Polyplexes were formed with plasmid pCMV-luc that encodes the gene for Photinus pyralis firefly luciferase under the control of a cytomegalovirus promotor and enhancer, using water as medium.(68) pDNA was chosen due to difficult polyplex formation, owing to large molecule size. In addition, efficiency of nucleic acid encapsulation can be better determined.
Every four-arm structure was formed with pDNA at three N/P ratios; 6, 12 and 20 to determine the quantity of oligomers for successful encapsulation. The N/P ratio is a proportion of the polymer’s positively chargeable amine groups and negatively charged phosphate groups of nucleic acid. By increasing the N/P ratio, the size had reduced, whereas zeta potential increased. PDI increased on account of agglomeration due to too a high concentration of four-arm structures. Assembly of polyplexes was accomplished with rapid pipetting, 15-times, and subsequent incubation for 40 min at RT.
47
Table 9: Four-arm structure ID, main motif of four-arm structure, molecular weight and amount of total and protonatable amines
# Motif MW [g/mol] Amines in total Protonable
Since the size of polyplexes is a critical parameter for successful gene delivery and bigger particles are often prone to lower stability, the polyplex size was measured using Zetasizer Nano ZS. For more reliable results, polyplexes were formed two times with size and zeta potential measured for each. At the end, an average of two measurements was calculated.
Regarding their size, polyplexes with the Sph building block were significantly smaller due to a higher number of total amines as well as protonable amines and thus superior to polyplexes containing a Stp building block. The range of the Sph ones was below 100 nm, whereas for polyplexes with Stp, it ranged mostly above 100 nm. Surprisingly, the zeta potential of was lower, which is quite interesting, especially regarding the high number of protonable amines. Arguably, Sph is not as protonated at a physiological pH where zeta potential was measured – but is more protonated at a lower pH due to its higher buffering capacity that can boost endosomal escape and decrease undesired interactions in blood circulation. Arginine as a building block did not show any mayor differences in size and zeta potential which were either surprisingly lower or not significantly higher. This could be due to the Pbf protection group, the deprotection of which calls for high concentrations of TFA.
For this reason, we assumed that the Pbf protection group was not fully removed, and the deprotection step was repeated. After the deprotection, zeta potential and polyplex size were measured again. The difference in zeta potential was in averaged between 1–2 mV, whereas the size predominantly remained the same. 1H-NMR or ESI-MS could also present a good
48
analytical method to determine if the Pbf protection group is still present on the arginine.
However, four-arm structures are too spiky for these analytical methods. Between the other building blocks, no substantial differences were observed when comparing zeta potential and size. Changes in the sequence of building blocks did not show any mayor differences as well.
Either way, this kind of evaluation, cannot guarantee that smaller particles with a more positive zeta potential are more efficient transfection agents. The only conclusion we can certainly draw is that four-arm structures are able to bind pDNA and form small particles in the range of 100 nm, which is the optimal range for the successful accumulation of nanoparticles within the tissue of interest. Nanoparticles can behave very differently from expectations when it comes to in vitro and in vivo experiments. The 606 four-arm structure is an example of such uncharacteristic behavior. While it has the worst zeta potential among the newly synthesized four-arm oligoaminoamides, and its size is also below average, the 606 nanoparticles so far displayed the best transfection results. Regardless, we can expect a promising in vitro result with further evaluation of the anew synthesized vectors, especially after linking them with ligands through DBCO click-chemistry. Furthermore, after the in vitro and in vivo evaluation, structure related activity relationships of the incorporated amino acids and their sequences could be discerned.
49
Table 10: Particle size (Z-average), polydispersity index (PDI) and zeta potential of four-arm structured polyplexes formed at different N/P ratios
# N/P
ratio Z-average [nm] PDI Zeta potential [mV]
1493
50
Figure 19: Polyplex size is in the range of 58 nm to 226 nm with mainly PDI below 0.2. PDI increases with N/P ratio, but this is because of agglomeration due to high concentration of oligoaminoamides. On the other hand, size reduce with increasing N/P ratio.
Figure 20: Bar chart showing the average zeta potential of four-arm polyplexes formed with pDNA at three different N/P ratios. From the chart, we can see that polyplexes are positive and that they are binding pDNA very well. Experiment was repeated two times to ensure more reliable results.
