How to screen non-viral gene delivery systems in vitro?

Journal of Controlled Release 154 (2011) 218–232

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Journal of Controlled Release j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j c o n r e l

Review

How to screen non-viral gene delivery systems in vitro? Ethlinn V.B. van Gaal a, Roel van Eijk a, Ronald S. Oosting b, Robbert Jan Kok a, Wim E. Hennink a, Daan J.A. Crommelin a, c, Enrico Mastrobattista a,⁎ a b c

Department of Pharmaceutics, Utrecht Institute of Pharmaceutical Sciences (UIPS), Utrecht University, 3584 CG, Utrecht, The Netherlands Department of Psychopharmacology, Utrecht Institute of Pharmaceutical Sciences (UIPS), Utrecht University, 3584 CG, Utrecht, The Netherlands Dutch Top Institute Pharma, Leiden, The Netherlands

a r t i c l e

i n f o

Article history: Received 9 July 2010 Accepted 1 May 2011 Available online 7 May 2011 Keywords: Gene delivery Transfection DNA Particle size Nanoparticles pEI

a b s t r a c t Screening of new gene delivery candidates regarding transfection efficiency and toxicity is usually performed by reading out transgene expression levels relative to a reference formulation after in vitro transfection. However, over the years and among different laboratories, this screening has been performed in a variety of cell lines, using a variety of conditions and read-out systems, and by comparison to a variety of reference formulations. This makes a direct comparison of results difficult, if not impossible. Reaching a consensus would enable placing new results into context of previous findings and estimate the overall contribution to the improvement of non-viral gene delivery. In this paper we illustrate the sensitivity of transfection outcomes on testing conditions chosen, and propose a screening protocol with the aim of standardization within the field. © 2011 Elsevier B.V. All rights reserved.

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Choice of plasmids . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Identity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Topology and size . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Source and quality . . . . . . . . . . . . . . . . . . . . . . . . Preparation of particles/complexes . . . . . . . . . . . . . . . . . . . . 3.1. Ratio of reagent to DNA . . . . . . . . . . . . . . . . . . . . . . 3.2. Medium used for complexation . . . . . . . . . . . . . . . . . . 3.3. Order of mixing . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Concentrations and volumes . . . . . . . . . . . . . . . . . . . . 3.5. Presence of excess reagent . . . . . . . . . . . . . . . . . . . . . Characterization and stability of particles . . . . . . . . . . . . . . . . . 4.1. Characterization . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Stability in salts and biological media . . . . . . . . . . . . . . . Choice of cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. Cell culture models . . . . . . . . . . . . . . . . . . . . . . . . 5.2. Non-dividing cells . . . . . . . . . . . . . . . . . . . . . . . . . Transfection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1. Maintenance of cells . . . . . . . . . . . . . . . . . . . . . . . 6.2. Confluency . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3. Incubation of samples with cells: effects of composition and volume of 6.4. Incubation of samples with cells: effects of dose and incubation time Read-out . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.1. Expression . . . . . . . . . . . . . . . . . . . . . . . . . . . .

⁎ Corresponding author. Tel.: + 316 22736567; fax: + 31 30 251 7839. E-mail address: [email protected] (E. Mastrobattista). 0168-3659/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jconrel.2011.05.001

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7.2. Toxicity . . . . . . 7.3. Data presentation . . 8. Choice of reference reagent 9. Discussion . . . . . . . . Appendix A. Supplementary data References . . . . . . . . . . .

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1. Introduction Gene therapy relies on successful delivery of therapeutic DNA into nuclei of target cells. The concept of introducing exogenous DNA in animal cells was first shown in 1979 by Mulligan et al., who reported successful transfection of cells with recombinant plasmid DNA using the calcium phosphate transfection technology [1]. By 1980 successful in vitro delivery of DNA by liposome-mediated gene transfer had been demonstrated [2], followed by polylysine (pLL)-based transfection in the late 1980s [3] and polyethyleneimine (pEI)-based delivery in the 1990s [4]. Based on these initial findings, the search for alternative delivery agents has moved into various directions and is ever expanding (depicted in Fig. 1). Despite the development of an extensive number of reagents varying in chemical composition and functionalization (for a comprehensive review see Mintzer et al. [5]), understanding of structure–activity relationships is limited. Screening of new gene delivery candidates regarding transfection efficiency and toxicity is usually performed by reading out transgene expression

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levels relative to a reference formulation after in vitro transfection. However, over the years and among different laboratories, this screening has been performed in a variety of cell lines, using a variety of conditions and read-out systems, and by comparison to a variety of reference formulations. This issue is easily illustrated by analyzing the materials and methods sections of a sample as small as 24 papers recently published in this journal on the topic of in vitro screenings of reagents for improved gene delivery (see Table 1). The large variability in experimental conditions observed in this limited sample is representative for gene delivery studies in general, and makes a direct comparison of results difficult, if not impossible. Besides a lack of consensus among in vitro studies, in vitro transfection screenings are often criticized for poorly correlating with in vivo results. Nevertheless, in vitro transfection screening is an indispensable tool as (a) testing of large numbers of reagents under various conditions directly in vivo is neither feasible nor ethical and (b) recent developments in chemical synthesis and (recombinant) technology enable generation of large libraries of synthetic or

Fig. 1. Progress in gene delivery research. Inside-out: the concept of transgene expression upon non-virally delivered DNA in target cells (center), the problems encountered (second ring), the mechanistic explanation for these problems (third ring) and the solutions investigated to improve success of non-viral gene delivery (outer part).

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Table 1 Parameters of variation in in vitro transfection screenings extracted from a sample of 24 papers [9–32]. Parameter Cell type HeLa HEK293T HEK293/A549/CHO/bone marrow-derived mesenchymal stem cells/NIH-3T3/MCF-7/HepG2 bEnd-3/HDFs/MA/K562/HT29/HT1080/B16F10/bone marrow dendritic cells/primary myoblasts/CHO-K1/Calu-3/COS/COS-7/mouse spleen-derived APC/BHK-21/SK-N-BE(2)C/mNPC Positive control 25 kDa b-pEI Lipofectamine™ 2000 Lipofectin pLL/Lipofectamine™ 22 kDa l-pEI/0.8 kDa b-pEI/pEI1800/pEI/Superfect/GenePorter™/ pDMAEMA/DOTAP No external reference Buffers used for complexation 20 mM Hepes pH 7.4 PBS 10 mM Hepes/10 mM Hepes, pH 7.4/10 mM Hepes, 150 mM NaCl, pH 7.4/10 mM Hepes, 1 mM NaCl, pH 7.4/10 mM Hepes, 5% glucose, pH 7.4/20 mM Hepes/20 mM Hepes pH 7.2/HBS pH 7.4/5% glucose/150 mM NaCl/α-MEM/MilliQ-water/DMEM Unclear Dose (μg DNA/cm2) 1/0.5/0.2 0.25/unclear 3.1/2/1.6/0.75/0.4/0.1

Times used 6 3 2 1

7 6 5 2 1 4

3 2 1

5

4 2 1

Cell confluency 70% 60–70%/70–80%/80%/unclear Reported as # of cells/well

3 1 17

Incubation of complexes with cells (h) 4 3 2 1/5/48

15 4 2 1

Incubation medium Without serum With serum With and without serum Unclear

12 6 4 2

Expression readout Luciferase (E)GFP Β-galactosidase Silencing

15 5 3 1

Toxicity readout MTT/XTT WST-1 LDH/Resazurin reduction assay/MTS proliferation assay/NADH Tetra Color ONE cell proliferation assay system/crystal violet assay None Physicochemical characterization Dynamic Light Scattering (DLS) Zetapotential Gel retardation Fluorescence displacement assay Microscopy (TEM/SEM/AFM) Nuclease resistance assay

