Controlled Mixing

Controlled mixing using a vertically mounted twin syringe pump, illustrated in Figure 3.5, was used in the production of all samples described in this report (Appendix 3). The device was purchased from Harvard Apparatus, Holliston, USA, and customised by the Biochemical Engineering workshop. University College London. The syringe pump utilises a micro-controller that controls a small step angle stepping motor that drives a lead screw and pusher block. This function allows the flow rate and target volume of both the refill and infuse options to be accurately controlled.

The assembly of the unit comprises two syringes tubing and adaptors. The device accommodates two syringes and has the facility to be altered to accommodate a range of syringe sizes. The syringes used for mixing were plastic disposable Becton- Dickinson syringes. The outlets of the syringes were, unless stated otherwise, fitted with three-way luer-lock adaptors and 0.8mm silicone tubing (Bio-Rad, Hercules, CA, USA). The outlet tubing of the syringes was joined together with a plastic T-connector, which was the site of mixing.

The process of mixing the formulation begins by loading, independently, equal volumes of DNA and complexing agent solutions, prepared to the desired

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co n c en tratio n s, in to the syringes using the re fill o p tio n . In the in fu se m o d e the pu m p then driv es the tw o syrin g es forcing the tw o so lu tio n s th ro u g h th e tubing. T he tw o so lu tio n s m eet at the sam e p oint in tim e an d sp a c e at the T -c o n n ecto r and passes through the m ix in g tube. T he m ix tu re w as then c o lle c te d in to a cu v e tte (S tarsted t, G erm an y ) o r th e ap p ro p riate co llectio n an d sto rag e vessel. T h e sam p les w ere then an aly sed w ith P C S and the Z eta siz er fo r size an d z e ta p o ten tial d e term in a tio n .

Figure 3.5. Twin Syringe Pump Mixing Device

(1) Carriage and drive shaft, simultaneously push syringe plungers; (2) Two syringes loaded with equal volumes o f DNA and complexing agent; (Sa&b) Stopcocks; (4) T- junction where DNA and complexing agent meet and mix; (5) Mixing tube; (6) Collection vessel.

DNA

agen t

^ lip op lex / p olyp lex / iip op olyplex

Claire Nicole Mount

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3.8 Particle Size and Zeta Potential

A fte r m ix in g in the syringe p u m p d ev ice th e zeta p o ten tial and size o f th e c o m p lex e s p re p are d u n d e r vario u s co n d itio n s w ere m easu red at 2 5°C usin g a M alv ern Z e ta siz e r (M odel: 3000, M alv ern In stru m en ts L td, U K ) as d e ta ile d in a p p e n d ix 4 (F ig u re 3.6).

Figure 3.6. ZE TASIZER 3000

(a) Sample entry port fo r zeta potential cell, (b) Sample exit port fo r zeta potential cell, (c) Access to the size and zeta potential cells, (d) Computer interface

T h e ze ta p o ten tial o f a disp ersio n o r em u lsio n is one o f the m a jo r p a ram eters th at control d isp ersio n stability. M o n ito rin g this p a ra m e te r can sh o rten p ro d u c t fo rm u latio n tim es, d isc o v e r the m ech a n ism s re sp o n sib le fo r the stab ility o f a d isp ersio n , and o p tim ise flo cc u latio n w here this is re q u ired . T he z e ta p o ten tial (Ç) o f the D N A c o m p lex e s w as d eterm in e d by laser D o p p le r v elo cim etry (L D V ) usin g th e M alvern Z e ta siz e r in “z e ta ” m ode. T h e zeta p o ten tial describ es th e su rfa ce p ro p e rtie s and ch a rg e o f co llo id al sy stem s given in m illi-v o lts (m V ). W ith in fiv e hours o f m ix in g m e asu rem en ts are p erfo rm ed on a 4m 1 sam p le o f th e su sp en sio n . T h e sam p les are in jected in to the d evice using ten m illilitre d isp o sab le sy rin g es (B ec to n -D ick in so n , M ad rid , S pain). P rio r to injectio n o f th e sam p le, the sy stem w as flu sh e d w ith d io n ised w a te r until the cou n t rate o f the in stru m e n t re g istered less then ten p articles. E ach m e asu rem en t read in g c o n sisted o f ten sub ru n s. T y p ica lly ten d u p lic a te re ad in g s o f each sam p le w ere taken in series allo w in g the g en e ratio n o f a m ean av e rag e ze ta p o ten tial an d stan d ard d ev iatio n from the m ean. C alib ratio n o f th e in stru m en t w as c o n d u c te d betw een m easu rem en ts u sin g stan d ard s (D T S 5 0 5 0 , M alv ern In stru m en ts L td) m e a su re d to be -55± 5m V ,

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The particle size and size distribution of the DNA complexes were measured using light scattering technique. Light scattering offers advantages over some of the other techniques for particle size analysis such as optical and electron microscope, sedimentation, centrifugation, filtration and diffusion. Firstly, the system under study can be observed in situ without significant perturbation. Secondly, the method is absolute in the sense that the theory permits the reduction of data directly to the final desired results without the need of secondary schemes for calibration. Finally, the measurements are almost instantaneous and can be recorded continuously so that rate processes may be followed (Kerker, 1969).

