Automated In-Line Mixing System for Large Scale Production of Chitosan- Based Polyplexes

Journal of Colloid and Interface Science 500 (2017) 253–263

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Journal of Colloid and Interface Science

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Regular Article Automated in-line mixing system for large scale production of chitosan- based polyplexes

Ashkan Tavakoli Naeini a, Ousamah Younoss Soliman a, Mohamad Gabriel Alameh a, Marc Lavertu b, ⇑ Michael D. Buschmann a,b, a Institute of Biomedical Engineering, Polytechnique Montreal, Montreal, Quebec, Canada b Department of Chemical Engineering, Polytechnique Montreal, Montreal, Quebec, Canada graphical abstract

article info abstract

Article history: Chitosan (CS)-based polyplexes are efficient non-viral gene delivery systems that are most commonly Received 5 February 2017 prepared by manual mixing. However, manual mixing is not only poorly controlled but also restricted Revised 5 April 2017 to relatively small preparation volumes, limiting clinical applications. In order to overcome these draw- Accepted 5 April 2017 backs and to produce clinical quantities of CS-based polyplexes, a fully automated in-line mixing platform Available online 07 April 2017 was developed for production of large batches of small-size and homogeneous CS-based polyplexes. Operational conditions to produce small-sized homogeneous polyplexes were identified. Increasing mix- Keywords: ing concentrations of CS and nucleic acid was directly associated with an increase in size and polydisper- In-line mixing sity of both CS/pDNA and CS/siRNA polyplexes. We also found that although the speed of mixing has a Chitosan siRNA negligible impact on the properties of CS/pDNA polyplexes, the size and polydispersity of CS/siRNA poly- Plasmid plexes are strongly influenced by the mixing speed: the higher the speed, the smaller the size and poly- Polyplex dispersity. While in-line and manual CS/pDNA polyplexes had similar size and PDI, CS/siRNA polyplexes Non-viral gene delivery were smaller and more homogenous when prepared in-line in the non-laminar flow regime compared to Bioactivity manual method. Finally, we found that in-line mixed CS/siRNA polyplexes have equivalent or higher silencing efficiency of ApoB in HepG2 cells, compared to manually prepared polyplexes. Ó 2017 Elsevier Inc. All rights reserved.