13 2 1 5

19 14 14 5 5 4

recombinant carriers, which warrants high-throughput screening assays [6]. Therefore, understanding and controlling in vitro transfection is of major importance to develop rational, reliable and fast

screening procedures that enable evaluation of structure–activity relations. The aim of this review is twofold. First, we want to illustrate the variability in screening procedures and sensitivity of transfection outcomes to testing conditions, based on published work and our own investigations (provided in the Supplementary information). Secondly, our objective is to improve future comparability between studies and laboratories by proposing the standardization of screening protocols and inclusion of a benchmark for inter-study comparison. Of note, this review is not intended to supply a complete overview of materials and methods used in gene delivery research (for such comprehensive reviews the reader is referred to for instance Shcharbin et al. [7,8]), but rather aims at highlighting parameters of interest based on selected examples. In this paper, the screening of gene delivery agents is discussed in a stepwise approach by addressing the following parameters in subsequent paragraphs: the choice of plasmid DNA, preparation of complexes, characterization and stability of particles (in biological media), the choice of cell types and transfection conditions, read-out systems, and the selection of reference reagents. Based on the literature discussed in these paragraphs (backed up with the experimental data provided in the supplementary information), critical factors for generating valid and comparable data are identified and a limited set of guidelines is proposed to substantiate the value of gene delivery screening data. Additionally, a protocol is drafted with the aim of initiating the discussion towards standardization of assays in the field. 2. Choice of plasmids 2.1. Identity Plasmids used for in vitro transfection screenings vary not only in reporter gene but also in vector backbone. The majority of in vitro transfection screenings is performed with plasmids in which a reporter gene is driven by a strong viral promoter, such as the cytomegalovirus (CMV), simian virus 40 (SV40), or Rous Sarcoma Virus (RSV) promoter. These promoters are chosen to ensure constitutive and high activity in a broad range of host cell types. Although plasmid design is not the scope of this paper, it is important to take note of differences in transfection efficiency related to differences among plasmids rather than delivery strategies. Transcription efficiency can be augmented through careful selection of promoter and enhancer sequences [33], codon optimization [34,35], removal of non-essential sequences and antibiotics resistance genes, addition of sequences that improve mRNA translation, avoiding extended palindromic sequences, sequences homologous to host genomic DNA, and sequences causing undesired targeting of the transgene product [35–40]. Additionally, certain sequences have been ascribed a role in facilitating active nuclear import depending on plasmid context and proliferative state of host cells [39,41–57]. When studying nuclear delivery by custom-made delivery agents, potential effects of such DNA nuclear Targeting Sequences (DTS) should be taken into account. When moving to in vivo testing, additional features such as minimizing viral/bacterial sequences and GC content through removal of antibiotics resistance genes and replacing viral promoters with eukaryotic alternatives can further enhance transgene expression profiles [38–40,58–60]. The choice for a plasmid identity is not critical per se (as long as it is compatible with the cell type used). Although absolute expression values will vary with plasmid construct, the relative expression levels for various transfection reagents will be related directly to the reagent and not to the plasmid. Nevertheless, it is worthwhile to consider the dynamic range: transgene expression may reach plateau upon transfection of easy-to-transfect cells with high-expression plasmids, resulting in loss of discriminative power of the assay.

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2.2. Topology and size Besides effects of plasmids at the sequence level, the overall size and tertiary structure of DNA is known to affect transfection efficiency. Plasmids can occur in a supercoiled (sc) open circular (oc) or linear form and conversion from sc to oc or linear forms can be induced by chemical, physical or enzymatic reactions. Sc DNA being the only intact and undamaged form is in theory superior to oc or linear forms in which damage at random gene locations may cause destruction of functional transcription cassettes [61]. Although controversial experimental results have been reported regarding the relative transfection potency of these topologies [39,61–69], a general trend towards assigning superior activity to sc plasmid DNA is recognized. Explanations given are increased intracellular mobility due to reduced hydrodynamic size, preferential perinuclear accumulation and effects on the template structure and interactions of regulatory sequences with host proteins [66,67]. Focusing on supercoiled isoforms seems sensible, one reason being that regulatory authorities recommend assessment of the proportion of supercoiled plasmid content and inclusion of minimal specifications thereof (preferably N80% according to the FDA) [70,71]. With regard to plasmid size, consensus exists that transfection efficiency is inversely related to plasmid size [39,40,64,72,73]. This effect was systematically studied by inserting 1–8 fragments of 0.65 kb stuffer DNA into a 4.8 kb pGL3-basic plasmid, which resulted in reduced transfection levels (up to 75%) with each extra insert [74]. 2.3. Source and quality Plasmids are generally produced in, and isolated from Escherichia coli (E.coli) bacteria using commercial plasmid purification kits or cesium chloride gradients. Quantification by absorption measurements and identification of plasmid DNA and its topology by electrophoretic analysis of undigested and digested plasmid on agarose gels are important and generally performed. In addition, presence of impurities such as genomic DNA, RNA, proteins and lipopolysaccharides (LPS) and endotoxins can affect both activity and safety and should therefore be minimized [61,75]. 3. Preparation of particles/complexes 3.1. Ratio of reagent to DNA A well-known critical parameter for the preparation of particles based on electrostatic interactions is the ratio of cationic reagent to negatively charged DNA. Complexes are generally formed by mixing reagent with DNA at a certain ratio, which can be expressed as a weight/weight (w/w) ratio, or given the electrostatic interaction as a basis for the reaction, as a charge ratio. Typically, incubation times of 10–30 min are used to allow complexes to mature. As for many reagents the charge of reagent and DNA is a function of the number of nitrogens (N) and phosphates (P), respectively, it is often preferred to express N/P ratios (assuming 3 nmol P/μg DNA) [4]. Since both physicochemical properties and biological activity of complexes are highly dependent on the reagent:DNA ratio, a range of ratios must first be screened to identify the minimal ratio at which DNA is condensed into small positively charged particles and is protected against degradation. Next, the optimal ratio to obtain maximal gene expression and minimal toxicity must be found in transfection studies. Usually, an excess of cationic reagent is required to obtain stable particles and to achieve gene expression. Taking pEI as an example, it has been found that an N/P ratio of 2–3 is required for complete charge neutralization. However, at such low N/P ratios complexes are large and unstable [76,77] and generally an N/P ratio of 6 is minimally required to obtain small stable particles [4,77,78]. At increasing N/P ratio, excess

