Inverse-Nanoemulsion-Derived Protein Hydrogels (NanoTrans-Gels) Can Outperform DOSPA/DOPE Lipid-Complex Transfection Agent

Abstract

Transfection of mammalian and human cell lines in medical research both are key technologies in molecular biology and genetic engineering. A vast variety of techniques to facilitate transfection exists including different chemical and nanoparticle-based agents as mediators of nucleic acid uptake, with nanoparticles composed of the lipids DOSPA/DOPE belonging to the established type of agents. We show that inverse-nanoemulsion-derived protein nanohydrogels (NanoTrans-gels), prepared by a simple synthesis protocol, are suited to transfect two model cancer cell lines (MCF7 and A549) with high efficiency. The transfection efficiency was analyzed in comparison to the DOSPA/DOPE-dependent protocols as a reference method. Since nanogel-based transfection outperformed the Lipofectamine-dependent technique in our experiments, we believe that the NanoTrans-gels loaded with plasmid DNA may open new avenues for simple and efficient transfection for humans and probably also other mammalian cell lines and may develop into a general tool for standard transfection procedures in cell biology laboratories.

1. Introduction

Transformation in the case of bacterial cells and—even more importantly—transfection of higher cells including human cell lines in medical research are both key technologies in molecular biology and genetic engineering [1,2]. Transfection is a procedure to deliver genetic material (either recombinant DNA constructs or RNA molecules) to living cells in a functional form, i.e., it allows for replication, expression of target/marker genes, and access to regulatory factors in the cell [3,4,5]. An impressive range of different techniques has been invented in the last 50 years (nicely reviewed in [6,7]), which can be summarized as belonging to (i) biological methods including virus-mediated gene transfer [8,9,10], (ii) physical methods (e.g., direct injection, electroporation, or biolistic particle delivery [11,12,13]), and (iii) chemical methods, which have developed into the best established standard techniques [14,15,16]. Although nucleic acid-based constructs appear to be promising structures for the manipulation and optimization of cellular functions (as single molecules or higher assembled complexes), naked nucleic acids have an intrinsic limitation in that they are prone to rapid degradation in vivo and their negative charges inhibit efficient binding to cell surfaces and their required subsequent passage through the cell membrane [17]. Hence, protective and efficient facilitators of nucleic acid uptake are generally needed [18,19,20,21]. Such carriers based on liposome-forming cationic lipids have been developed as generally used assemblies of complex nucleic acids prior to their transfective uptake into living cells [22,23]. Among them, defined mixtures of the lipids DOSPA (2,3-dioleoyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate) and DOPE (1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine) commercialized under the name Lipofectamine [24] can be regarded as a “gold standard” with mentions in currently over 3580 scientific publications listed in the NCBI PubMed literature database (accessed 16 May 2024). In the background of the complex physiology of mammalian and human cells with different susceptibilities for uptake of foreign nucleic acids (i.e., mediated uptake and intracellular processing of the foreign DNA), efficiencies of such manipulations can be expected to be very different. Depending on the cell line used or the growth conditions applied in the experiment prior to and after transfection, Lipofectamine has received recognition for its broad spectrum of cell lines accessible by this agent [25]. Nevertheless, its cytotoxicity appeared to be considerable [26].

Protein hydrogels based on crosslinked serum albumin (human or bovine; “HSA” or “BSA”) have been developed for applications as different as matrices for 3D cell culture, as functional layers in novel composite wound dressings and as novel passaging tools in cell culture. Moreover, they have proven their low cytotoxicity in these applications [27,28,29,30,31,32,33]. From these materials, nanometer-sized spheres (“nanogels” (Ng)) can be fabricated in an inverse-nanoemulsion synthesis technique that we have recently introduced [34]. The nanogels were shown in initial experiments to be suitable for delivering fluorescent dyes, model drug molecules, aptamers, and plasmid DNA to selected human cell lines [34]. Delivery of the GFP-encoding plasmid pEGFP-c3 was demonstrated only with a single breast cancer cell line (MDA-MB-231 (ATCC CRM-HTB-26) [35]), suggesting a principal capability of the nanogels to serve as a transfection agent [34], and we tentatively suggest the nomination “NanoTrans-gels” for these functional nanostructures. Here, we present additional evidence for the functionality of nanogel-mediated transfection with the breast cancer cell line MCF7 (ATCC HTB-22) [36,37] and the lung adenocarcinoma cell line A549 (ATCC CRM-CCL-185) [38,39] as probably two of the best described and established model human cell lines [40,41]. The efficiency of nanogel-based transfection was analyzed in comparison to the transfection efficiency using DOSPA/DOPE-dependent protocols as a reference method. Since nanogel-based transfection outperformed the Lipofectamine-dependent technique, in our experiments, we believe that the NanoTrans-gels loaded with plasmid DNA may open new avenues for simple and efficient transfection for humans and probably also other mammalian cell lines and may develop into a general tool for standard transfection procedures in cell biology laboratories.