1. Introduction

⇑ Corresponding author at: Department of Chemical Engineering, Polytechnique Montreal, Montreal, Quebec, Canada. Non-viral gene delivery mainly relies on the use of cationic E-mail address: [email protected] (M.D. Buschmann). lipids and cationic polymers to bind and condense negatively http://dx.doi.org/10.1016/j.jcis.2017.04.013 0021-9797/Ó 2017 Elsevier Inc. All rights reserved. 254 A. Tavakoli Naeini et al. / Journal of Colloid and Interface Science 500 (2017) 253–263 charged nucleic acids (NAs). Complexes formed between a nucleic connected via an adjustable regulator to a compressed air supply acid and polymers are referred to as polyplexes. Upon mixing, [36]. They were the first to reporting an in-line mixing device for these oppositely charged species bind to each other by means of the preparation of polyplexes (PEI/pDNA), however, their device attractive electrostatic interactions, a process favored by a con- was not easily scalable. In 2011, Kasper et al. claimed reproducible comitant increase in entropy due to the release of low molecular production of small and homogeneous PEI-pDNA polyplexes by weight counter-ions. In very dilute solutions, complex formation means of an up-scaled micro-mixer system, consisting of two syr- leads to nanoparticle suspensions at the colloidal level, while mix- inges and a T-connector [33]. They reported a reduction in the size ing in a concentrated regime results in macroscopically flocculated of polyplexes by increasing the mixing speed and reducing the con- systems [1–4]. Nonetheless, higher mixing concentrations result in centration of pDNA. Finally, a chaotic mixer specified as ‘‘staggered higher dose of NA in a given volume of the suspension, hence herringbone microfluidic mixing device” was designed based on increasing the deliverable doses. microfluidic mixing in a laminar regime to produce well-defined Chitosan (CS), a naturally derived polycation, is a biocompatible lipid nanoparticles (LNPs) [39–40]. This staggered herringbone pat- and biodegradable linear polysaccharide that has gained interest tern creates transverse flows in the microchannels to induce chao- for safe delivery of NA. It has been demonstrated that CS-based tic mixing at a low speed of the laminar regime. Belliveau et al. delivery systems are efficient for delivery of plasmid DNA (pDNA), investigated the ability of SHM to produce monodisperse LNP- [5–12] and siRNA [13–24] both in vitro and in vivo. Manual mixing siRNA complexes [31]. While the size of LNPs remained fairly con- is the most commonly used method to produce CS-based poly- stant over the range of the tested flow rates, the lowest PDI for plexes. However, this manual method varies in implementation LNP-siRNA complexes was obtained at the highest tested mixing which raises some concerns regarding comparability of the proper- flow rate. In addition, reducing the concentration of siRNA resulted ties of such complexes. Classical pipetting is a frequently reported in a reduction in polydispersity. Although the above-mentioned technique in the literature, where CS is promptly added into NA methods were introduced for up-scaled mixing platforms, there solution followed by pipetting the mixture up and down for has been no systematic study to date on the operational parame- homogenization [5,7–9,11,13,25–27]. One other manual approach ters of a fully automated in-line mixing system that can repro- is addition of NA into excess CS solution under stirring condition ducibly prepare large batches of CS-based polyplexes with to allow formation of polyplexes [17–19,24]. Another technique defined physico-chemical properties, and transfection efficiency. is drop-wise addition of CS solution into equal volume of NA solu- The main objective of this study was to develop a computer tion followed by quick mixing, [28] while similar method was controlled in-line mixing platform for reproducible production of reported with reverse order of addition [16]. Addition of siRNA into small sized homogeneous CS-based polyplexes, and examine the CS followed by vortexing of the complex for a short period (30 s) is influence of mixing parameters on the properties of polyplexes. also a reported technique [14,21]. Properties of final polyplexes We hypothesized that reducing the NA concentration and increas- depend upon the mixing technique, experience of the operator, ing mixing speed would reduce the size and polydispersity of CS- [29] and order of addition of the two polyelectrolytes [30]. Further- based polyplexes. Another objective was to assess the in vitro more, independent of the adopted approach, conventional manual silencing efficiency of the CS/siRNA polyplexes, where we hypoth- mixing is not only a poorly controlled method which may result in esized equal or even better bioactivity for in-line CS/siRNA com- irreproducibility, [29,31–32] but it is also restricted to relatively pared to manually prepared polyplexes. small preparation volumes, that seriously limit applicability. Small scale manual preparation of complexes poses risks of batch to 2. Materials and methods batch and inter-user variability, [29,33–34] thus a controlled and repeatable production process of polyplexes in large quantities is 2.1. Materials a prerequisite for their clinical applications [32]. Several mixing techniques and devices have been proposed in Chitosan was obtained from Marinard. Trehalose dehydrate (Cat the literature to prepare large volumes of complexes. These tech- #T0167), L-histidine (Cat #H6034) were from Sigma. HepG2 (hep- niques rely on in-line mixing in Y or T-shaped connectors, [33– atocellular carcinoma) cells (Cat# HB-8065), and Eagle’s Minimum 36] microfluidics, [29–32,37–40] as well as jet mixing [41–42]. Essential Medium (Cat #30-2003) were from American Type Cul- To the best of our knowledge, the very first in-line mixing system ture Collection (ATCC). Dulbecco’s Modified Eagle Medium, high for complex production was designed by Zelphati et al., where syr- glucose pyruvate (DMEM-HG, Cat #12800-017) was from Gibco. inges were used to drive pDNA and cationic lipids into a T- Fetal bovine serum (FBS, Cat #26140) was from Thermo Fisher Sci- connector [34]. Later, John and Prud’homme invented a Confined entific. Plasmid EGFPLuc (Cat #6169-1) was from Clontech Labora- Impinging Jet mixing (CIJ) apparatus to generate two opposing tories. siRNA targeting ApoB mRNAs (siRNA ApoB) contains sense high velocity linear jets of polyelectrolyte solutions, where the sequence of 50-GUCAUCACACUGAAUACCAAU-30 and antisense 50- two streams collide in a chamber for a few milliseconds (rapid AUUGGUAUUCAGUGUGAUGACAC-30 was obtained from GE (Cus- mixing takes place in a time less than the nucleation and growth tom synthesis, A4 scale). Double stranded oligodeoxynucleotide time of complexes) [42]. Ankerfors et al. applied the CIJ system (dsODN, 21 bp) encoding the same sequences and mimicking to investigate the influence of mixing time on the size of produced siRNA ApoB physico-chemical properties was obtained from Inte- Polyallylamine hydrochloride (PAH)/polyacrylic acid (PAA) com- grated DNA Technologies Inc, Coralville, IO. The commercially plexes. They reported production of larger particles at reduced available liposome, DharmaFECTTM2 was from Dharmacon RNAi speeds of the polyelectrolyte streams [41]. While establishment Technologies and Diethyl pyrocarbonate (DEPC) from Sigma of a jet mixer is sophisticated, mixing in a Y or T-shaped connector Aldrich (Cat #D5758). is a simpler design. In 2005, Clement et al. introduced an in-line mixing platform (but not computer controlled) based on two peristaltic pumps and a Y-connector. They showed no difference in 2.2. Preparation of CS, plasmid DNA and siRNA for mixing transfection efficiencies of manual and in-line mixed complexes, [35] however, the impact of mixing conditions (e.g., speed and The plasmid eGFPLuc stock solution was prepared and charac- NA concentration) on physico-chemical properties of complexes terized by UV spectrophotometry, as described in Lavertu et al. was not studied. In 2010 Davies et al. designed a novel mixing [9]. The plasmid DNA (pDNA) stock solution was diluted to 10, device based on a static mixer and a cylindrical pneumatic actuator 50, 100, 200, and 300 lg/mL with Milli-Q water as well as sterile A. Tavakoli Naeini et al. / Journal of Colloid and Interface Science 500 (2017) 253–263 255