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polymer is thought to stabilize complexes through electrostatic repulsion between positively charged particles [79]. 3.2. Medium used for complexation Behavior of complexes also depends on the composition of the medium used for complexation. In case of pEI, a trend towards increased transfection efficiency at decreasing pH was reported, explained by the pH-dependency of nitrogen protonation [4]. Being driven by electrostatic forces, complexation is affected by ionic strength of the medium. Linear pEI (l-pEI) has been observed to form small (50–70 nm), stable particles with DNA when mixed in low-ionic strength buffers (5% glucose in hepes buffer (HBG)), and to aggregate in time when prepared in physiological salt (150 mM NaCl in hepes buffer (HBS) or in water) (see Supplementary information Table S1 and [4,77,80–83]). Subsequent exposure of complexes prepared at low-ionic strength to physiological salt concentrations (e.g. serumfree cell culture medium) induces particle growth [80,84]. This process was not observed in the presence of serum, which is believed to act through shielding of complexes (see Supplementary Fig. S1 and [77,83]). Since the dispersion medium in which particles are prepared affects particle size (stability) and transfection efficiency, it should be chosen with care. Gene delivery reagents are developed for therapeutic purposes and formulations should ultimately be regarded as pharmaceutical products. From this point of view, preparation of formulations in uncomplicated well-defined dispersion media is preferred over cell culture medium or minimal media such as Optimem. Use of low ionic strength and physiological pH buffers supplemented with glucose at iso-osmotic concentrations (such as HBG) is promoted for the formation of electrostatic complexes, as the absence of salts and the isotonicity make them compatible with characterization as well as transfection studies. To evaluate effects of ionic strength on a formulation's characteristics (and stability), additional formulations prepared in high ionic strength buffers can be tested. 3.3. Order of mixing The electrostatic interaction between two extensively charged components such as polycationic reagents and polyanionic DNA occurs very rapidly, such that hardly any rearrangement occurs after initial contact. For this reason, complexation differs when adding reagent to DNA (polymer will bind to DNA until charge neutralization is achieved) or vice versa (complex formation varies strongly in time due to decreasing concentration of unbound polymer). Adding reagent to DNA was shown to be 10-fold more efficient than the reverse order, with regard to transfection efficiency [4,85] and is preferred over the reverse sequence of addition. However, this parameter may become redundant with the development of more sophisticated and automated mixing devices that allow instantaneous mixing. 3.4. Concentrations and volumes Another aspect that is of special interest in anticipation of the need for upscaling for in vivo testing is the concentration of DNA used. Increasing DNA concentrations have been associated with increases in particle size and polydispersity [77,78,86,87]. Increasing the DNA concentration by modifying the compaction volume was reported to have no effect for volumes ranging from 10 μL to 1 mL [4], but to yield larger and more heterogeneous particles when increasing from 500 μL to 5 mL [88]. Mixing is generally performed either by rigorous pipetting or vortexing. More recently, efforts are being made to replace these difficult-to-standardize methods by reproducible and up-scalable mixing methods using micofluidic hydrodynamic

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focusing [89] or a micro-mixer [88]. Comparison of these methods shows an inverse relation between mixing speed on the one hand, and size, polydispersity and transfection efficiency on the other hand [88,89].

particles is to use low ionic strength buffers set at physiological pH and adjusted to iso-osmotic concentrations.

3.5. Presence of excess reagent

Measurements in plain solvents directly after preparation is useful for product characterization, but biological activity will ultimately depend on the particle characteristics in biological fluids, being (serum-supplemented) cell culture medium for in vitro and ex vivo studies, and full blood for in vivo studies and ultimately therapeutic application. Albumin, an abundant protein in biological fluids, was shown to induce aggregation of pEI/DNA complexes at concentration below 2 mg/mL, but not at higher concentrations, where particles adopt a negative charge through extensive albumin adsorption [94]. Serum components have been described to affect nucleic acid containing particles in various ways. Firstly, adsorption of negatively charged serum proteins onto positively charged particles can lead to changes in size, zetapotential, surface characteristics [96–102] and consequentially to altered clearance and biodistribution [78,99,103– 106], cellular uptake and processing [107–109] and toxicity [110]. Secondly, charged compounds can induce dissociation of complexes, followed by release and degradation of the nucleic acid [80,96, 111,112]. Thirdly, the large fraction of polymer that is present in free form [90] can interact with negatively charged serum proteins and form new particles [63,113]. Despite their importance, studies regarding nanoparticle behavior in biological fluids are scarce and often omitted when evaluating gene delivery reagents. One reason why these studies are lacking is that methods to measure submicron particles in complex fluids are still limited. The heterogeneous nature of biological fluids (e.g. serum) can give rise to significant background signal, whereas sizing techniques based on conventional light scattering (i.e. DLS) require minimum levels of background noise for generation of accurate results. Moreover, samples of polymer/DNA complexes are typically dilute in nature and often contain large fractions of free polymer [90], which can interact with serum proteins and form aggregates that strongly interfere with size measurements by conventional light scattering techniques [63,113]. Imaging techniques such as electron microscopy and atomic force microscopy allow studying individual particles, but are laborious and suffer from poor statistical power. Moreover, risks of artifacts introduced by sample preparation (e.g. drying, fixation or cutting) should be acknowledged. Analytical ultracentrifugation techniques can be used to fractionate and analyze submicron particles, but are laborious, time-consuming, result in complex data analysis and require purification (dialysis) prior to fraction analysis in case of isopycnic sedimentation [102,114]. More recently, new methods based on fluorescence fluctuation spectroscopy [112], flow cytometry [113] and fluorescence nanoparticles tracking analysis by Nanosight [115] have been developed and applied to study the integrity of siRNA-carrier complexes in full human serum [112] and analysis of size distributions of DNA-containing nanoparticles in serum-supplemented cell culture medium [113]. Additionally, fluorescence single particle tracking (fSPT) was used as the first method to study time-dependent aggregation of various liposomes in full blood [116]. Breunig et al. report similar aggregation behavior of pEI/DNA complexes prepared at low ionic strength upon exposure to either serum-free or serum-supplemented cell culture medium based on confocal microscopy images [80]. In contrast, other studies report aggregation of such complexes upon exposure to physiological salt concentrations in absence, but not in presence of serum, as measured by DLS [77,83,85] and flow cytometry [113] (see Supplementary information Table S1 and Fig. S1). An explanation for this observation is that salt present in medium without serum induces aggregation caused by reduced electrostatic repulsion between particles. When using serum-supplemented medium, adsorption of serum proteins

Since excess reagent is required to stabilize particles, especially in high-ionic strength media, final formulations contain free reagent. For example, the amount of unbound pEI was reported to be N80% for 25 kDa pEI/DNA complexes prepared at N/P ratios of 6 and 10 [90]. Removal of excess reagent prior to physicochemical characterization and assessment of transfection activity and cytotoxicity is not common practice, but would allow a cleaner assessment of toxicity of formulations, as the formulated complexes are in general far less toxic than free reagent. Removal of free pEI can be performed by size exclusion chromatography (SEC) and was shown to not affect particle size or zetapotential and to significantly reduce toxicity [91]. However, transfection efficiency was also reduced, most likely due to decrease of the amount of pEI below threshold levels required for proton-sponge mediated endosomal escape [91]. These results indicate that removal of free pEI allows higher dosing of DNAcomplexes. Alternatively, the DNA-dose can be kept constant while maintaining high total pEI-concentrations by diluting active DNA with inactive (junk) DNA prior to complexation [92,93]. The observed effects of reagent:DNA ratio, medium composition, mixing order and method, concentrations, volumes, incubation time and purification indicate that minor differences in protocols generate different complexes and prevent reliable comparison of results from different laboratories. 4. Characterization and stability of particles 4.1. Characterization When developing reagents for non-viral gene delivery, the ideal scenario is to have well-defined nanoparticles, preferably b100 nm, which are stable in physiological media and efficiently transfect cells with minimal toxicity. In practice, DNA-complexes are mostly formed via electrostatic interaction with positively charged polymers, lipids or peptides, are sensitive to dispersion media (i.e. ionic strength) and are subject to changes upon incubation in physiological media. Particle characteristics are well-known determinants in cell transfection and a set of complementary tools is available to characterize formed complexes. Parameters of interest are size and stability, surface charge, condensation and protection of DNA, which can be studied with techniques including DLS, zetapotential measurements, gel retardation and fluorescence displacement assays (in presence and absence of displacing polyanions such as heparin) and nuclease resistance assays. Imaging techniques such as electron microscopy and atomic force microscopy can provide supplementary insight into shape, morphology and size of particles. Performing characterization experiments under both realistic and rational conditions requires attention. Quite frequently, characterization studies are performed in a plain buffers (or even water) while a buffer of a different composition is used for in vitro studies for isotonicity reasons. The effect of ionic strength on particle size shown here and by others [77,94] indicates that characterization studies can only be linked to in vitro results when in both cases complexes are prepared in corresponding dispersion media. At the same time, compatibility of dispersion media with analytical techniques should be considered. For example, measuring zetapotentials in aqueous media without stabilizing and defining pH creates meaningless data [95]. The high conductivity of high ionic strength media causes substantial heat development and complicates obtaining reliable data. A straightforward approach for reliable (preparation and) analysis of