The binding ability of the various synthesized oligoaminoamides was evaluated by the agarose electrophoretic mobility shift assay. This assay is widely used for the determination of the binding ability of nucelic acids to the proteins. If the nucleic acid binds the protein, thenn it stays at the postion of the well. Otherwise, it travels down the gel.(69) In this assay, free pCMV-luc migrated into the gel without hindrance, whereas sufficient polyplex formation resulted in a complete loss of electrophoretic mobility which can be seen by a
0
1493 1519 1494 1518 1495 1516 606 1513 1514 1515 1517
[Stp-H-R] R][Stp-H-W]
1493 1519 1494 1518 1495 1516 606 1513 1514 1515 1517
[Stp-H-R] [Sph-H-R] [Stp-H-W] [Sph-H-W] [R-R-R] [R-R-R] Standard
Sph 606 + KN3 [Y-Y-Y] [W-W-W] [Sph-H-K]
Zeta potential [mV]
Zeta potential
51
sharp band at the position of the samples' well. The results show that oligoaminoamides are binding pDNA very well and that there is no free pDNA which could move through the gel.
Binding was sufficient even at a lower nitrogen/phosphate ratio (N/P ratio) of 6. The electrophoretic mobility shift assay was repeated two times and the obtained results were the same.
Figure 21: Agarose electrophoretic mobility shift assay was performed to evaluate binding ability of oligomers to pCMV-luc. Polyplexes were containing 200 ng of pCMV-luc at different N/P ratios. For visualization of pCMV-luc, 150 μL GelRed™ was added to the 1 % agarose gel.
52 5 CONCLUSION
Over the last years, various nucleic acid therapeutics have been developed, representing an innovative class of biopharmaceuticals with versatile modes of action. However, due to rapid degradation and glomerular filtration, as well as poor cellular uptake, nucleic acid therapeutics must be delivered by a proper delivery system. So far, viral vectors have excelled as the most efficient delivery systems. Yet, most of them exhibit high immunogenicity. For this reason, non-viral delivery systems present a promising alternative, but pose a challenge due to their inefficient gene transfer into the cell. Thus, using a common Fmoc strategy, a four-arm oligoaminoamides library was generated by a solid-phase-assisted synthesis (SPAS). They consist of C-terminal alanine and two lysine branching points, each coupled with histidine for improved buffering capacity, followed by the elongation of the four arms with three Stp and Sph artificial amino acids repeats, respectively, and subsequent additional three repeats of explicit amino acid such as arginine, tyrosine, histidine, tryptophan, or lysine, and finally ending with cysteine and N-terminal azido lysine. Arginine and lysine were introduced to improve the DNA binding ability, tyrosine and tryptophan to improve hydrophobic interaction and to stabilize polyplexes, cysteine to stabilize polyplexes through covalent disulfide cross-linkage that can be reduced in the reductive cytosol and thereby facilitate payload release, and finally azido lysine for further attachment of shielding agents or ligands using DBCO click-chemistry strategy. All synthetized oligoaminoamides were purified using size-exclusion chromatography (SEC). Stp and Sph were synthesized due to their very efficient nucleic acid binding and endosomal buffering ability. For the first time, Sph was synthesized according to the improved protocol that significantly reduces the amount of used solvents, making its synthesis more environmentally friendly. After the evaluation of formed nanoparticles, Sph showed to be superior to Stp due to the formation of smaller particles. Overall, all synthesized four-arm structures exhibited very efficient pDNA binding, positive zeta potential and consequent formation of polyplexes in the size range of 100 nm that correspond required size between 10 and 400 nm. After the electrophoretic mobility shift assay, no pDNA could penetrate out of the particles. Regarding the effect of incorporated amino acids and their sequences, we cannot draw any clear-cut structure-activity relationships and further in vitro and in vivo evaluations still need to be carried out.
53 6 Appendix
O N H
N
O O O
N N
O O
O O NH
O
OH O
a
a
b c d
e f
i j
k l
m p
q s
s s
Figure 22: 1H-NMR spectrum of Stp building block recorded in D2O
54 Figure 23: ESI-MS spectrum of Stp building block
55
Figure 24: 1H-NMR spectrum of Sph containing impurities recorded in D2O. In the range of 7.75 ppm and 8.25 ppm, Integrals of Fmoc group are 4 what is 2 times more than it should be. That indicate the presence of Fmoc-PEHA(Boc4)-Fmoc adduct.
56
Figure 25: ESI-MS spectrum of Sph containing impurities. At 955.54 g/mol, the peak of Sph can be seen, whereas an intensive peak at 1099.55 g/mol correspond to the molar mass of Fmoc-PEHA(Boc4)-Fmoc.