The particle size and distribution of the polyplexes and lipopolyplexes analysed in this investigation were measured by dynamic light scattering (DLS) using the Malvern Zetasizer in “size” mode. Each measurement consisted of ten sub measurements generating a distribution curve. Typically five duplicate readings of each sample were taken in series. The size data derived using the zetasizer is a function of the light scattered by the particles (Appendix 2). The intensity of light scattered at a given angle from a visible laser beam is related to the Brownian motion of the scattering particles in suspension and is measured by a photon detector. This temporal fluctuation is analysed by a correlator that computes, in real time the autocorrelation function to yield the effective translational diffusion coefficient, Ôt r a n s- The apparent

hydrodynamic diameter, D h , of the particles is then determined from the Stokes-

Einstein relation:

Strans — _______

371|XDh

where k is the Boltzmann’s constant, T is the absolute temperature o f the sample, and p is the solution viscosity. In the investigations detailed here the data were interpreted using the automatic analysis software generating a mean or “z-average” for each sample along with a standard deviation and polydispersity.

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3.9 Variables Studied

3.9.1 M ix in g F lo w R ate

In itia lly a series o f c o n tro lled m ixing ex p e rim en ts w ere p e rfo rm e d in w hich the flow ra te at w hich the c o m p lex e s w ere p re p are d w as v arie d fro m 15m lm in'* to the h ig h est ca p a c ity o f the d ev ice, lOOmlmin'*. S u b seq u en t e x p e rim e n ts w ere then p erfo rm ed at a m ix in g flow rate o f 6 0 m lm in ‘ unless stated o th erw ise .

3.9 .2 M e th o d o f C o llectio n

T h e ex te n t to w hich the collection m eth o d affec ts the p h y sical c h a ra c te ristic s o f the c o m p le x e s w as assessed. T he m eth o d o f co llec tio n w as in v e stig a te d by v arying both the v essel used to co llec t the p o ly p lex es and the v o lu m e o f p o ly p lex so lu tio n co llec te d in to the vessels. T h e v essels used ra n g ed from cu v e tte s and falco n tu b es to g lass bea k ers an d v o lu m e co llec te d w as v arie d from 12ml to 50m l (F ig u re 3.7).

Figure 3.7. Variation in the Method o f Collection

C u v e t t e 2 m 1 X 12

T

I

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3.9.3 Tubing Characteristics

The characteristics of the tubing used to bring the two components of the complex together and to deliver the complexes to the collection vessel were varied in experiments. These included the tube material, tube internal diameter and the tube length. The tubing used was of two descriptions, silicon, used in all initial work, and PharMed. Each of the tubing types has defined characteristics (Table 1). The tubing internal diameter was also varied between 0.8mm, the smallest available and the size used in previous work, 1.6mm and 3.2mm. The internal diameter of a pipe is a factor in the equation used for the calculation of flow Reynolds numbers, which describes flow characteristics. Varying tube internal diameter changes the Reynolds number and therefore the flow regime (Appendix 6). The residence time o f the complexes in the mixing tube, that is the tubing that extends from the T-junction, where the materials come together down to the collection vessel, was also investigated (Appendix 7). In previous research all complexes were prepared using a constant mixing tube length of 2cm. In this investigation however it was important to ensure that fully developed flow was created under all conditions varied in the experiments to ensure thorough mixing occurred. The minimum length required to achieve fully developed flow was therefore calculated for all combinations of variables under investigation and the minimum tubing length varied accordingly (Appendix 8). The tube lengths investigated ranged from 0.5cm to 80cm.

Table 3.1. Comparison of Tubing Material

Quality Silicone PharMed

Appearance Translucent Off-white

Flexibility Excellent Excellent

Autoclavable Yes Yes

Chemical compatibility Fair Good

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3.9.4 Degree of Mixing

A mixing time experiment was performed using the syringe pump. An acid, (IM HCl), alkali, (IM NaOH) plus indicator (Bromocrysol blue - Sigma Aldrich) were used in a titration type experiment in which the acid and alkali were in separate syringes with the acid containing the indicator. The two solutions were mixed using the syringe pump and the time taken to see a colour change upon mixing was recorded.