4.2. Stability in salts and biological media

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onto the particle surface may lead to shielding of particles, thereby preventing severe aggregation [77]. With the set of techniques that enable reliable size measurements in biological fluids which have become available in the past years, such size analyses should become routine in gene delivery screenings. 5. Choice of cells 5.1. Cell culture models The majority of transfection studies investigating the potential of gene delivery are performed in adherent monolayer cell culture systems. An extensive number of different cell types have been used, which vary in species (e.g. human, murine, rabbit, rat or monkey), tissue type (e.g. liver, kidney, endothelium, muscle) and proliferation characteristics (e.g. cancer cell lines, virally-transformed cell lines, primary cells). Specific cell types are selected for in vitro transfection screenings because they are a well-established model cell line (e.g. HeLa CCL-2 cells), are easy to transfect (e.g. COS-7, HEK293), and/or represent a likely target for gene therapy (e.g. breast, lung, kidney, colon cells). Transfection efficiencies are highly variable among different cell lines (see Supplementary information Fig. S2). Boussif et al. compared the transfection efficiency of 800 kDa branched pEI and Transfectam in 25 different cell types, including cells of animals and tissues of various origin, immortalized and primary cells, and adherent and suspension cells [117]. Luciferase expression levels were found to vary five orders of magnitude depending on cell type, without a clear correlation between transfection efficiency and tissue origin or rate of cell division. Focusing on a specific tissue, immortalized cells were generally easier to transfect than primary cells (e.g. expression levels in HepG2 cells were two orders of magnitude higher than those in primary hepatocytes). Suspension cells were at the lower range of expression levels. Differential transfection efficiency and toxicity in different cell types was also reported in studies comparing various commercially available transfection reagents [85,118–120] (and Supplementary information Fig. S2), comparing non-viral chemical versus physical and/or viral delivery methods [121,122], and evaluating a specialized Tat-based delivery reagent [123]. All these studies show both differences in overall expression levels and toxicity of reagents in different cell types (and subclones) as well as differences in the relative transfection efficiencies of reagents compared to one another. Additionally, effects of serum, reagent: DNA ratio and chloroquine varied among cell types. Possible explanations for cell type dependency are discriminative expression patterns resulting in different membrane characteristics, intracellular transport mechanisms and molecules, and DNA-degrading enzymes. One of the reasons why high expression levels are observed in COS-7 may be that COS-7 cells constitutively express the Simian Virus (SV40) large T antigen. Interaction of this antigen with SV40 origin of replication regions often present in reporter plasmids leads to plasmid replication [124]. Transfection efficiencies obtained in COS-7 (and other SV40-transformed cell lines) are therefore likely to be an overestimation of actual transfection in normal tissues. 5.2. Non-dividing cells Although in vitro/in vivo correlation continues to be a matter of debate, some useful efforts to maximize predictive value of in vitro transfection studies can be made, including screening in various cell types with different characteristics and screening in a model for nondividing cells. An important discrepancy between cell lines and the in vivo situation is the proliferation state of cells. Cells in culture divide rapidly (doubling times of ~24 h) whereas cells in tissues, the targets for gene therapy, are mostly quiescent. This has a huge impact on transfection outcomes because temporary breakdown of the nuclear

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envelope during mitosis allows bulk access of DNA (-complexes) to the nucleus whereas active import via nuclear pore complexes is essential in absence of mitosis [125–128]. The ultimate test would be to study transfection in vivo. However, additional barriers would be encountered such as stability in blood and biodistribution. In order to isolate the parameter of cell division and assess the potential of gene delivery systems to transfect cells in the absence of mitosis, in vitro models are needed. Primary cells are harvested from a donor organ and reach senescence after a limited amount of doublings (the number varies by species, cell type and culture conditions) and therefore represent an important model. For some, but not all, (immortalized) cell types, growth to confluency is sufficient to reach quiescence. Alternatively, several methods are available to arrest cells in a certain phase within the cell cycle, including serum deprivation and chemical treatment with growth inhibitors such as thymidine block, synchronization with hydroxyurea, lovastatin, heparin or aphidicolin. However, success of such treatments varies with cell types and should be verified by cell cycle analysis based on flow cytometry analysis of DNA content following cell staining with propidium iodide. Additionally, secondary effects of the treatment such as toxicity and effects of inhibitors on physicochemical properties and behavior of DNA-complexes should be evaluated. In our hands, arresting HeLa CCL-2 cells in S-phase with aphidicolin (an inhibitor of DNA polymerase α) proved most useful as it combined efficient (unlike serum deprivation) and continuous synchronization with acceptable toxicity (unlike hydroxyurea). Overall expression levels dropped roughly 10-fold for l-pEI complexes and to 0 for b-pEI and Lipofectamine™ 2000 (Lipofectamine) in arrested HeLa CCL-2 cells compared to normal HeLa CCL-2 cells (see Fig. 2). Clearly, transfection of non-dividing cells represents a major bottleneck and should be part of screening procedures for gene delivery agents with final applications in vivo. 6. Transfection Transfections are performed to evaluate the efficiency with which reagents can induce transgene expression in host cells, but this efficiency is also dependent on cell passaging and seeding procedures, medium composition and the exact protocol used for incubation of samples with cells (e.g. transfection volume and incubation time). 6.1. Maintenance of cells A critical factor in high-efficiency and reproducible transfection is a consistent number of healthy proliferating cells [129]. The majority of in vitro transfection screening studies is performed in immortalized cells that are passaged one or multiple times per week and seeded into well-plates 24 h prior to an experiment. Cell cultures should be passaged frequently and at regular intervals to maintain cells in midlog growth, which is optimal for transfection [129]. Cell cultures cannot be used indefinitely, as sub-culturing changes cell morphology, response to stimuli, growth rates, protein expression, transfection and signaling in time [130]. Exhaustive passaging may decrease transfection efficiency. On the other hand, Jacobsen et al. reported increased transfection efficiency in NIH-3T3 cells at 56 passages compared to 2, whereas transfection efficiency in CHO-K1 cells was consistent from 2 to 26 passages [131]. Cultures of cells used for transfection should be regularly monitored for evidence of mycoplasma, as mycoplasma infection can alter cell growth, function and transfection efficiency [132–134]. 6.2. Confluency Since both transfection efficiency and toxicity are sensitive to cell confluency (defined as the density of cells on the seeding surface, generally expressed as the % of surface which is covered by cells),

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standardized seeding protocols are required to obtain consistent results among different experiments. Cells should be harvested, counted and seeded in dedicated cell culture medium (supplemented with serum) at fixed numbers per well and at fixed times prior to an experiment to reach 50–95% confluency on the day of transfection. Cells require a certain confluency to grow and perform naturally, and too low confluencies lead to decreased growth rates and increased sensitivity to treatment. When confluence is chosen too high on the other hand, cells become (over)confluent throughout the assay, potentially causing cell cycle arrest, decreased metabolic activity and detaching of cells. Differences in cell densities at the time of seeding have been associated with differences in cell doubling times [135], cell death and cell cycle arrest [136], which in turn can affect transfection. Indeed, confluencydependent transfection efficiency has been observed by us (Supplementary information Fig. S3) and others. Gebhart et al. [85] reported increasing transfection efficiency and toxicity of pEI/DNA complexes when increasing the confluency from 40 to 90%. Another commercially available reagent, Superfect™ was less sensitive to cell densities. Wiseman et al. [137] tested the transfection efficiency of linear pEI, branched pEI and liposomes in a human bronchial epithelial cell line (16HBE) at different culture conditions. The efficiency of all reagents was reduced in confluent and in differentiated polarized cells compared to subconfluent (70% confluent) cells. In our hands, maximum transfection and viability for l-pEI/DNA in Hela CCL-2 cells was obtained at a seeding density resulting in 80% confluency on the day of transfection (Supplementary information Fig. S3).