2. Materials and Methods

2.1. General Procedure

In this study, we utilized the following chemicals and reagents as received from specific suppliers: BSA (Albumin Fraktion V, neoFroxx GmbH, Einhausen, Germany), 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC·HCl, ≥99%, Carl Roth GmbH + Co. KG, Karlsruhe, Germany), MES (99.5%, Carl Roth GmbH + Co. KG, Karlsruhe, Germany), n-dodecane (99%, Alfa Aesar, Haverhill, MA, USA), Span 80 (Carl Roth + Co. KG, Karlsruhe, Germany), cyclohexane (≥99%, VWR International, LLC, Radnor, PA, USA), ethanol (99.5%, VWR International, LLC, Radnor, PA, USA), TRIS hydrochloride (≥99%, Carl Roth GmbH + Co. KG, Karlsruhe, Germany), and Lipofectamine 3000 (Invitrogen, Waltham, MA, USA). For all experiments, demineralized water of MilliQ quality (resistivity: 18 MΩ cm) is used. For cell experiments, DMEM (Gibco/Thermo Fisher Scientific, Waltham, MA, USA), FBS (Gibco/Thermo Fisher Scientific, Waltham, MA, USA), penicillin–streptomycin (100 U mL⁻¹, Gibco/Thermo Fisher Scientific, Waltham, MA, USA), MEM NEAA (Gibco/Thermo Fisher Scientific, Waltham, MA, USA), and Accutase (Merck Millipore, Burlington, MA, USA) are utilized.

2.2. Nanogel Preparation and Analyses

The nanogels are synthesized as described in the literature [34]. In a 30 mL screw-top jar, 16.5 mL n-dodecane containing 125 mg Span 80 is ultrasonicated (Branson Digital Sonifier W-450 D; 40% amplitude; ¼ inch tip; titanium horn) under ice cooling while 250 µL BSA solution (200 mg mL⁻¹ in MES buffer (100 mM, pH 5)) containing the desired amount of plasmid DNA and 250 µL EDC·HCl solution (100 mg mL⁻¹ in MES buffer) are mixed, added to the continuous oil phase immediately, and sonicated for another 30 s. The emulsion is then left at room temperature for 12 h. To change the continuous phase to a more polar solvent, the nanogel dispersion was transferred in a 50 mL conical centrifuge tube and centrifuged at 13,400× g for various indicated time intervals. DLS measurements were performed in relevant pure solvents to surveil the size of the nanogels and ensure successful redispersion. The supernatant was discarded and the nanogels were redispersed in the new solvent by vigorous shaking and ultrasonication (Elma TI-H-5; 25 kHz). After n-dodecane (5 min), the continuous phase is changed to cyclohexane (5 mL, 5 min), cyclohexane/ethanol (3.5 mL each, 4:20 min), ethanol (5 mL, 5 min), and ethanol/water (2.5 mL each, 20 min) and then washed twice with water (5 mL, 20 min) before the final uptake in TRIS·HCl buffer (10 mM, pH 7.5) as a ready-to-use product. The experiments were conducted in triplicate (n = 3) and plotted as mean values ($\overline{x}$ = ${\displaystyle \frac{1}{n}}\sum _n^{x=1}{x}_i$) with error bars representing the standard deviations (SD = $\sqrt{{\displaystyle \frac{\sum {\left(x-\overline{x}\right)}^2}{n}}}$).