filtered trehalose and histidine (pH 6.5) solutions for having final media with moderate to high electrolyte concentration. For ZP concentration of 0.5% and 3.5 mM, respectively (these are opti- measurements, each sample was diluted 8-fold to a final volume mum concentrations of excipients for freeze-drying and stability of 800 mL by adding 700 mL of buffer (trehalose 0.5%, and 3.5 mM of complexes over time that have been previously established) histidine) for a final ionic strength of about 1 mM. Each sample [27]. The siRNA ApoB stock solution was prepared and character- was analyzed for three consecutive runs at 25 °C. ized by UV spectrophotometry, then diluted to 10, 50, 100, 200, 300, and 400 mg/mL with RNase/DNase free water as well as the 2.6. Transmission electron microscopy imaging excipients as described for plasmid DNA. Commercial chitosan was first heterogeneously deacetylated to 92% using concentrated Morphology of both inline and manually produced polyplexes sodium hydroxide and was depolymerized to 10 kDa and 2 kDa was studied using Transmission Electron Microscopy (TEM). On a using nitrous acid, as established and previously reported by our carbon coated copper grid (200 mesh, Electron microscopy group [9]. It has been reported that the 10 kDa chitosan with sciences), 5 mL drop of polyplex suspension was pipetted and DDA of 92% (CS92-10) form stable complexes when prepared at allowed to evaporate for 30 min. To avoid drying artifacts, remain- molar ratio of CS amine to NA phosphate ratio of 5 (N:P = 5), and ing solvent was removed by blotting using a filter paper. 5 mLof2% also dissociates effectively upon release from lysosomes, maximiz- phosphotungstic acid was placed on the grid. After 10 min the stain ing the level of transfection [12]. Degree of deacetylation (DDA) was removed as described above. The grid was washed two times and number-average molar mass (Mn) of chitosan was confirmed with deionized water to avoid any salt contamination from the by 1H NMR, [43] and gel permeation chromatography multi- sample and air dried. TEM images were obtained using a Tecnai angle light scattering [44]. A chitosan stock solution at 5 mg/mL T12 electron microscope operating at 120 keV. For each sample was prepared from dry powder dissolved in 28 mM HCl overnight four random areas on the grid were imaged at different at room temperature (RT). The stock solution was sterile filtered, magnifications. then diluted with Milli-Q or RNase free water (for siRNA applications), as well as excipients as described before for N:P = 5, based 2.7. Sterile and RNase free production via AIMS on the concentration of nucleic acid (either pDNA or siRNA). The AIMS including the upstream vessels, collecting containers, 2.3. Preparation of CS/NA polyplexes by manual mixing (classical as well as the tubing set were autoclaved for sterility. In order to pipetteing) maintain the sterility of AIMS, the entire tubing network was iso- lated from the environment where each vessel was open to atmo- m CS/NA polyplexes were prepared by manual addition of CS sphere via a 0.22 m syringe filter in order to maintain pressure (100 lL) to equal volume of NA (diluted to maintain N:P = 5), fol- during pumping and mixing. When working with siRNA, the closed lowing by immediate pipetting up and down repeated 10 times. system was treated with diethylpyrocarbonate (DEPC) in order to Samples were then incubated for 30 min at RT before analyses or inactivate contaminant nucleases. transfections. 2.8. Cell culture and transfection

2.4. Preparation of CS/NA polyplexes by the Automated In-line Mixing HepG2 cells were cultured in EMEM supplemented with 8% System (AIMS) fetal bovine serum (FBS). One day prior to transfection, cells were seeded in a 24 well plate at 300,000 cells per well to reach 80% In addition to the manual mixing, both types of polyplexes (CS/ confluence on the day of transfection. Prior to transfection, media pDNA and CS/siRNA) were produced via AIMS in the exact same over cells was aspirated and replenished with serum free DMEM- formulation as for the manual mixing. Both initial and advanced HG media (pH 6.5) supplemented with 0.976 g/L MES and 0.84 g/ versions of AIMS (configuration a and b of Fig. 1) were tested. Pre- L NaHCO . Inline and manually mixed polyplexes were prepared pared samples were incubated in the original collecting vessel for 3 as described above and a specific volume was pipetted into each 30 min at RT before analyses or transfections. well to reach a target siRNA concentration of 100 nM/well (0.66 mg/500 mL). FBS was added 5 h post-transfection to reach a 2.5. Polyplex size, polydispersity, and surface charge analysis final concentration of 8%. Transfection media was aspirated 24 h post transfection, replenished with complete EMEM and incubated Ò Hydrodynamic size and polydispersity index of the produced for an extra 24 h. DharmaFECT 2/siRNA lipoplexes were used as polyplexes were measured using dynamic light scattering (Zeta- positive control and were prepared as per manufacturer recom- sizer Nano ZSP-ZEN5600, Malvern Instruments, Worcestershire, mendation. Lipoplex and naked siRNA transfection were performed UK). Samples were diluted by 4–5-fold by adding Milli-Q or RNase at a final concentration of 100 nM/well. Chitosan only and free water, then analyzed for three consecutive runs at 25 °C. Zeta untreated cells were used as negative controls. potential (ZP) measurements were made with the Malvern Zeta- sizer Nano ZSP using folded capillary cells. ZP value was calculated 2.9. Assessment of silencing efficiency from the measured electrophoretic mobility uE by applying the ¼ = ð Þ Henry equation: ZP 3guE 2ef ja , where UE is the velocity of Polyplex bioactivity was assessed using MIQE compliant quan- the particle in the applied electric field (referred to as the elec- titative real time PCR (qPCR) [45]. All primers and probes were trophoretic mobility), e and g are the dielectric constant and the intron spanning to avoid amplification of potential genomic DNA viscosity of the medium, respectively. f (ja) is the Henry’s function, contamination and in silico validated using the NCBI blast tool for where a is the particles radius, and 1/j is the Debye length, thus ja specificity (Supp info). Total RNA extraction was performed 48 h is the ratio of particle radius to Debye length. For calculation of the post transfection using the EZ-10 Spin column Animal total RNA ZP from electrophoretic mobility, a value of 3/2 for the Henry func- extraction kit (BioBasic). Total RNA was extracted as per manufac- ¼ guE tion was used so that ZP e was used. This limiting case is turer protocol and subjected to in-column DNase digestion (Qia- referred to as the Smoluchowski equation, an approximation that gen, Cat#79254). The purity of the eluate was assessed using UV/ is valid when the size of the particle is much larger than the Debye VIS spectrometry at 260, 280, 230 and 340 (Nanodrop 8000). The length (ja 1), a condition generally satisfied in an aqueous integrity of extracted RNA was determined using the Agilent 256 A. Tavakoli Naeini et al. / Journal of Colloid and Interface Science 500 (2017) 253–263

Fig. 1. Schematic of Automated In-line Mixing System (AIMS): initial version (conﬁguration a), and advanced version (conﬁguration b).