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Fig. 2. Transfection efficiency in dividing versus non-dividing cells. Transfection of dividing cells was performed in HeLa CCL-2 cells in the absence of aphidicolin (A) and transfection in non-dividing cells was studied by transfecting HeLa CCL-2 cells in the presence of 15 μM aphidicolin, a reagent which arrests cells in early S-phase (B). Cells were transfected with DNA (0.25 μg DNA/well in 96-well format) complexed with l-pEI (N/P = 6), b-pEI (N/P = 5) or Lipofectamine (2 μL/μg DNA) in the indicated dispersion medium for 4 h in the absence (white bar/circle) or presence of serum (gray bar/ asterisk). Cells were analyzed for gene expression (bars) and metabolic activity (circle and asterisk) 48 h after transfection. Arrest of HeLa CCL-2 cells in S-phase was confirmed by cell cycle analysis. (C): untreated cells (gray) and cells incubated for 24 h with 15 μM aphidicolin (transparent overlay) were fixed, stained with propidium iodide and analyzed for total DNA content by flow cytometry. (More experimental details can be found in the Supplementary information).

Exposure of cells to samples is either obtained by adding sample to transfection medium that was already present in the wells containing cells (with or without a washing step), or by diluting or preparing complexes in the transfection medium and replacing the standard cell culture medium with this mixture. The first procedure prevents risks of excessive drying of cells during execution. The composition of the transfection medium is critical. As explained before, presence of salts and proteins can affect physicochemical properties of complexes, and their transfection activity and toxicity as a consequence. In addition, detrimental effects have been ascribed to antibiotics, as their uptake may be facilitated when cell permeability is increased during transfection, causing toxicity [138]. Antibiotics-related reductions in transfection levels of up to 25% have been reported for COS-1 and CHO-K1 cells [131]. Additionally, an extensive list of ingredients including EDTA, citric acid, phosphate, RPMI, chondroitin sulfate, hyaluronic acid and sulfated proteoglycans are indicated to potentially inhibit Lipofectamine-based transfection [139]. Obviously, in vivo application will require a robust delivery system that is less affected by the composition of its surrounding fluids. Having selected a medium composition in which transfection studies are performed, the dilution effect upon addition of sample should be acknowledged. By keeping the sample: medium volume ratio low (e.g. ≤1:4), effects of changes in medium composition on cell functioning are minimized. The total volume of transfection medium may also be of interest, particularly for small particles. In their studies regarding salt-induced aggregation and increased transfection, Ogris et al. [77] hypothesized that the increased in vitro transfection efficiency of larger complexes is related to their faster sedimentation onto the cells adhered to the bottom of the wells. Indeed, small (but not large) complexes were shown to benefit 14-fold from a 6-fold reduction in transfection volume. The relevance of facilitating contact of complexes with cells is further supported by the observation that gentle plate centrifugation immediately after addition of complexes augmented transfection levels up to 50-fold [117].

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6.4. Incubation of samples with cells: effects of dose and incubation time The final dose of DNA reached per cell in in vitro experiments is determined by the number of cells per well (confluency), the dose (and volume) applied per well and the time during which complexes are incubated with cells. Most transfection experiments are performed at a confluency of 60–80%. In contrast, large variations in incubation times and DNA dose are used. Incubation of complexes with cells ranges from 1 h to continuous exposure until time of analysis and DNA doses vary from 0.2 to 1 μg DNA/well (96-well format) (see Table 1). The optimal time of incubation of complexes with cells should balance detectable expression with limited toxicity. Toxicity (if present) increases with increasing incubation time and this effect is augmented at higher doses. In general, expression increases steeply with increasing incubation times up to a certain time point (around 4 h), after which expression levels may either reach a plateau, continue to increase less steeply, or decrease, depending on the type and dose of complexes and cell type used (see Supplementary information Fig. S4). In CHO-K1 expression levels were observed to continuously increase at incubations from 0.5 up to 48 h upon transfection with Fugene, whereas in NIH-3T3 maximum levels were already reached after 4 h [131]. In case of small particles (pEI/DNA prepared in HBG), increasing the incubation time from 4 to 72 h gave a 20-fold increase in expression in Neuro2A cells, whereas expression levels of large particles (pEI/DNA prepared in HBS) were unaffected [77]. The faster sedimentation of larger particles may lead to increased uptake and gene expression [77] in an in vitro setup and cause them to play a dominant role in transfection outcomes when using short incubation times. This phenomenon should be considered as experimental bias and must be taken into account when interpreting data and drawing conclusions regarding the potential of reagents for in vivo gene delivery where sedimentation is irrelevant and aggregation is undesired. The amount of DNA to incubate with cells should be chosen such that expression can be detected while toxicity remains limited. Increasing the dose of DNA may yield higher expression levels, but at the cost of increased toxicity (unless incubation times are kept very short) and potential loss of discriminative power. At high doses of plasmids in which transcription is controlled by a strong promoter (such as the CMV promoter) the system can become saturated, possibly due to saturation of transcription and/or translation [93]. To place chosen doses into perspective, a human cell contains 7.1 pg chromosomal DNA. A dose of 0.25 μg per 8000 cells seeded per well corresponds to a dose of 31 pg DNA/cell. Assuming that approximately 10% of the applied dose is taken up by cells [140], this translates into delivery of a mass of exogenous DNA corresponding to ~40% of a cells chromosomal DNA. Since both expression and toxicity increase with increasing dose (see Supplementary information S4), a positive dose–response relation may be inverted depending on the incubation time, when dose-dependent toxicity outweighs dose-dependent transfection. At fixed incubation times where toxicity is limited, expression levels increase with increasing dose until a plateau is reached. The dose at which this plateau is reached, is dependent on formulation and cell type. Huh et al. [118] tested 25 kDa l-pEI/DNA complexes (prepared with pEGFP-C1 plasmid in 150 mM NaCl at an N/P ratio of 40) in four different cell types at doses ranging from 1 to 3 μg DNA/well in a 6 well-format. In human endothelial kidney (HEK) cells, expression levels were generally high and only slightly increased from 1 to 3 μg. In COS-7 cells, transfection efficiency increased until reaching an optimum at 2 μg, after which efficiency decreased. In HeLa and NCCIT cells, dose-dependent increase in efficiency appeared to reach plateau around 3 μg. Kichler et al. [92] investigated dose-dependency of gene expression upon transfection of HepG2 cells with complexes of 22 kDa pEI and a luciferase-expressing plasmid (prepared at an N/P ratio of 13 in 150 mM NaCl and tested in absence of serum). At doses