2.3. PCR

Before the PCR, the NanoTrans-gels are digested by adding 2 µL Proteinase K (600 U/mL, Thermo Fisher Scientific, Waltham, MA, USA) to a 20 µL sample, incubating at 55 °C for 3 h, and deactivating at 95 °C for 10 min. Afterwards, the Mastermix containing 14.13 µL PCR H₂O, 5 µL Herculase buffer (Agilent Technologies, Inc., Santa Clara, CA, USA), 0.625 µL dNTPs (25 mM each, Agilent Technologies, Inc., Santa Clara, CA, USA), 0.0625 µL per primer (100 µM, biomers GmbH, Ulm, Germany), 0.125 µL Herculase II (Agilent Technologies, Inc., Santa Clara, CA, USA), and 5 µL of the sample is used for PCR with an initial denaturation step at 95 °C for 2 min before 25 cycles of denaturation at 95 °C for 30 s, annealing at 56 °C for 30 s, elongation at 72 °C for 10 s, and a final elongation step at 72 °C for 2 min (Sensoquest labcycler gradient). The amplification of the DNA of interest is shown by agarose gel electrophoresis with a 4% agarose gel in 0.5× TBE buffer.

2.4. Cell Culture

The breast cancer cells MCF 7 (ACC HTB-22) and lung cancer cells A549 (ATCC CRM-CCL-185) are cultivated in a DMEM medium supplemented with 10% FBS, 1% penicillin–streptomycin (100 U mL⁻¹), and 1% MEM NEAA at 37 °C with 5% CO₂ in a T25 flask.

2.5. Transfection Experiments

For detachment, the medium is removed, and the cells are treated with 2 mL Accutase for at least 10 min prior to cell counting using a Neubauer counting chamber. Then, 20,000 cells per well are seeded in a 96-well plate and incubated in a total volume of 200 µL medium for 24 h as described above. A total of 10 µL of NanoTrans-gels are added and incubated for a further 48 h at 37 °C with 5% CO₂. For comparison with Lipofectamine, 5 µL DMEM without supplements and 0.3 µL Lipofectamine 3000 are mixed and shortly vortexed. In another vessel, 0.8 µL P3000 reagent and the appropriate amount of plasmid are added to 5 µL DMEM and mixed well. Both mixtures are combined and incubated for 10 min before adding 10 µL per well to the cells and incubation for 48 h at 37 °C with 5% CO₂.

2.6. Flow Cytometry

After incubation for 48 h, the medium is removed and 100 µL Accutase is added for detachment of the cells. The cells are transferred in a 1.5 mL reaction vessel and 400 µL PBS is added prior to centrifugation at 2000× g for 3 min. The supernatant is removed, and the pellet is resuspended in 1 mL PBS and transferred in a FACS tube for flow cytometry measurements (Sysmex CyFlow Cube 6, Sysmex Deutschland GmbH, Bornbach, Norderstedt). The data are analyzed with the FlowLogic software (FlowLogic 8.7, Inivai Technologies Pty. Ltd., Mentone, VIC, Australia).

3. Results

Preparation of DNA-loaded NanoTrans-gels included the addition of the model plasmid pEGFP-c3, with a molecular size of 4691 base pairs carrying the egfp gene for the enhanced GFP protein under the transcriptional control of the CMV promotor to the BSA/EDC-based nanogel inverse-nanoemulsion synthesis reaction, as described earlier [34]. The plasmid contained binding sites for analytical oligonucleotide primers allowing semiquantitative PCR-based analysis of the DNA loading. The loaded nanogels were then incubated with defined numbers of both model cell lines for transfection, and subsequently, GFP-producing cells were detected and quantified by flow cytometry (Figure 1).

The inverse-nanoemulsion synthesis workflow included six individual steps in n-dodecane as the continuous phase, subsequent transfer into solvents of increasing polarity (n-dodecane < cyclohexane < ethanol) to finally reach an aqueous environment (washing with pure water), and storage as ready-to-use DNA-loaded spheres in TRIS-HCl buffer (pH 7.5). The resulting NanoTrans-gels had diameters ranging from 600 nm to 800 nm and a polydispersity (PDI) between 0.1 and 0.4 determined by dynamic light scattering (DLS). During this procedure, the nanogels showed the earlier described solvent-dependent swelling and agglomeration behavior [34] resulting in size changes from 200 nm to over a maximum of 900 nm for the final sizes, independent of the presence or concentration of the cargo/plasmid DNA (Figure 2a). The empty nanogels of pure crosslinked BSA showed the expected zeta-potentials of around +30 mV as shown before by Sihler et al. [34]. Slightly reduced zeta-potentials of loaded nanogels may be the result of the negative charges of the cargo DNA molecules (Figure 2b). Nanogel DNA samples were subjected for qualitative analysis by conventional PCR and subsequent electrophoretic analysis in traditional agarose gels to prove loading with DNA of the NanoTrans-gels by the presence of a distinct 118-base-pair PCR product (Figure 2c).