2100 Bioanalyzer (Agilent technologies). Total RNA concentration grammed via LabVIEWTM to drive CS and NA solutions through was determined using the Qubit reagent (Life technologies, Cat# either silicon or PharmaPure tubings that were connected to each Q10210) as per the manufacturer’s protocol. For each condition other with a connector (Y, T or cross connector). The mixing system tested, a total RNA quantity of 200 ng/20 mL reaction was reverse also comprises digital balances that record the masses of buffers transcribed using the SuperScript VILO cDNA synthesis kit (Life (e.g., water) for calibration of the pump(s), and to monitor the vol- technologies, Cat#11754250). Primer and probe efficiency was ume/mass of payloads during mixing process. The whole closed experimentally determined using the standard curve method on tubing network was designed to be primed in order to remove the same plate where target gene knockdown is being assessed air pockets trapped in streams as well as to pre-wet the inner walls (Supp info). Reference gene stability was validated using the GeN- of the tubings prior to the actual mixing. Pinch valves were pro- orm statistical package (Biogazelle NV) (Supp info) cDNA was grammed to switch from one stream to the other (i.e., in the down- amplified using TaqMan Fast Advanced Master Mix (Life technolo- stream: from waste line to the collecting vessel, as well as in the gies, Cat# 4444557) on a QuantStudio 12 K flex system (Thermo- upstream: from buffer line to the payloads after priming, and vice Fisher Scientific). All reactions were performed in a 384 well versa). Calibration of the pump(s) was done automatically via a plate (ThermoFisher Scientific, Cat# 4309849) using a final volume sub-program developed in house by LabVIEWTM. Configuration of 10 mL (10 ng of cDNA) and the following cycling conditions: (a) of Fig. 1 displays an overview of the initial version of AIMS 2 min hold at 55 °C, then 10 min hold at 95 °C followed by 40 which features: (1) two separate but inter-connected peristaltic cycles at 95 °C for 15 s and 60 °C for 1 min. Data was exported in pumps where one drives NA and the other dispenses the CS solu- RDML and analyzed using the Biogazelle qBase + software package. tion, simultaneously, at the same flow rate; (2) priming of the tubings is done using the NA and CS solutions; (3) mixing takes place 2.10. Statistical analyses both during and after the acceleration phase of the pumps (acceleration phase refers to the period of time during which the pump All experiments were repeated once with technical replicates head starts from stationary and reaches the target speed). Config- within each for the transfections. Values are given as aver- uration (b) of Fig. 1 displays the advanced version of the mixing age ± standard deviation. For statistics and plotting, SigmaPlot system that features some technical, hardware and software 13.0 package was used. One way ANOVA (at a significance level improvements: (1) only one peristaltic pump with a dual channel of 0.05) was applied and normality and other assumptions were head drives both CS and NA, simultaneously; (2) priming of the validated. tubings is done with the buffer solution rather than the NA and CS solutions in order to minimize consumption of NA; (3) mixing takes place after the acceleration phase of the pump, hence, poly- 3. Results and discussion plexes are mixed only at the target speed by switching the pinch valve from waste to the collection vessel once the final target speed 3.1. Design and establishment of the AIMS was reached.

