EJECUCIÓN Y SEGUIMIENTO DE LOS PLANES DE INVERSIÓN DE

The experiments detailed here were designed to investigate the influence o f aspects of the twin syringe pump assembly on the physical stability o f DNA/PLL complexes. The twin syringe pump device arrangement, used to mix DNA complexes in this investigation, consists of the main pump plus tubing, syringes and tubing connectors (Figure 3.1). It is possible that the nature of any one o f these components could influence the physical characteristics o f the complexes formed. Experiments were

Chapter 5._______________________Manufacture o f Non-viral DNA Complexes: Results a nd Discussion

performed in order to investigate this possibility with the aim of establishing an optimum arrangement for future work.

The type of flow experienced during the mixing of complex formation has the potential to affect the way in which the components come together and level of aggregation. The internal diameter of a pipe and the mixing flow rate (velocity), are two of the main factors in the equation used to calculate Reynolds numbers thereby influencing the type of flow within the pipe (Appendix 6). The Reynolds number (Re number) is a dimensionless number used to describe the fluid dynamics within a pipe. There are three types of flow, laminar, turbulent and transitional, each represented by a specific range of Reynolds numbers. In this investigation the tubing internal diameter was varied between 0.8mm, the smallest available and the size used in previous work, 1.6mm and 3.2mm. The mixing flow rates used ranged from 15mlmin'^ to the highest capacity o f the device, lOOmlmin \ The Reynolds numbers, calculated on the basis of the internal diameter and mixing flow rates used in the investigation, ranged between Re = 265 and Re = 2652. Laminar flow in tubes is usually assumed for Reynolds numbers o f less than 2000. Mixing is known to affect the characteristics of complexes and therefore creating different, yet defined, flow regimes should reflect the level of influence each o f the factors involved including tube ID and mixing flow rate.

A series of controlled mixing experiments were performed to test the affect o f varying the tube internal diameter and the mixing flow rate on the size and zeta potential characteristics of ctDNA/PLL complexes. The polyplexes analysed in these experiments were prepared at a charge ratio of +2.0 and a DNA concentration of 25p-gmf ^ in 20mM HEPES pH7.2. The tubing used in the processing o f the polyplexes was o f three different tubing internal diameters (ID), 0.8mm, 1.6mm and 3.2mm. The flow rate through the tubing was varied ranging from 15mlmin'^ up to 100m lm in'\ The size data generated in these experiments illustrates the effect of three key process variables, mixing flow rate, tubing internal diameter but also the flow regime in the tubing represented by the Reynolds number (Figures 5.2 to 5.7).

Chapter 5. Manufacture o f Non-viral DNA Complexes: Results and Discussion 120 100 E c N 13

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Figure 5.2. A verage p article size o f ctDNA/PLL 29300 com plexes p rep a red using a tube internal diam eter o f 0.8mm, as a function o f mixing flo w rate (velocity and residence time). The complexes were p rep a red at a charge ratio o f + 2.0 in 20mM HEPES pH7.2.

T he re su lts d em o n strate that fo r sam p les p re p a re d usin g tu b in g o f three d iffe ren t internal d iam eters both the averag e p article d ia m e te r (F ig u re 5.2, 5.4 and 5.6) and d istrib u tio n (F igure 5.3, 5.5 and 5.7) in cre ase as the flow rate, an d th ere fo re Re n u m b er, increases. F o r ex am p le co m p lex e s p re p are d u sin g tu b in g w ith an internal d ia m e te r o f 0 .8 m m h ad an average size o f 9 1 .6 n m at 4 0 m lm in '' w h ile at lO O m lm in'' the av e rag e size h ad in cre ased to 1 1 1.2nm. A co m p ariso n o f the th ree d istrib u tio n plots in fig u re s 5.3, 5.5 and 5.7 also d em o n strate s that, at equal flow rates, the R e n u m b ers d ec rea ses as the tu b in g ID increases from 0 .8 m m to 3.2m m .

Chapter 5. Manufacture o f Non-viral DNA Complexes: Results and Discussion 14 12 10 8 6 4 2 0 0 50 100 150 200 25 0 3 0 0 3 5 0 c ■40ml/m in - Re 1061 S iz e (nm ) ■60ml/min - Re 1591 lOOml/min - Re 2 6 5 2

Figure 5.3. Typical size distribution profiles f o r com plexes p rep a red using a tube internal diam eter o f 0.8mm, as a function o f mixing flo w rate (residence time). The distributions represent the profde o f ctDNA/PLL 29300 complexes p rep a red at a charge ratio o f + 2.0 in 20mM HEPES pH 7.2 and mixed at a flo w rate o f either 40m lm in'\ ôOmlmin^ o r lOOmlmin \

T h e tu b in g internal d ia m e te r (ID ) and re su ltin g R e n u m b e r e v id e n tly affect the c o m p le x e s, as there is a clea r trend o f n arro w in g size d istrib u tio n as the tu b in g internal d ia m e te r in cre ases and the R e n u m b e r d ec rea ses (F ig u res 5.3, 5.5 and 5.7). A lth o u g h the sm a lle st p articles w ere o b tain ed using tu b in g w ith a 3 .2 m m ID the tren d o f red u ctio n in size and the narro w in g o f the d istrib u tio n w ith d e c re a sin g flow rate w as n o t as p ro n o u n c e d as fo r co m p lex es p ro d u ced usin g tu b in g w ith an in ternal d ia m e te r o f 0 .8 m m o r 1.6m m . A s a resu lt o f these fin d in g s the e x p e rim e n ts th at fo llo w e d w ere p e rfo rm e d usin g tu b in g w ith an ID o f 1.6m m a n d a flow ra te o f 6 0 m lm in ' unless stated oth erw ise.

Claire Nicole Mount

Chapter 5. Manufacture o f Non-viral DNA Complexes: Results and Discussion 120 100 E c 0 20 4 0 6 0 8 0 100 120 M i x i n g f l o w R a t e ( m l / m i n )

Figure 5.4. A verage p article size o f ctDNA/PLL 2 9 3 0 0 complexes p rep a red using a tube internal diam eter o f 1.6mm, as a function o f mixing flo w rate (residence time). The complexes were p rep a red at a charge ratio o f + 2 .0 in 20mM HEPES pH7.2.

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Eigure 5.5. Typical size distribution profiles fo r com plexes p rep a red using a tube