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below 0.4 μg DNA/well (24-well format) no expression was observed, followed by a dose-dependent increase between 0.4 and 1 μg and saturation at 1–2 μg. In HeLa CCL-2 cells, we observed the optimal incubation time and dose to vary with the formulation and presence of serum, but overall an incubation time of 4 h at a dose of 0.25 μg DNA/well (96-well format) was considered optimal (Supplementary information Fig. S4). Dose-dependent increases in transfection efficiency likely reflect the amount of nuclear-delivered plasmids [141], whereas the plateau phase reflects either excessive toxicity or saturation of transcriptional and/or post-transcriptional processes [142–144]. At this point it should be mentioned that strikingly different patterns have been observed depending on the method of dose reduction, being dilution of complexes in buffer, dilution by mixing with inactive complexes, or mixing of active DNA with inactive DNA prior to complexation [92,93,141,143,145]. 7. Read-out 7.1. Expression Transfection efficiency is often determined with plasmids containing reporter genes under the control of strong, constitutively active promoters (e.g. CMV). Three of the most abundantly described reporter genes are β-galactosidase [146–148], luciferase [149,150] and (enhanced) green fluorescent protein ((E)GFP) [151]. Read-out of transgene expression can be direct (detection of fluorescent proteins, e.g. EGFP) or indirect based on enzymatic conversion of an added substrate to a colored, fluorescent or luminescent product. Detection of the fluorescent protein or colored/luminescent products can be at the single cell level (flow cytometry or microscopic analysis) or a batch analysis (detection of collective protein/product from all cells in a well). Each assay is characterized by differences in sensitivity, signal-to-noise ratio, kinetics of expression and analytical techniques required for detection. Comparison of the three readout systems by transfection of HeLa CCL-2 cells with either pCMV-LacZ, pCMV-luc or pCMV-EGFP and analysis at several timepoints after transfection did reveal differences in onset, level and peak of expression, but the overall observed trends were similar. Data obtained for l-pEI-, b-pEI-based and Lipofectamine-based complexes are shown in the Supplementary information, Fig. S5. The β-galactosidase assay had a slower onset (~8 h) and peak (48 h) than either EGFP or luciferase (onset 4 h; maximum at 24–48 h). The choice for a reporter gene assay proved not crucial for the experimental outcome and can therefore be made based on other parameters such as desired sensitivity or costs as listed in Table 2. An added value is assigned to those read-out assays that allow quantification of overall expression levels as well as fractions of transfected cells (e.g. flow cytometry analysis of EGFP-expressing cells). Thereby it is possible to discriminate whether transfection efficiency is due to a small population of cells expressing high levels of protein or due to many cells expressing low levels of proteins. This information gives insight into the mechanism of transfection. In addition, with EGFP it is possible to measure transgene expression in living cells without requirement for substrates. βgalactosidase and luciferase reporter proteins can only be detected indirectly by conversion of an added substrate into a colored, fluorescent or luminescent product. An advantage of enzyme/substrate based detection is that sensitivity can be improved by increasing substrate incubation time. Moreover, these assays are better suited for highthroughput screenings. 7.2. Toxicity Toxic effects of gene delivery reagents are mostly related to their cationic nature. Strong interaction between cationic polymers or lipids with outer and inner cell membranes affects the integrity of these membranes [155–159], and interactions with negatively charged cell components such as proteins, DNA and RNA alters natural protein

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Table 2 Characteristics of reporter gene assays (partially based on [152–154]). Advantages

Disadvantages

β-galactosidase + ONPG → colorimetric detection + Cost effective and easy + Variety of substrates available allowing different and more sensitive analysis: fluorescence (MUG, FDG, DDAOG), luminescence (1,2-dioxetane-β-gal, AMPGD) + Possibility for histochemical staining with X-gal substrate → single cell analysis

- Slower onset and peak of expression detection - Lower sensitivity (when using non-luminescent assays) - Background signal from endogenous β–galactosidase in some cell types (especially in vivo)

+ Stable reporter protein + X-gal and fluorescence based assays are suitable for in vivo Luciferase + luciferin → luminescence detection + Fast onset and peak of expression detection + Sensitivity

- Expensive

+ Bioluminescence assay in live cells can be used in vivo

- Only batch analysis possible

- No histology possible - Relatively labile reporter protein (short half-life)

EGFP → fluorescence detection - Relative insensitivity of fluorimetry assay makes it less suitable for high throughput screening

+ No substrate required + No apparent toxic effects of GFP in eukaryotic cells + Cost effective, easy + Analysis at single cell level and batch analysis possible + Allows various types of analysis: flow cytometry, microscopy or fluorimetry

- Microscopy and flow cytometry require expensive equipment and are less suitable for high-throughput screening - Protein is stable and continues to emit fluorescence long after the host cell has died

+ Various color variants available + Direct fluorescence detection is possible in vitro and in vivo AMPGD: 3-(4-methoxyspiro(1,2-dioxetane-3,2′-tricyclo(3,3,1,1(3,7))decan)-4-yl)phenylgalactopyranoside; DDAOG: 9H-(1,3-dichloro-9,9-dimethylacridin-2-one-7-yl) β-D-galactopyranoside; FDG: fluorescein di-β-D-galactopyranoside; (E)GFP: (enhanced) green fluorescent protein; MUG: 4-methylumbelliferyl-β-D-galactoside; ONPG: o-nitrophenyl-β-D-galactopyranoside.

expression, function and cell cycling [144,158,160–162]. Charge density, molecular weight, presence of free carrier and degree of biodegradability are important factors influencing toxicity and are topics of investigation in new carrier development [163]. To evaluate the biocompatibility of new reagents, several assays are available for which detection is either based on cell death (cytotoxicity assays, e.g. lactate dehydrogenase leakage (LDH) and trypan blue exclusion assays) or on living cells (cell viability assays, e.g. protein quantification, neutral red uptake and mitochondrial activity) (see Table 3). Different assays can give different results depending on the test reagent, cell type, experimental conditions and cytotoxicity assay used [164,165]. A Table 3 Assays used to evaluate biocompatibility of reagents. Assay

Principle

Ref.

Toxicity assays LDH

Release of intracellular LDH into cell culture [168] medium as an indication of cell membrane damage. [169,170] Exclusion of trypan blue Dyes can penetrate through apoptosisdamaged membranes, but are excluded and fluorescent dyes (i.e. propidium iodide) from viable cells with functional membranes. Viability assays MTT, XTT

Neutral red uptake ATP Protein quantification

In mitochondria of metabolically active cells tetrazolium salts are reduced to colored products that are unable to cross membranes and accumulate in viable cells. Living cells take up neutral red, which is concentrated in lysosomes. Quantification of ATP as an indication of metabolic activity of cells Quantification of protein content of viable cells that are left after washing away detached cells as an indirect measure of viability.

[171]

[172] [173] [174]

ATP: adenosine triphosphate; LDH: lactate dehydrogenase leakage; MTT: 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; XTT: 2,3-bis-(2-methoxy-4nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide.

rational selection of assays and/or employing at least two independent methods is therefore warranted to gain insight in the mechanism of toxicity and to prevent underestimation of toxicity. The most widely used assay in gene delivery research is based on the detection of dehydrogenase activity in active mitochondria by the MTT/XTT assay. In a comparative study performed by Fotakis et al., the MTT and neutral red assays proved to have superior sensitivity to LDH and protein assays [166]. However, the MTT assay is not appropriate for cells with unchanged redox fluctuation (e.g. CHO cells) [165]. The LDH assay is based on detection of LDH released from cells with compromised membranes and since the half-life of released LDH is approximately 9–10 h [167], this assay is particularly suitable to study immediate toxicity. MTT/XTT assays are useful to study toxicity at the intracellular level and efficiency of gene delivery in parallel at later time points after treatment. The presence of enzyme inhibitors (e.g. chloroquine) may interfere with the enzymatic reactions essential to the MTT/XTT and LDH assays and therefore require enzyme-independent viability tests such as the neutral red uptake assay [164].