The in-line mixing setup uses peristaltic pumps to drive fluids based on pulsatile motion, where the natural oscillation of the flu- 3.2. Influence of mixing speed on the properties of CS/NA polyplexes ids provides a wavy and stretched interface between the two streams, thus enhancing the mixing [46–47]. A schematic of the In order to examine the impact of mixing speed on the size and in-line mixing is shown in Fig. 1. Digital peristaltic pumps are pro- polydispersity of final complexes, CS/NA polyplexes were mixed A. Tavakoli Naeini et al. / Journal of Colloid and Interface Science 500 (2017) 253–263 257 via AIMS using flow rates ranging from 2 to 300 mL/min (1.7 to [40,46]. The speed of molecular diffusion is a constant and intrin- 253 cm/s given 1/1600 as the tube inner diameter, ID). 300 mL/ sically slow process; hence relying on diffusion results in incom- min is the maximum flow rate achievable using tubing plete mixing before fluid enters the collecting vessel, and non- ID = 1/1600. Equal volumetric flow rates between the inlets was homogenous complexes due to concentration gradient. As men- used in all mixing experiments. Production of polyplexes was done tioned previously, the natural peristaltic actions of the pump(s) in duplicates for every mixing flow rate tested. The dimensionless promote(s) diffusion by stretching the interfacial area [46–47]. Reynolds number (Re) was calculated in the outlet as Re = UD/m, The stretched contact area is associated with shorter diffusion path where U is fluid velocity, D is the tube inner diameter and m repre- so that at each snapshot of the mixture throughout the outlet tub- sents the fluid kinematic viscosity. The Re range covered in this ing, the two polyelectrolytes are brought closer to each other to study was 26–4000. In a cylindrical/tubular geometry with smooth bind quickly and form the complex [42]. Increasing the frequency walls, Re <2000 and Re >4000 are indicative of laminar, and of rotation of the peristaltic pump (faster mixing), augments the turbulent flow regime, respectively [48]. 2000 < Re < 4000 corre- intensity of pulsatile effect which enhances the overall mixing pro- sponds to transitional flow regime, which has characteristics of cess by further stretching the interface area which favors produc- both laminar and turbulent regime. However, these values may tion of smaller and more homogenous polyplexes. Reduction in not accurately translate the flow regimes in our system, due to size and PDI of CS/siRNA polyplexes could be also due to introduc- the presence of peristaltic motions. tion of chaotic advection (irregular/random transport of matter by Literature reveals that the size and polydispersity of manually flow) in the mixing process at high mixing flow rates. Chaotic mixed CS/pDNA and CS/siRNA polyplexes both increase signifi- advection can be introduced to a system by either implementing cantly at NA concentrations higher than about 0.1 mg/mL, and obstacles in the flow channel, [40] or non-aligned inputs that gen- 0.2 mg/mL, respectively, [9,27,49–50] hence, these concentrations erates vertical flow in the mixer, [51] or turbulence and vortex by were taken to optimize the speed of mixing in AIMS. Both initial high speed streams [41–42]. By folding the streams, chaotic advec- and advanced versions of AIMS were used for producing polyplexes tion offers enhanced mixing performance due to transverse com- by mixing 2 mL of CS and 2 mL of NA with either LS14 (ID = 1/1600) ponents of flow. Not only intertwinement of the two streams by or LS13 (ID = 1/3200) tubing. For comparison purposes, manual mix- chaotic advection generates additional contact area, but also ing was performed through classical pipetting by addition of quickly alters and refreshes the interfacial area that accelerates dif- 100 mL of CS into 100 mL of NA followed by pipetting up and down fusion. Here, it was revealed that by increasing the speed ten times (Fig. 2). (Re > 2140), mixing efficiency increases and translates into smaller and more homogenous CS/siRNA polyplexes in non-laminar regime (Fig. 2a). 3.2.1. Faster mixing results in smaller and more homogenous CS/siRNA AIMS led to better reproducibility in production of CS/siRNA polyplexes polyplexes versus the manual method except for the very low Re As shown in Fig. 2a, a marked reduction in size and polydisper- of 26. AIMS produced much smaller and more homogenous CS/ sity of CS/siRNA polyplexes was observed when Re increased. Z- siRNA polyplexes in the non-laminar regime (Re between 2140 average diameter and PDI dropped from 235 to 75 nm, and from and 4000) compared to manual polyplexes, with Z-average diame- 0.40 to 0.17, respectively, using the initial version of AIMS in the ter and PDI from 84 nm to 75 nm and, 0.18 to 0.17 in the initial ver- Re range from 26 to 4000. Similar trend was observed using the sion of AIMS, and 65 nm to 47 nm and, 0.18 to 0.12 using the advanced version of AIMS in the same range of Re where Z- advanced version of AIMS, versus 101 nm and 0.2, respectively average diameter and PDI were reduced further from 230 to for manual mixing. The largest improvement was at the highest 47 nm, and from 0.44 to 0.12, respectively. Re = 4000 with the advanced version of AIMS to produce CS/siRNA In a purely laminar flow regime with smooth walls (e.g., using polyplexes with Z-average of 47 nm, and PDI of 0.12, while manual syringe pumps), where the streams move in parallel with no trans- mixing led to Z-average of 101 nm, and PDI of 0.2. This suggests verse movements or vortex, mixing relies solely on diffusion