57
Figure 26: 1H-NMR spectrum of pure Sph recorded in D2O
58
Figure 27: ESI-MS spectrum of pure Sph (MW 955.16 g/mol)
59
Figure 28: 1H-NMR spectrum of 606 four-arm structure, recorded in D2O. δ (ppm) = 0.68-0.72 (m, 3 H, βH alanine); 1.31- 1.42 (m, 18 H, βγδH lysine); 2.44-2.64 (m, 48 H, -CO-CH2 -CH2-CO- succinic acid); 2.76-3.75 (m, 290 H, -CH2- PEHA, βH cysteine, βH histidine, εH lysine); 4.18-4.26 (m, 8 H, αH alanine, αH cysteine, αH lysine); 4.68-4.75 (m, 18 H, αH histidine); 4.76 (s, HDO); 7.29-7.37 (m, 18 H, imidazole); 8.60-8.71 (d, 18 H, imidazole)
606
60
Figure 29: 1H-NMR spectrum of 1493 four-arm structure, recorded in D2O. δ (ppm) = 0.45-1.01(m, 3 H, βH alanine); 1.39-1.98 (m, 90 H, βγδH lysine, βγH arginine); 2.47-2.67 (m, 48 H, -CO-CH2-CH2-CO- succinic acid); 2.7-3.64 (m, 274 H, -CH2- TEPA, βH cysteine, βH histidine, εH lysine, δH arginine); 4.04-4.36 (m, 24 H, αH alanine, αH cysteine, αH lysine, αH arginine); 4.49-4.72 (m, 18 H, αH histidine); 4.78 (s, HDO); 7.29-7.39 (m, 18 H, imidazole); 8.61-8.71 (d, 18 H, imidazole)
1493
61
Figure 30: 1H-NMR spectrum of 1494 four-arm structure, recorded in D2O. δ (ppm) = 0.68-0.71 (m, 3 H, βH alanine); 1.13-2.13 (m, 42 H, βγδH lysine); 2.43-2.64 (m, 48 H, -CO-CH2 -CH2-CO- succinic acid); 3.09-3.76 (m, 274 H, -CH2- TEPA, βH cysteine, βH histidine, εH lysine, βH tryptophan); 3.94-4.28 (m, 12 H, αH alanine, αH cysteine, αH lysine); 4.4-4.65 (m, 30 H, αH histidine, αH tryptophan); 4.70 (s, HDO); 7.0-7.37 (m, 18 H, imidazole); 7.44-7.52 (m, 12 H, indolaromat); 8.47-8.71 (d, 18 H, imidazole)
1494
62
Figure 31: 1H-NMR spectrum of 1495 four-arm structure, recorded in D2O. δ (ppm) = 0.62-0.79 (m, 3 H, βH alanine); 1.38-2.06 (m, 90 H, βγδH lysine, βγH arginine); 2.51-2.61 (m, 48 H, -CO-CH2-CH2-CO- succinic acid); 3.04-3.74 (m, 274 H, -CH2- TEPA, βH cysteine, βH histidine, εH lysine, δH arginine ); 4.05-4.40 (m, 24 H, αH alanine, αH cysteine, αH lysine, αH arginine); 4.47-4.71 (m, 18 H, αH histidine); 4.80 (s, HDO); 7.28-7.37 (m, 18 H, imidazole); 8.61-8.70 (d, 18 H, imidazole)
1495
63
Figure 32: 1H-NMR spectrum of 1513 four-arm structure, recorded in D2O. δ (ppm) = 0.67-0.79 (m, 3 H, βH alanine); 1.30-1.71 (m, 42 H, βγδH lysine); 2.50-2.60 (m, 48 H, -CO-CH2 -CH2-CO- succinic acid); 3.19-3.61 (m, 298 H, -CH2- PEHA, βH cysteine, βH histidine, εH lysine); 4.02-4.25 (m, 12 H, αH alanine, αH cysteine, αH lysine); 4.28-4.65 (m, 18 H, αH histidine); 4.83 (s, HDO); 7.30-7.37 (m, 18 H, imidazole), 8.63-8.68 (d, 18 H, imidazole)
1513
64
Figure 33: 1H-NMR spectrum of 1514 four-arm structure, recorded in D2O. δ (ppm) = 0.68-0.72 (m, 3 H, βH alanine); 1.21-1.48 (m, 42 H, βγδH lysine); 2.49-2.63 (m, 48 H, -CO-CH2 -CH2-CO- succinic acid); 2.85-3.73 (m, 322 H, -CH2- PEHA, βH cysteine, βH histidine, εH lysine, βH tyrosine); 3.95-4.10 (m, 12 H, αH alanine, αH cysteine, αH lysine); 4.34-4.61 (m, 18 H, αH histidine); 4.81 (s, HDO); 6.72-6.85 (m, 48 H, phenole); 7.0-7.14 (m, 12 H, αH tyrosine); 7.23-7.33 (m, 18 H, imidazole); 8.57-8.67 (d, 18 H, imidazole)