7.3. Data presentation Expression and toxicity can be expressed as absolute values or calculated relative to control values. In case of toxicity data, untreated cells serve as a negative control representing 100% cell viability. Since this value is known and fixed, data can be presented relative to negative control values. In contrast, expression data must be compared to a positive control. Expression levels for positive controls vary with the reagent chosen as well as with the experimental conditions used (as discussed in more detail in the next paragraph). Since the control values are variable, presentation of expression data calculated relative to these control values results in loss of insight in the order of magnitude of expression levels. Alternatively, independent presentation of the absolute expression data of both test formulations and control formulations allows interpretation of the overall assay while maintaining the desired possibility of comparing samples to controls.

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modification with polyethyleneglycol (PEG) or targeting ligands, or by modifying molecular weight, crosslinking or charge density, the unmodified polymer is usually included for reference. Including independent reference reagents is however valuable to enable comparison among experiments and laboratories. Several studies have been performed to compare various commercially available non-viral reagents (including Exgen500, 25 kDa lpEI, 50 kDa l-pEI, 25 kDa b-pEI, Lipofectamine 2000, Lipofectamine Plus, Lipofectin, Effectene, Fugene6 and Superfect), viral vectors (adeno-associated virus and lentiviral transfections) and physical transfection methods (nucleofection) in various cell types [85,118,119,121,122] (see also Supplementary information Fig. S2). The most important conclusion to be drawn from such comparative studies is that the optimal reagent varies with cell type, and that for a given reagent optimal conditions (reagent:DNA ratio, presence of serum) differ per cell type. An elegant approach was followed by Gebhart et al. [85], who first selected the four best performing reagents out of seven tested in COS-7 cells (in the presence of serum), and then analyzed the relative transfection efficiencies of these four reagents in 10 different cell lines. Ranking based on a Kruskal–Wallis test identified Exgen500 as the best transfection reagent for a large number of cell types (8/10), closely followed by Superfect, while pEI (25 kDa) and P123-g-pEI (2 kDa) appeared less effective (see Table 4). This conclusion is supported by Huh et al., who selected Exgen500 as the overall best performing reagent in comparison to 25 kDa l-pEI, Lipofectamine 2000 and Effectene when tested in HEK293T, COS-7, HeLa and NCCIT cells [118]. Besides dependency of transfection efficiency and toxicity on cell types, this paper has also highlighted sensitivity of reagents to exact formulation parameters and transfection conditions. In order to compare gene expression and toxicity of three frequently described reference reagents under various conditions, we transfected HeLa CCL-2 cells in the presence and absence of serum with complexes of DNA and either 22 kDa l-pEI, 25 kDa b-pEI or Lipofectamine™ 2000

Table 4 Ranking of effectiveness of four different transfection reagents in various cell lines. A panel of polyplexes was tested with each cell line in at least three separate experiments. The results were analyzed using Kruskal–Wallis ranking test. This analysis shows, for each cell line, which polyplex more frequently performs better than others in a series of repeated evaluations of the polyplex panel. As a result, even if in one individual experiment a polyplex shows higher transfection efficacy than the other members of the panel, it is not ranked the best for a given cell line if it is not the most frequent best performer in the series of repeated evaluations. n/d indicates that the given condition was not tested. Reprinted from [85]. Cell line

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Importantly, interpretation of transfection efficiency data of new reagents requires comparison to reference reagents. Although this may seem a trivial issue, selection of a suitable universal reference reagent is a difficult task. In literature, a great variety of control reagents included for estimation of transfection efficiency of a gene delivery reagent compound under investigation are described, including naked plasmid DNA, 22 kDa linear pEI (or linear pEI of other molecular weights), 25 kDa b-pEI (or branched pEI of other molecular weights), Lipofectamine, Lipofectin, Superfect, GenePorter, Fugene, Effectene, pDMAEMA, polylysine, DOTAP, and others. In studies aiming at improving a certain polymer through for example

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Fig. 3. Gene expression and cell viability obtained after transfection of HeLa CCL-2 cells with frequently used control formulations prepared in various buffers. Gene expression (bars) and cell viability (circle and asterisk) after transfection of HeLa CCL-2 cells in the absence (white bar/circle) or presence (gray bar/asterisk) of 10% serum with complexes prepared in the indicated dispersion medium (l-pEI N/P = 6, b-pEI N/P = 5, Lipofectamine 2 μL/μg DNA). Complexes were incubated 4 h with cells (0.25 μg DNA/well) and gene expression and cell viability were measured 48 h after transfection. Dashed line indicates 100% viability. (More experimental details can be found in the Supplementary information).

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○ Optimize ratios of reagent:DNA based on particle stability, transfection efficiency and toxicity. ○ Include 22 kDa l-pEI/DNA at N/P = 6 in HBG and HBS as reference formulation. Characterization ○ Measure condensation, protection, size and stability of formulations in HBG and HBS (e.g. by gel retardation, fluorescence displacement, DLS). ○ Measure zetapotential in HBG. ○ Evaluate effect of medium without and with serum on particle size (e.g. by DLS, microscopy and flow cytometry or single-particle tracking analysis). Cell transfection ○ Seeding: 24 h prior to experiment, seed cells at a density to reach 80% confluency on day of transfection. ○ Treatment: Immediately prior to transfection replace medium with fresh medium with and without serum. Dropwisely add 25 μL sample per well (100 μL medium and 0.25 μg DNA/well in 96-well format). After 4 h incubation replace medium with normal cell culture medium. ○ Selection of cell type: ▪ Commonly used, readily available and well-known model cell line(s) (e.g. HeLa CCL-2) ▪ Cell line relevant for disease/application studied ▪ Model for non-dividing cells (e.g. aphidicolin-arrested cells, primary cells) Readout and data presentation ○ Expression assay: Use expression plasmids with an optional reporter gene under control of the CMV promoter (readout: β-galactosidase 48 h/luciferase 24 h/EGFP 24 h post-transfection). Present absolute data (e.g. mU/well) for samples and positive controls. ○ Toxicity assay: e.g. XTT. Present data as % viability (e.g. metabolic activity) relative to untreated cells.

scientist in the field. We now take the initiative by proposing a first version of a universal standard procedure as presented in Table 6 and Fig 4. The proposed screening protocol offers a useful starting point for evaluating the potential of novel gene delivery reagents. The scope of this paper is limited to plasmid based transfection systems, but a similar

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EI l-p

• Provide full disclosure of all parameters known to affect transfection efficiency and toxicity. • Prepare formulations in simple and well-characterized buffers rather than complex media. • Use equal buffers to prepare complexes for characterization and transfection studies. • Evaluate complex stability and size in biological fluids (e.g. cell culture medium). • Always include transfection studies in the presence of serum, unless this is irrelevant for the application in mind (e.g. does not involve exposure to blood). • Back up results obtained in dividing cell culture models by studies in a model that more closely resembles the in vivo situation, such as primary or arrested cells. • Present gene expression data independently for samples and (positive control) references, and present viability or toxicity data calculated relative to untreated cells.

○ Prepare working stocks of reagent and of plasmid DNA in both HBG and HBS. Prepare polyplexes by adding 4 volumes of reagent to 1 volume of plasmid DNA (50 μg/ml) and mix by pipetting 10×. Incubate 30 min at room temperature.

H

Table 5 Proposed guidelines for in vitro transfection screenings.