Fig. 2. Influence of mixing speed on size and polydispersity of CS/siRNA polyplexes (a) and CS/pDNA polyplexes, (b) produced by initial and advanced versions of AIMS. pDNA and siRNA concentration prior to mixing were 0.1 mg/mL, and 0.2 mg/mL, respectively, at molar ratio of CS amine to NA phosphate ratio of 5 (N:P = 5). In-line mixing was done using 2 mL of CS and 2 mL of NA. Manual mixing was done by addition of 100 mL of CS into 100 mL of NA, followed by pipetting up and down ten times. Error bars represent standard deviation between the duplicates. 258 A. Tavakoli Naeini et al. / Journal of Colloid and Interface Science 500 (2017) 253–263 that in-line mixing in a non-laminar regime is more efficient than speed, Re = 4000. We found that the mixing pattern and mixing manual mixing, which is likely due to the role of chaotic advection length have no effect or a very slight effect on polyplex properties which offers enhanced mixing performance due to transverse com- (data not shown), suggesting that pulsatile actions and transverse ponents of flow. advection are sufficient for mixing over 25 cm. Moreover, as shown in Fig. 2a, smaller and more homogenous CS/siRNA polyplexes were obtained using the advanced version 3.4. Influence of NA concentration on the properties of CS/NA of AIMS. In order to better understand the source of this reduction polyplexes in size and polydispersity of polyplexes, each of the three modifica- tions of the advanced version of AIMS (as described above) was iso- Based on the results of Fig. 2, the highest Re (4000) was chosen lated and studied separately, where oligonucleotide (ODN) was for further mixings and investigation of the impact of NA concen- used to mimic siRNA as a lower-cost alternative. It was found that tration on properties of polyplexes. Here, NA concentration was mixing during the acceleration phase of the pump was the source varied from 0.01 to 0.4 mg/mL. Equal flow rate between the inlets of increase in size and PDI of CS/ODN polyplexes (see Supporting was maintained. Both configurations of AIMS were tested. For com- Information, Fig. S1). This can be attributed to the fact that CS/ parison purposes, manual mixing was done at every concentration siRNA polyplexes are sensitive to the speed (Fig. 2a) and mixing by pipetting, as described in the method section. Polyplexes were in the acceleration phase of the pump, where the mixing takes prepared in duplicate for each mixing condition. place at different speeds results in the formation CS/siRNA polyplexes with various sizes. 3.4.1. Mixing in dilute regime results in small and homogeneous polyplexes 3.2.2. CS/pDNA polyplexes properties are not very sensitive to mixing As shown in Fig. 3, regardless of the type of NA (siRNA or pDNA), flow rate mixing method (manual or in-line), and configuration of AIMS (ini- For 26 < Re < 4000, Z-average diameter and PDI of CS/pDNA tial or advanced), concentration of NA prior to mixing increases the polyplexes varied only slightly between 186 and 112 nm, and size and PDI of polyplexes. The increase in polyplex size and PDI between 0.25 and 0.23, respectively. As shown in Fig. 2b, size with increasing NA concentration has been reported in the litera- and PDI of CS/pDNA polyplexes change very smoothly throughout ture [31,33–34]. Z-average diameter for CS/siRNA polyplexes pro- the tested range of Re. Davies et al. reported the same behavior for duced with AIMS varied from 53 to 95 nm in the concentration PEI/pDNA polyplexes in the moderate range of 568 < Re < 2840 range of 0.01–0.4 mg/mL (Fig. 3a), while for CS/pDNA Z-average when a static mixer was applied in the downstream [36]. This sug- diameter increased from 80 to 261 nm in the concentration range gests that the pulsatile action of the peristaltic pumps produces of 0.01–0.3 mg/mL (Fig. 3b), respectively. The small increase in Z- sufficient perturbation and interface stretching for efficient mixing average and PDI of polyplexes prepared at the very lowest NA con- and production of homogeneous small sized pDNA/CS polyplexes, centration (0.01 mg/mL) compared to 0.05 mg/mL can be even at very low speeds. Due to its high MW (4 MDa, with explained by the low intensity of the scattering signal. When the 6.4 kbp), pDNA has a low diffusivity, hence, while pDNA has lim- number of particles is very low, the particle count (particles recog- ited mobility, mostly CS molecules diffuse to contribute to complex nized by the DLS instrument) is too low for the instrument to formation. Once association starts on a pDNA supercoil, the chance derive a precise size distribution. Independent of the mixing of collision with subsequent CS is much higher than colliding with method, macroscopic flocculation in CS/pDNA polyplexes was another pDNA molecule, which limits random bridging of poly- observed at 0.4 mg/mL, however, such behavior was not found plexes and heterogeneity. Rapid saturation of pDNA binding sites for CS/siRNA polyplexes up to the highest concentration tested by CS molecules limits the heterogeneity of the CS/pDNA poly- (0.4 mg/mL). It is already reported in the literature that for produc- plexes, even when advection is limited as pDNA does not diffuse tion of stable and small size homogenous complexes, the mixing of quickly. two polyelectrolytes has to be done in dilute regime [9–10,19,52]. On the other hand, the stronger dependence of CS/siRNA poly- In fact, there is a significant difference between the characteristics plexes size upon the mixing speed is likely due to high diffusivity of a dilute polymer solution (where polymer chains are completely of siRNA molecules. siRNA as a much smaller molecule separated) and a semi-dilute to concentrated solution (where poly- (MW 14 kDa with only 21 bp duplex) has a characteristic diffu- mer chains are overlapped). Generally, the transition concentration sion time scale much shorter than that of pDNA. Hence, in addition of polyelectrolyte solutions from dilute to semi-dilute regimes is to CS, siRNA diffusion significantly contributes to the complex for- defined as the overlap concentration (C⁄) [53]. The closer the start- mation. Thus, in order to achieve efficient mixing, stronger advec- ing concentration of polyelectrolytes to their C⁄, the bigger the size tion is required to eventually saturate siRNA with excess CS and increased polydispersity of the final complexes. molecules rather than colliding with another siRNA. The maximum concentration of pDNA for the preparation of It was also observed that in-line CS/pDNA polyplexes (regard- small size uniform polyplexes is reported as 0.1 mg/mL, [50] less of the AIMS configuration) have similar properties and similar while production of small sized lipoplexes using relatively high reproducibility to the manually prepared polyplexes (Fig. 2b). This concentration of siRNA (0.59 mg/mL) was reported by Belliveau could be again explained by low diffusivity of pDNA and limited et al. [31]. As shown in Fig. 3b, regardless of the mixing method sensitivity of CS/pDNA polyplexes to the mixing speed, suggesting (manual vs in-line) and configuration of AIMS (initial vs advanced), that even the low advection provided by manual mixing is suffi- the onset pDNA concentration for increasing size and PDI of CS/ cient for having an efficient mixing. pDNA polyplexes is clearly 0.1 mg/mL. However, with siRNA AIMS allowed production of relatively small and homogeneous CS/siRNA 3.3. Mixing patterns and tube length have no impact on the properties polyplexes at increased concentrations of 0.3 mg/mL and even of CS/NA polyplexes 0.4 mg/mL. The possibility of producing CS/siRNA polyplexes at higher concentrations could be attributed to the smaller size of Two other mixers were tested at relatively low speed (Re = 266), siRNA (higher C⁄) as compared to pDNA. Several techniques are as well as in the highest mixing speed (Re = 4000): Y-connector used to measure the overlap concentration C⁄ of polymeric solu- was replaced by either a T-connector or a cross connector. More- tions, such as small angle light scattering, osmometry, viscometry, over, five mixing lengths with the output downstream after the light scattering and nuclear magnetic resonance [54–55]. Accord- connector, were tested (25, 35, 45, 55, and 65 cm) at the highest ing to our calculations, while C⁄ for CS 92-10 and pDNA are close A. Tavakoli Naeini et al. / Journal of Colloid and Interface Science 500 (2017) 253–263 259

