Efectos sociopolíticos

evaluated to determine if utilizing a degradable scaffold yielded materials with a low toxicity profile. To do this,

100, and PEI at concentrations r

toxic compared to the nondegradable material, with PII toxicity at concentrations greater than 50

than PII-60, which has less cationic sites. This was not expected since toxicity of gene delivery materials is usually associated with the charge density, and future studies will focus on evaluating the toxicity of the nonfunctionalized monomer (D3).

0 20 40 60 80 100 120 1 C e ll V ia b il it y ( % ) 77

Cytotoxicity of PII-60, PII-100, and PEI; Values represent mean ± SD (n = 3)

3.2 Polyplex/Biological Characterization. The in vitro cytotoxicity was evaluated to determine if utilizing a degradable scaffold yielded materials with a low

an MTS assay (Figure 3.2) was performed with PII

100, and PEI at concentrations ranging from 1 to 1000 µg/mL. The polyesters were less toxic compared to the nondegradable material, with PII-100 only showing significant toxicity at concentrations greater than 50 µg/mL. Interestingly, PII-100 was less toxic cationic sites. This was not expected since toxicity of gene delivery materials is usually associated with the charge density, and future studies will focus on evaluating the toxicity of the nonfunctionalized monomer (D3).

10 50 100 500 1000

Concentration(µµµµg/mL)

PII-60 PII-100 PEI

100, and PEI; Values represent mean ± SD (n = 3)

The in vitro cytotoxicity was evaluated to determine if utilizing a degradable scaffold yielded materials with a low ) was performed with PII-60, PII-

g/mL. The polyesters were less 100 only showing significant 100 was less toxic cationic sites. This was not expected since toxicity of gene delivery materials is usually associated with the charge density, and future studies will

1000

78

Table 3.2. Complex size and surface charge measurements

PEI PII-60 PII-100

N/P Size a (nm) ζ-potential a Sizea (nm) ζ-potential a Sizea (nm) ζ-potential a 1 146 -25.6 309 -30.5 402 -31.7 2 314 -19.6 220 7.6 325 1.9 4 347 20.9 272 31.8 332 29.1 10 346 22.6 222 45.0 363 33.9 20 358 27.2 322 42.6 464 35.1 a

determined by a 90Plus Particle Size Analyzer

The properties of the polyplexes were then evaluated for surface charge (ζ- potential) and complex size (Table 3.2). For nontargeted transfection, it is important for the complex to maintain a positive surface charge to interact with the cell surface. Complexes were formed between the TEG materials (PII) or PEI and a 6000 base pair plasmid, pCMV-Luc. The ζ-potentials revealed that both of the polyesters reached a near neutral charge around an N/P ratio of 2, and PEI was between 2 and 4. Requiring a lower concentration to create a positive surface for the PII materials was promising since they could potentially transfect at lower concentrations than PEI. Complex sizes were then measured by dynamic light scattering. The average diameter of the unbound plasmid was ~750 nm, and it was shown that each material was able to complex DNA and form a more compact structure.

Figure 3.3. Transfection experiments of PII

of 20 for the polyesters and N/P of 4 for PEI

Based on the results of low toxicity, ability to compact DNA, and positive surface charge at low concentrations, the materials were evaluat

ability. For the transfection experiments, the same pCMV luciferase reporter gene was used

Figure 3.3, at an N/P of 20 the polyesters showed poor tra

performing 1000 fold lower than PEI (whose optimum performance was taken at an N/P of 4). Despite concentrations well above charge neutrality and complexation, there was no bioactivity. The DLS experiments

formation without distinguishing

characterize the complex formation, DNA binding gels were used.

1 10 100 1000 10000 100000 lo g R L U / µµµµ g p ro ti e n 79

riments of PII-60, PII-100, and PEI with HeLa cells at an N/P ratio of 20 for the polyesters and N/P of 4 for PEI

the results of low toxicity, ability to compact DNA, and positive surface charge at low concentrations, the materials were evaluated for their transfection ability. For the transfection experiments, the same pCMV-Luc plasmid containing the luciferase reporter gene was used, and PEI served as the positive control. As seen in

, at an N/P of 20 the polyesters showed poor transfection ability, with PII performing 1000 fold lower than PEI (whose optimum performance was taken at an N/P of 4). Despite concentrations well above charge neutrality and complexation, there was no bioactivity. The DLS experiments, however, only gave limited insight into complex

ation without distinguishing the nature of the cation/anion interaction characterize the complex formation, DNA binding gels were used.

PII

PII

PEI

100, and PEI with HeLa cells at an N/P ratio

the results of low toxicity, ability to compact DNA, and positive ed for their transfection Luc plasmid containing the and PEI served as the positive control. As seen in nsfection ability, with PII-100 performing 1000 fold lower than PEI (whose optimum performance was taken at an N/P of 4). Despite concentrations well above charge neutrality and complexation, there was ave limited insight into complex anion interaction. To further

PII-60

PII-100

80

Figure 3.4. Agarose gel electrophoresis assay; lane L) DNA molecular ladder; lane P: Naked

DNA; lane A) 5:10 (polymer/DNA w/w ratios); lane B) 10:10; lane C) 20:10; lane D) 40:10; lane E) 50:10

The DNA binding ability was investigated using agarose gel electrophoresis to determine the polymer concentrations that PII-100 could effectively bind DNA (Figure 3.4). In this test when a current was applied, naked DNA migrated down the gel. As aliquots of cationic material were added, the electrostatic interactions resulted in charge neutralization and migration of the DNA bind was quenched. Polymer/DNA complexes were formed at ratios from 0 µg to 50 µg of polymer with 10 µg of DNA. As seen in Figure 3.4, even at high polymer concentrations, there was no effective DNA binding, or complex formation. With a molecular weight of the repeat unit near 500 g/mol, a material with only a total molecular weight of 4000 g/mol would only have eight amines per polymer chain. Transfection efficiency is greatly dependent on the amine density in

81

order to complex DNA and to serve as a ‘proton sponge’ for endosomal release, and the poor results of these materials may stem from their low amine density.

4. Conclusions

A facile Diels Alder reaction between a functionalized diene and fumaric acid was exploited to synthesis an amine functionalized monomer. Previously, materials utilizing this chemistry were limited to an upper limit of 50% incorporation of the amine monomer with higher feed ratios resulting in crosslinking. A new monomer was developed that utilized a tetrasubstituted double bond to prevent crosslinking. After successful synthesis of this monomer, a series of degradable tertiary amine containing copolymers and a homopolymer were synthesized. Installment of the methyl group on the double bond was successful in limiting crosslinking and permitted development of the amine homopolymer. Two types of polyesters were synthesized, the first containing octanediol, and the second containing tetraethyleneglycol. The former was found to have poor solubility, and the latter materials were used in the biological characterization experiments. It was shown that the degradable system was much less toxic than the control (PEI), but only formed weak complexes with DNA and ultimately had low transfection efficiency. Only low molecular weight material was synthesized, resulting in low amine density. This most likely led to the materials poor delivery performance.

82 References

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Chapter 4. Developing Guanidine Functionalized Materials for