1514
65
Figure 34: 1H-NMR spectrum of 1515 four-arm structure, recorded in D2O. δ (ppm) = 0.67-0.72 (m, 3 H, βH alanine); 1.54-2.07 (m, 42 H,βγδH lysine); 2.48-2.61 (m, 48 H, -CO-CH2 -CH2-CO- succinic acid); 2.91-3.77 (m, 322 H, -CH2- PEHA, βH cysteine, βH histidine, εH lysine, βH tryptophane); 4.21-4.33 (m, 12 H, αH alanine, αH cysteine, αH lysine); 4.37-4.46 (m, 12 H, αH tryptophan); 4.62-4.73 (m, 18 H, αH histidine); 4.79 (s, HDO); 7.15-7.23 (m, 12 H, indolaromat); 7.26-7.34 (m, 1 8H, imidazole); 7.46-7.56 (m, 12 H, indolaromat);
8.51-8.68 (d, 18 H, imidazole)
1515
66
Figure 35: 1H-NMR spectrum of 1516 four-arm structure, recorded in D2O. δ (ppm) = 0.62-0.77 (m, 3 H, βH alanine); 1.47-2.08 (m, 90 H, βγδH lysine, βγH arginine); 2.48-2.61 (m, 48 H, -CO-CH2-CH2-CO- succinic acid); 3.8-3.63 (m, 322 H, -CH2- PEHA, βH cysteine, βH histidine, εH lysine, δH arginine); 4.28-4.39 (m, 12 H, αH alanine, αH cysteine, αH lysine);4.53-4.67 (m, 12 H, αH arginine);4.67-4.73 (m, 18 H, αH histidine); 4.77 (s, HDO);
7.28-7.36 (m, 18 H, imidazole); 8.60-8.70 (d, 18 H, imidazole)
1516
67
Figure 36: 1H-NMR spectrum of 1517 four-arm structure, recorded in D2O. δ (ppm) = 0.69-0.71 (m, 3 H, βH alanine);1.33-1.71 (m, 114 H, βγδH lysine); 2.48-2.65 (m, 48 H, -CO-CH2 -CH2-CO- succinic acid); 2.96-3.55 (m, 322 H, -CH2- PEHA, βH cysteine, βH histidine, εH lysine); 4.14-4.31 (m, 24 H, αH alanine, αH cysteine, αH lysine); 4.71-4.75 (m, 18 H, αH histidine); 4.75 (s, HDO); 7.28-7.36 (m, 18 H, imidazole); 8.60-8.71 (d, 18 H, imidazole)
1517
68
Figure 37: 1H-NMR spectrum of 1518 four-arm structure, recorded in D2O. δ (ppm) = 0.67-0.68 (m, 3 H, βH alanine); 1.20-1.44 (m, 42 H, βH βγδH lysine); 2.44-2.60 (m, 48 H, -CO-CH2-CH2-CO- succinic acid); 3.11-3.53 (m, 322 H, -CH2- PEHA, βH cysteine, βH histidine, εH lysine, βH tryptophane); 3.87-4.08 (m, 12 H, αH alanine, αH cysteine, αH lysine); 4.42-4.67 (m, 30 H, αH histidine); 4.74 (s, HDO); 7.11-7.15 (m, 12 H, indolaromat); 7.21-7.26 (m, 18 H, imidazole); 7.44-7.52 (m, 12 H, indolaromat); 8.58-8.68 (d, 18 H, imidazole)
1518
69
Figure 38: 1H-NMR spectrum of 1519 four-arm structure, recorded in D2O. δ (ppm) = 0.58-0.78 (m, 3 H, βH alanine); 1.09-2.24 (m, 90 H, βγδH lysine, βγH arginine); 2.52-2.63 (m, 48 H, -CO-CH2-CH2-CO- succinic acid); 3.14-3.60 (m, 322 H, -CH2- PEHA, βH cysteine, βH histidine, εH lysine, δH arginine); 4.23-4.30 (m, 12 H, αH alanine, αH cysteine, αH lysine);
4.51-4.67 (m, 18 H, αH histidine); 4.70-4.74 (m, 12 H, αH arginine); 4.78 (s, HDO); 7.30-7.35 (m, 18 H, imidazole); 8.63-8.68 (d, 18 H, imidazole)
1519
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