3.9.5 Maturation Time

Complexes formed under different experimental conditions were in some cases stored for extended periods of time. The time periods used varied from thirty minutes up to periods of three months. The storage took place at both room temperature and 4°C. Following the defined period o f storage the size and zeta potential of the complexes were assessed.

3.9.6 Buffer

Different buffers were used to alter environmental conditions. All experiments were prepared in 20mM HEPES, unless stated otherwise. The pH of the HEPES buffer was varied within the range of pH 6 and pH 8.5 by titrating with NaOH. Adding concentrated NaCl, prepared in deionised water, to the appropriate buffer, produced the buffers containing different salt molarities. The type of buffer was also varied between HEPES buffer and ultra-pure water. W ater used in the preparation o f buffers was obtained from a M illipore (18.2M-cm) Milli-Q water system (Millipore Ltd., Bedford, MA, USA).

3.9.7 DNA Concentration

A series of experiments were conducted in which the DNA concentration was varied, in combination with other factors, between 12.5pgml'^ to 300|Xgmr^ in order to characterise the affect on both the size and zeta potential of the DNA complexes. It has been suggested that stabilisation of complexes can be achieved by three main techniques, one of which is temperature stabilisation. Size measurements were therefore also made on a series of complexes formed at various DNA/PLL

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concentrations and repeated again 24 hours and 72 hours after preparation with storage at either room temperature or 4°C.

3.9.8 Statistical Studies

Response surface methodology (RSM) is a mathematical technique that is used to obtain engineering equations between a measured (response) parameter and a set of defined variables (factors) that affect it. The methodology, which is described in greater detail in appendix 9, consists of a number of steps including:

1) Recognition o f the problem and development o f all the ideas about the objective o f the experiment that is to be designed.

2) Establish the factors and the levels and range at which they are to be varied. 3) Select o f response variable(s).

4) The choice o f experimental design involves the consideration o f the sample size, that is the number o f replicates, selection o f a suitable run order fo r the experimental trials and establishment o f any blocking, other randomisation restrictions or identification o f any additional design features to be applied.

5) Set up a mathematical model to describe the experiment.

6) Data collection can then begin with the experiment perform ed in run and block order.

The identification of each factor and the range over which it will be allowed to change in the experiments relies on the experimenters knowledge o f the process under investigation. Initially, particularly in the absence of prior knowledge about the extent of influence of each factor, the number o f potential factors may be too high to allow an engineering solution to emerge. In practice therefore, an initial screening design is performed to determine the relative influence of each factor. Analysis of the results provides statistically significant information on the importance o f each factor so that it is possible to select those factors considered to have the largest effects. The most influential factors are then used in a response surface design (Kalil et al., 2000; Lundstedt et a l , 1998). In this experiment the most influential factors are allowed to vary in accordance with a pre-determined pattern defined by the design while the value

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of each of the remaining less influential factors, identified through the screening experiment, is maintained constant throughout the experiments.

In all statistical designs presented in this study two levels are assigned to each variables and represent the allowable limits, that is the maximum and minimum values set on the basis of preliminary trials. The final relationship that is determined must hold within these limits. Additionally, in all the designs centre repetitions are carried out in order as this allows us to derive the experimental error variance and to test the predictive validity of the model (Ficarra et a l , 2002b). In the case of the plasmid DNA complexes, two response parameters (the z-average size and zeta potential) and either six or seven independent factors were included in the initial screening experiment, which was designed and analysed using Design-Expert 5® (Stat-Ease, Inc., Minneapolis, USA). For each response parameter statistically significant factors were selected using analysis of variance (ANOVA) and generated a model equation and response surfaces to describe the process as described in chapters 6 and 7.

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3.9.8.1 Six-Factor Statistical Study of a Polyplex Formulation

A half-factorial design was created as a screening experiment using six factors, from the pDNA/PLL polyplex formulation process. The factors were varied at two levels, high (+1) and low (-1) with the addition of twelve centre-points (0). The information generated from the screening experiment was used to identify the significant factors to include in the follow up RSM design. A D-optimal RSM design was created using three of the initial six factors. The factors were again set at two levels and centre points were included. The completed RSM design was analysed using a quadratic model in the DX5® software. The factors and specific levels from each experiment can be seen in table 3.2 and 3.3 respectively and the full details of the design are displayed in appendix 10. The trials in each design were randomised and carried out in run order. Six responses were measured in both designs including the average particle size and the zeta potential of the pDNA/PLL complexes measured immediately after mixing, and re-measured after one week and two weeks with storage at 4°C.

Table 3.2. Six-Factor Half-Factorial Experiment: Factors and Levels