilization can affect cell viability and lead to cytotoxicity, and is also nondegradable.⁽⁴²⁾ Cytotoxicity triggers necrosis and apoptosis, defects in cell surface and mitochondria or nuclear membranes, and inhibits mitochondrial ATP synthesis. Furthermore, polycations such as PEI, PAMAM, or polylysine demonstrated in vitro triggering of the innate immune system and activation of the complement. This can be avoided with PEGilation.⁽¹⁹⁾ Introduction of artificial amino acid Stp that is comprised of tetraethylene pentamine and succinic acid showed efficient delivery and lower toxicity. In one study, an oligomer with 30 Stp units was synthesized and compared with PEI (around 25 kDa). It showed that the oligomer containing Stp units exceeded transfection efficiency of PEI by a sixfold and exhibited tenfold lower cytotoxicity.⁽⁴³⁾ To go further, the idea was to lengthen Stp by one 1,2-diaminoethane motif and design a so-called Sph. In comparison with Stp, this novel building block exhibited higher buffering capacity, better binding, and transfection of pDNA with no notable cytotoxicity at high N/P ratios as well. However, for siRNA, both showed a good binding to nucleic acid, with Stp exhibiting a higher level of gene silencing.⁽⁴⁴⁾ Other polymers, worth mentioning, are PAMAM dendrimers, dendritic polyamidoamines, which have a high charge density and consequently possess strong proton sponge activity. Nevertheless, they show high generation-dependent cytotoxicity. Linear 10 kDa amphoteric PAMAMs were found to be the most biocompatible and express favorable transfection activity. Conjugation with a large protein ligand such as transferrin can reduce cytotoxicity and preserve the transfection activity that also enables in vivo

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systemic delivery.⁽¹⁹⁾ To avoid cytotoxicity, biodegradable polymers were designed. The toxicity of such polymers is highly dependent regarding molecular weight and subsequent poor elimination. With modification of the polymer’s backbone with hydrolyzable ester bonds in extra- and intracellular space, endosomally cleavable acetal or imine bonds or bioreducible disulfide bonds in cytosol, cytotoxicity can be reduced, and furthermore, transfection efficiency can be retained or even enhanced. Bioreversible cross-linkage of ester bonds, disulfides, imines, polyglutamic acid amide, ketals and other amide linkages can also be applied.⁽⁴⁵⁾ Nature-derived polymers such as protein based ones that comprise chromosomal proteins such as unmodified or modified histones or HMG, chitosan, cyclodextrin or collagen-derived gelatin and atelocollagen, can be advantageous because of biodegradability and increased biocompatibility.⁽¹⁹⁾

1.4.3 Sequence-defined oligomers

In previous chapters, many facts were stated, considering minor changes in nanocarrier structures that can significantly increase cell transfection. As in nature, precise and unique nucleotide or amino acid sequences build macromolecules such as nucleic acids or proteins which play a crucial role in organisms. The slightest change in their sequence or substitution of a cornerstone can totally change the function of protein or gene. This observation could be also exploited for design of polymeric carriers. Properties such as size, monodisperse molecular weight, exact monomer order, orientation, topology (linear, branched, comb, dendritic), number, and attachment sites of subunits can play crucial role in biological activity. To achieve this, classical solid-phase peptide synthesis can be used, developed by Robert Bruce in 1950s and 1960s, enabling precise and highly efficient assembly of different building blocks into sequence-defined oligomers. This precise synthetic strategy can provide answers to structure related activity relationships.⁽¹⁹⁾ Various building block can be used, ranging from artificial polymeric components, artificial amino acids (Stp, Sph, Gttp), natural protected amino acids, to lipids.^(42,46) So far, a lot of research has been done about using natural amino acids such as lysine, histidine, arginine or proline,based on that some promising delivery vehicles have been developed.^(47,48)

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Figure 2: Sequence-defined oligoaminoamide architectures: linear, two-arm, three-arm, four-arm, and comb

In studies of peptide-based transfection agents, some structure related activity was observed.

Natural amino acids such as lysine, arginine, or ornithine, both linear and branched, showed very efficient nucleic acid binding. Additionaly, lysine with two amino groups (α, ε) can introduce branching points and provide more branched oligoaminoamide-sequences.⁽⁴⁰⁾ Stability can be improved with incorporation of cysteine through covalent disulfide cross-linkage that can improve transfection up to 60-fold.⁽⁴⁴⁾ Furthermore, disulfides can be reduced in the reductive cytosol, which can facilitate payload release from the vehicles at the target site.⁽⁴⁰⁾ bis (acyl)-modified lysine tryptophan and tyrosine can improve the compacting of payload through hydrophobic interactions.^(19,49,50) With histidine, based on its imidazole ring with pKa of ∼ 6, buffering can be increased because of protonation in acidifying endosomes and the transfection efficiency can be increased from 10- to 100-fold.^(38,40,51) These building blocks, as mentioned before, can be assembled by solid-phase-assisted synthesis with common Fmoc strategy, and furthermore many Fmoc protected α-amino acids are commercially available on the market. In such a way, many linear, two-arm, three-arm, four-arm, and comb architectures can be synthesized. Four-arm oligomers showed that they are very effective pDNA-compacting carriers. Because of their large cationic backbone, the compaction is much better compared to two-arms oligomers.