Particle preparation

% Metabolic activity (rel. to untreated)

This paper has highlighted the sensitivity of the outcome of transfection experiments to various variables, which makes interpretation and comparison of result from literature troublesome, especially if detailed descriptions of applied methods and conditions are lacking and expression data are calculated relative to highly variable controls. Given the fact that experimental conditions can already account for 1000-fold differences in transfection efficiency when testing a single reagent in a single cell type, and that the differences obtained upon optimization of the delivery agent itself are within this same range [117], it is obvious that there is a need for standardization of protocols in order to enable meaningful comparisons of non-viral gene delivery systems. This review was therefore written with two aims: (1) to provide an overview of parameters that can affect transfection efficiency and toxicity by presenting a collection of illustrative examples from literature as well as our own datasets, and (2) to initiate standardization of in vitro transfection screening procedures in the field of non-viral gene delivery research. It may not be realistic nor desired that all laboratories follow the exact same protocol for their specialized experiments. However, following a limited set of guidelines as proposed in Table 5 would already substantiate the value and understanding of obtained results significantly. Additionally, we suggest the introduction of a universal standard of measure. By including testing of new reagents according to a standardized procedure (in parallel to user-defined testing conditions relevant to a specific study), inter-experimental and interlaboratory comparison of transfection results would be facilitated. It is acknowledged that reaching consensus and implementation of a standardized protocol requires time and thorough discussion among

Suggested standardized procedure for in screening non-viral gene delivery systems in vitro

EI

9. Discussion

Table 6 Suggested standardized procedure for in vitro transfection screenings of non-viral gene delivery systems (Detailed procedures are supplied in the materials and methods section in the Supplementary information).

l-p

prepared in HBG, HBS or Optimem (see Fig. 3). Transfection efficiencies of l-pEI, b-pEI and Lipofectamine™ 2000 all proved highly sensitive to the buffer in which complexes were prepared as well as the presence of serum during incubation with cells. When comparing these transfection outcomes to characterization results (obtained by DLS and flow cytometry, see Supplementary information Table S1 and Fig. S1), a tendency towards increased expression levels upon aggregation appears present (see Supplementary information Fig. S6). Altogether, it can be concluded that transfection efficiencies of transfection reagents, being either reagents under investigation or used as a positive reference, are highly dependent on experimental conditions chosen, which hampers comparative investigations. For the selection of a positive reference reagent, none of the commercially available reagents is ideal. Each reagent suffers from drawbacks of colloidal instability, inactivity in the presence of serum, a strong cell type dependency, substantial toxicity and/or undefined (or undisclosed) composition. Based on comparative studies by others and ourselves, it is our opinion that 22 kDa l-pEI is the best (though not ideal) option at current.

β-galactosidase (mU/well)

228

Fig. 4. Gene expression and cell viability obtained using the suggested standard protocol. Gene expression (bars) and cell viability (circle and asterisk) after transfection of HeLa CCL-2 cells in the absence (white bar/circle) or presence (gray bar/asterisk) of 10% serum with complexes prepared in the indicated dispersion medium. Complexes were incubated 4 h with cells (0.25 μg DNA/well) and gene expression and cell viability were measured 48 h after transfection. Means of 6 independent experiments.

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approach would be valuable for studies on siRNA delivery. Based on outcomes of this screening, directions for follow-up studies can be decided upon. For example, if characterization studies in buffers and physiological media reveal insufficient colloidal stability, a primary focus on improving particle properties is desired before moving to cell (and in vivo) studies. In case of unfavorable activity/toxicity ratios, subsequent efforts can be made to modify the molecular structure of the reagent and/ or removing excess free reagent. Stable particles that exhibit limited toxicity can be selected for mechanistic follow-up studies. Understanding what makes one reagent successful whereas another fails to mediate transgene expression will contribute to the rational design of novel formulations. Recent technological developments including improved cellular subfractionation methods, advanced microscopic techniques (reviewed in [175,176]) and quantitative PCR have extended the toolbox and enable studying cellular uptake pathways, endosomal transport [177,178], intracellular trafficking [54,179,180], cytosolic degradation [181], nuclear uptake [41,140,142,182] and efficiency of transcription/ translation [183,184]. Progressive implementation of these techniques and methods in the field of gene delivery is expected to yield important findings and substantial progress. However, as these studies require sophisticated, laborious and expensive techniques it is not feasible to apply them to each and every reagent under investigation. The screening protocol suggested in this paper (Table 6) provides a relatively simple, cost- and time-effective procedure for the pre-selection of potential candidate reagents. Reaching consensus on screening procedures within the field will enable placing individual results into a broader context, thereby accelerating the identification of structure/activity relations and facilitating to estimate significance of new findings to progress in nonviral gene delivery. This review article is an attempt to develop a standardized protocol for testing the efficacy of non-viral gene delivery systems, and readers are invited to make any further suggestions and contribute to the development of such a globally accepted standardized procedure. Appendix A. Supplementary data Supplementary data to this article can be found online at doi:10.1016/j.jconrel.2011.05.001. References [1] R.C. Mulligan, B.H. Howard, P. Berg, Synthesis of rabbit beta-globin in cultured monkey kidney cells following infection with a SV40 beta-globin recombinant genome, Nature 277 (1979) 108–114. [2] R. Fraley, S. Subramani, P. Berg, D. Papahadjopoulos, Introduction of liposomeencapsulated SV40 DNA into cells, J. Biol. Chem. 255 (1980) 10431–10435. [3] G.Y. Wu, C.H. Wu, Receptor-mediated in vitro gene transformation by a soluble DNA carrier system, J. Biol. Chem. 262 (1987) 4429–4432. [4] O. Boussif, F. Lezoualc'h, M.A. Zanta, M.D. Mergny, D. Scherman, B. Demeneix, J.P. Behr, A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine, Proc. Natl. Acad. Sci. U. S. A. 92 (1995) 7297–7301. [5] M.A. Mintzer, E.E. Simanek, Nonviral vectors for gene delivery, Chem. Rev. 109 (2009) 259–302. [6] D.G. Anderson, W. Peng, A. Akinc, N. Hossain, A. Kohn, R. Padera, R. Langer, J.A. Sawicki, A polymer library approach to suicide gene therapy for cancer, Proc. Natl. Acad. Sci. U. S. A. 101 (2004) 16028–16033. [7] D. Shcharbin, E. Pedziwiatr, J. Blasiak, M. Bryszewska, How to study dendriplexes II: transfection and cytotoxicity, J. Control. Release 141 (2010) 110–127. [8] D. Shcharbin, E. Pedziwiatr, M. Bryszewska, How to study dendriplexes I: characterization, J. Control. Release 135 (2009) 186–197. [9] Y. Arthanari, A. Pluen, R. Rajendran, H. Aojula, C. Demonacos, Delivery of therapeutic shRNA and siRNA by Tat fusion peptide targeting bcr–abl fusion gene in Chronic Myeloid Leukemia cells, J. Control. Release (2010), doi:10.1016/ j.jconrel.2010.04.011. [10] A. El-Sayed, T. Masuda, I. Khalil, H. Akita, H. Harashima, Enhanced gene expression by a novel stearylated INF7 peptide derivative through fusion independent endosomal escape, J. Control. Release 138 (2009) 160–167. [11] T. Higashi, I.A. Khalil, K.K. Maiti, W.S. Lee, H. Akita, H. Harashima, S.K. Chung, Novel lipidated sorbitol-based molecular transporters for non-viral gene delivery, J. Control. Release 136 (2009) 140–147. [12] W.C. Hung, M.D. Shau, H.C. Kao, M.F. Shih, J.Y. Cherng, The synthesis of cationic polyurethanes to study the effect of amines and structures on their DNA transfection potential, J. Control. Release 133 (2009) 68–76.

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