Fig. 3. Influence of NA mixing concentration on size and polydispersity of CS/siRNA polyplexes (a) and CS/pDNA polyplexes, (b) produced manually and by AIMS (using both initial and advanced versions), at molar ratio of CS amine to NA phosphate ratio of 5 (N:P = 5). In-line mixing was done using 2 mL of CS and 2 mL of NA at Re = 4000. Manual mixing was done by addition of 100 mL of CS into 100 mL of NA, followed by pipetting up and down for ten times. CS/pDNA polyplexes severely aggregate at 0.4 mg/mL. Error bars represent standard deviation between the duplicates. to each other and fairly low (C⁄ 3.6 mg/mL and 1.36 mg/mL for PDI, and production of small size homogeneous polyplexes at ele- chitosan 92-10 and pDNA, respectively), C⁄ for siRNA is relatively vated concentrations. high at 690 mg/mL, which permits increased siRNA mixing concentration before reaching flocculation (see Supporting Informa- 3.5. Surface charge density of CS/NA polyplexes increases with NA tion for C⁄ calculations). mixing concentration In order to further investigate the impact of C⁄ on the highest possible mixing concentrations, a smaller CS molecule (Mn = 2kD) Independent of the type of NA, concentration of polyelectrolytes was used to prepare CS/siRNA polyplexes. Being a smaller chain, prior to mixing also influenced the zeta potential (ZP) of poly- CS92-2 has a smaller radius of gyration (Rg), hence, has a higher plexes: the higher the NA concentration prior to mixing, the higher C⁄. It was shown that CS92-2 allows for production of small size the electrophoretic mobility/calculated ZP of final complexes homogeneous polyplexes at increased concentrations of siRNA, (Fig. 4). In-line CS/siRNA had ZP varying between +10.5 and up to 0.6 mg/mL (see Supporting Information, Fig. S2), confirming +21 mV in the concentration range of 0.01–0.4 mg/mL Fig. 4a), the direct impact of chain length on the mixing concentrations. whereas for in-line CS/pDNA the recorded ZP was from +5 to Using manual method, the maximum siRNA concentration for pro- +16.5 mV in the concentration range of 0.01–0.3 mg/mL (Fig. 4a). ducing relatively small size polyplexes is clearly 0.2 mg/mL, where As shown previously in Fig. 3, polyplex size increases with increas- a sharp increase in size of polyplexes was observed at higher con- ing NA concentration, so both ZP and size increase with mixing centrations. However, using the advanced version of AIMS small concentration. It is worth mentioning that for the polyplexes tested and monodisperse polyplexes were successfully prepared at ele- and analysis conditions used in this study, the ratio of the particle vated concentration of 0.4 mg/mL. size to the Debye length changes with particle size and in all conditions, does not strictly meet the criteria for Smoluchowski model 3.4.2. CS/siRNA polyplexes are smaller than CS/pDNA polyplexes to apply precisely (i.e. ja 1, and the Henry function is not At a given concentration, CS/siRNA polyplexes were generally exactly 1.5 as explained in the methods). In order to examine if size smaller than CS/pDNA polyplexes. For example, at an NA mixing variations could account, at least partially, for the observed ZP vari- concentration of 0.1 mg/mL, CS/siRNA and CS/pDNA polyplexes ations, calculations using the complete Henry function were also have Z-average of 40 nm and 112 nm, respectively. This is most performed (data not shown). These calculations reveal that even probably due to the fact that pDNA is a larger molecule compared if the calculated ZP changes slightly versus that obtained with the to siRNA, which naturally leads to larger complexes. Yet, the larger Smoluchowski equation, the general trend observed is unchanged, size of CS/pDNA polyplexes could be attributed to the slower diffu- namely, ZP increases as mixing concentration increases. Therefore, sion of pDNA molecules that leads to broader distribution of pre- although ZP is not a direct measurement of surface charge, [56] our complexes which therefore induces formation of polyplexes with results indicate that the surface charge density of the polyplexes more than one pre-complex [41]. As shown in Fig. 3b, the configu- increases with mixing concentration, suggesting that more chi- ration of AIMS has no significant influence on the properties of CS/ tosan is incorporated in polyplexes when mixing concentration is pDNA polyplexes. This was also observed previously in Fig. 2b higher. This could be the result of faster association kinetics at where the properties of CS/pDNA polyplexes are independent of higher mixing concentrations. the AIMS configuration. However, in the case of CS/siRNA polyplexes (Fig. 3a), the advanced version of AIMS produced smaller 3.6. Morphology of the CS/pDNA and CS/siRNA polyplexes and more monodisperse CS/siRNA polyplexes at the given concentrations, which again confirms their sensitivity to mixing speed as Morphology of polyplexes was assessed by Transmission Elec- described in Fig. 2a. Compared to manual mixing, production of CS/ tron Microscopy (TEM). CS/pDNA, and CS/siRNA polyplexes were siRNA polyplexes with AIMS at Re = 4000 (regardless of the AIMS prepared both manually and in-line using the advanced version configurations) provided better reproducibility, smaller size, lower of AIMS at NA starting concentrations of 0.1 mg/mL and 0.2 mg/ 260 A. Tavakoli Naeini et al. / Journal of Colloid and Interface Science 500 (2017) 253–263