However, two-arm oligomers showed better compaction for siRNA.⁽⁵¹⁾ In addition, precise PEGylation and a targeting ligand can be attached.^(19,40)

14 1.4.4 Functional delivery domains

As mentioned before, dissociation and aggregation of nanoparticles and blood cells, unspecific interactions with complement and other blood components can lead to acute toxicity. To avoid that, applying hydrophilic macromolecules can shield the surface of polyplexes, prevent undesired interactions, dissociation, aggregation and prolong blood circulation time. The predominantly used shielding agent is PEG, with some alternatives such as pHPMA, hydroxyethyl starch, hyaluronic acid available.⁽³³⁾ To improve targeting specificity, selectivity and efficiency, some targeting ligands, specific to certain tissues, can be attached, especially concering cancer tissues that have upregulated receptors on its surface. The main groups of targeting ligands are antibodies, antibody fragments, aptamers, glycoproteins, peptides, and small molecules.⁽³⁸⁾ So far, the following ligands were used as targeting domains: cMBP, GE11, cRGD, VEGFR, EGFR ligands, transferrin and folate to name a few.⁽³⁰⁾ To conjugate shielding or targeting domain to the carrier, click-chemistry can be applied. The DBCO motif enables post-functional copper free click-chemistry with azido-lysines, which were introduced in oligoaminoamides systems, via strain-promoting alkyne-azide cycloaddition. Moreover, this linker increases the distance of the two units for facilitating a crosslinking reaction with a second oligomer and faster disassembly in the reductive cytosol.^(52,53)

15 2 AIMS OF THE THESIS

Delivery efficiency of non-viral carriers remains the main obstacle for successful delivery of nucleic acids as medical agents. While viral vectors show several magnitudes higher overall delivery efficiency, they exhibit high levels of immunogenicity. Hence, non-viral vehicles present a promising alternative to viral vectors due to their high-level of flexibility in structural design and many possibilities for further improvements. Recent developments in the solid-phase synthesis method allow us to assemble amino acids and similar agents quickly and easily into cationic oligomers. The latter are very efficient at complexing and stabilizing nucleic acids by forming sub-micrometer complexes called polyplexes that can protect them from degradation and facilitate delivery of nucleic acids into cells.

The following three aims were set for this master’s thesis:

First, two novel artificial amino acids were to be synthesized, based on PEI with repeating 1,2-diaminoethane motifs which is well known for its nucleic acid binding and endosomal buffering abilities. The first artificial amino acid Stp comprises four 1,2-diaminoethane motifs and the second Sph five 1,2-diaminoethane motifs. To connect the oligoamine segments in a more biodegradable manner and to enable the use of a building block in SPAS, succinic acid needs to be coupled on one terminal primary amine of four and five 1,2-diaminoethane sequences, respectively.

Second, a small library of eleven well-defined four-arm oligoaminoamides with different architectures and functionalities had to be generated via the SPAS approach to identify structure related activity relationships of DNA delivery carriers. In addition to the synthesis of Stp and Sph artificial amino acids, a different combination of amino acid sequences, for which a positive effect for transfection was reported, had to be incorporated into the four-arm structures. Four-four-arm oligoaminoamides were synthesized in a way where a good comparison between Stp and Sph artificial amino acids could be observed.

Third, four-arm structured polyplexes had to be formed with pDNA in three N/P ratios, and biophysical parameters had to be evaluated, such as pDNA complexation and binding ability, along with polyplexes’ size, and zeta potential.

16 3 MATERIALS AND METHODS

3.1 Materials

Table 1: Solvents used for experimental procedures

Solvents CAS-No. Supplier

Acetone⁶ 67-64-1 LMU chemical supply center; Faculty of chemistry and pharmacy

Acetonitrile¹ 75-05-8 VWR Int. (Darmstadt, Germany) Chloroform⁶ 67-66-3 VWR Int. (Darmstadt, Germany)

Chloroform-d² 865-49-6 Euriso-Top (Saint-Aubin Cedex, France) DCM anhydrous³ 75-09-2 Acros organics (Geel, Belgium)

DCM³ 75-09-2 Fisher Scientific (Schwerte, Germany) Deuterium oxide² 7789-20-0 Euriso-Top (Saint-Aubin Cedex, France) DMF⁴ 68-12-2 Iris Biotech (Marktredewitz, Germany) Ethanol absolute³ 64-17-5 Vwr Int. (Darmstadt, Germany)

Ether⁶ - LMU chemical supply center; Faculty of

Ether⁶ - LMU chemical supply center; Faculty of