Fig. 4. Inﬂuence of NA mixing concentration on zeta potential of CS/pDNA polyplexes (a) and CS/siRNA polyplexes, (b). In-line mixing was done using the advanced version of AIMS: 2 mL of CS and 2 mL of NA at Re = 4000. Manual mixing: pipetting 100 mL of CS into 100 mL of NA. Zeta potential measurements were done in 0.5% trehalose and 3.5 mM histidine (Ionic strength = 1 mM). Error bars represent standard deviation between the duplicates. mL, respectively. TEM images show CS/pDNA polyplexes with (Fig. 5d) were smaller and more uniform than those prepared man- mixed morphology (spherical, toroidal and rod like) when pro- ually (Fig. 5c), in agreement with DLS results. duced both manually and using the AIMS (Fig. 5a and b). This morphology of CS/pDNA polyplexes was reported in literature [57–58]. 3.7. CS/siRNA polyplexes produced in-line are as bioactive as manual On the other hand, CS/siRNA polyplexes were generally spherical polyplexes regardless of the method of preparation (Fig. 5c and d). The spherical shape of CS/siRNA polyplexes was also reported previously In order to test the suitability of AIMS to produce bioactive CS/ [16–18]. However, CS/siRNA polyplexes produced using AIMS siRNA polyplexes, the hepatocellular carcinoma cell line HepG2

Fig. 5. Transmission Electron Microscopy (TEM) images of CS/pDNA polyplexes (a and b) and CS/siRNA polyplexes (c and d), produced both manually (a and c) and using the advanced version of AIMS (b and d). pDNA and siRNA concentration prior to mixing were 0.1 mg/mL and 0.2 mg/mL, respectively. In-line mixing was done by mixing 2 mL of CS and 2 mL of NA at 150 mL/min. Manual mixing was done by pipetting 100 mL of CS into 100 mL of NA. A. Tavakoli Naeini et al. / Journal of Colloid and Interface Science 500 (2017) 253–263 261

Fig. 6. Bioactivity of Inline and manually mixed CS/siRNA polyplexes. HepG2 cells were transfected with either inline or manually mixed anti-ApoB CS/siRNA polyplexes at a final siRNA concentration of 100 nM/well. Apob mRNA knockdown was assessed by qPCR using geometric averaging of the most stable reference genes (GAPDH and B2 M) and expressed relative to non-treated cells. Data represent the mean of two independent experiments (N = 2) with two technical replicate per experiment (n = 2). Analysis of Variance (ANOVA) was performed using the SigmaPlot 13.0 statistical package. was transfected in vitro and Apolipoprotein B (ApoB) mRNA knock- mixing siRNA concentrations on gene knockdown. Compared to down assessed using quantitative real time polymerase chain reac- the inline mixed polyplexes, manually prepared polyplexes tion (qPCR). Cells were transfected with polyplexes prepared using resulted in slightly lower ApoB mRNA knockdown (30–45%). the advanced version of AIMS at Re = 4000, and different siRNA These results are consistent with a previous study where CS/siRNA mixing concentration (0.05–0.4 mg/mL). Manually prepared poly- polyplexes achieved ApoB mRNA knockdown of 50% in HepG2 plexes were also transfected for comparison purposes. when prepared manually at N:P 5 and siRNA concentration of Ò DharmaFECT 2, a commercially available lipoplex, was used as 0.05 mg/mL [5]. Although manually prepared polyplexes at siRNA positive control. Naked siRNA, mock chitosan and non-treated cells concentration of 0.2 mg/mL showed the lowest transfection effi- were used as negative controls. ciency (30%), analysis of variance did not show statistical signif- ApoB mRNA knockdown reached 40–55% when produced via icance compared to other siRNA mixing concentrations. CS-based AIMS (Fig. 6). Statistical analysis revealed no significant effect of polyplexes achieved comparable knockdown efficiency to

Fig. 7. Correlation between bioactivity and physico-chemical parameters of inline and manually prepared polyplexes. (a) Correlation between ApoB mRNA knockdown (average) and polyplexe size (average). (b) Correlation between ApoB mRNA knockdown (average) and polyplexe zeta potential (average). Regression and Pearson correlation analysis was performed with SigmaPlot 13.0 statistical package. 262 A. Tavakoli Naeini et al. / Journal of Colloid and Interface Science 500 (2017) 253–263

Ò DharmaFECT 2(Fig. 6), independent of the mixing method. We Appendix A. Supplementary material have previously shown that manually prepared polyplexes achieve Ò comparable knockdown efficiencies to DharmaFECT 2 but with Supplementary data associated with this article can be found, in lower toxicity [7,13,59]. Nanoparticle size and surface charge are the online version, at http://dx.doi.org/10.1016/j.jcis.2017.04.013. cited in the literature to be important parameters for nanoparticle uptake and bioactivity [60–61]. In our study, polyplex size corre- 2 lated moderately (r = 0.376) with knockdown efficiency (Fig. 7a), References with smaller sized polyplexes inducing higher knockdown efficiency. This trend can be seen from results presented in [1] H. Dautzenberg, J. Hartmann, S. Grunewald, F. Brand, Stoichiometry and Figs. 3a and 6 where inline mixed polyplexes demonstrate a structure of polyelectrolyte complex particles in diluted solutions, Ber. Bunsen. Phys. 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