After transfection, cells were allowed to express the egfp gene and hence the fluorescent phenotype for 48 h. Subsequently, they were submitted to flow cytometric analysis using measurements of the side scatter against fluorescence intensity either with cells alone, cells with free plasmid, or cells with empty nanogels as the essential controls for both cell lines (A549 and MCF7), respectively. The actual experiments, in contrast, were then based on nanogels loaded with increasing amounts of 5, 10, and 20 ng of pEGFP-c3 per transfection reaction or the same amount of DNA in solution, but in the presence of Lipofectamine to assist transfection of the naked soluble DNA. Upon appropriate gating, the resulting side scatter (“SSC”; y-axis) against fluorescence intensity (“FL1”; x-axis) plots allowed for the discrimination, as expected, of fluorescent cells from non-fluorescent cells and hence the differentiation of transfected and non-recombinant wild-type cells (Figure S1 in SI). The transfection efficiency can be derived from the difference between the percentage of transfected cells in the actual samples and the respective control samples (Figure 3a). While the NanoTrans-gels in the case of MCF7 cells only slightly improved the transfection efficiency for the lower DNA concentrations compared to the Lipofectamine reference transfections (red columns), efficiencies of A549 transfection reactions were drastically improved using the NanoTrans-gels resulting in an up to 95-fold increase in fluorescent and hence transfected cells (green columns) (Figure 3b).

4. Discussion

The uptake of nucleic acids into living cells with the aim to provide them with extra genetic information, e.g., for the recombinant production of foreign proteins, is an essential experimental task in cell biology. A variety of tools and techniques for this process known as transfection has been invented in recent decades [7,42]. In addition to chemical compounds as transfection facilitators like cationic polymers [14], calcium phosphate [16], or cationic lipids [15], a category in which the reference transfection agent Lipofectamine used here can be subsumed, different classes of nanoparticles have been described including gold nanoparticles [43], organically modified silica (ORMOSIL) nanoparticles [42,44], biologically degradable polymer nanoparticles (i.e., poly(lactic acid)) [45]; poly(β-amino ester) [46]), or chitosan-based nanoparticles [47]. With the recent introduction of inverse-nanoemulsion-derived BSA nanohydrogels, we have added a simple protein-based nanoparticle to the available portfolio of cell uptake-mediating nanostructures [34]. The existing robust and simple synthesis protocol for these nanogels was shown here to allow for easy experimental modification by simply adding increasing amounts of plasmid DNA as an example of functional nucleic acids without disturbing the nanogel quality delivering indistinguishable key parameters like size, swelling, agglomeration behavior, and zeta-potential. These NanoTrans-gels can be stored in a ready-to-use manner in simple TRIS·HCl buffer (pH 7.4) at 4 °C, and addition to the target cells allows for conditions resembling those of standard transfection protocols as they are used, e.g., with Lipofectamine (i.e., incubation time, temperature, expression time, etc.). The principal functionality of transfection with calcium phosphate nanoparticles has been shown, but a large study with a variety of cells found that this method resulted in notoriously low reproducibility of transfection efficiencies generally below those obtained with the commercial DOSPA/DOPE transfection agent Lipofectamine 2000 [24,48]. Lipofectamine has been optimized with the introduction of Lipofectamine 3000, which is a kit that contains a lipid mixture and a so-called P3000 reagent with a composition that is not fully known. However, this has been claimed by the manufacturer and described in a series of studies to improve transfection efficiencies for a range of different cell lines including “hard to transfect” cells like A549 [49,50,51,52,53]. In the context of such a technological breakthrough, it is of special remarkability that the inverse-nanoemulsion-derived BSA/EDC nanohydrogels, as carriers of DNA in our preliminary study using, so far, protocols that are not further optimized, instantaneously allowed for improvements in A549 transfection efficiencies of up to 95-fold compared to Lipofectamine 3000/P 3000 dependent protocols as the reference. Differences in transfection efficiencies principally depend on genetic and cell physiological conditions and are influenced by a vast variety of parameters that are hard to control in experiments. The respective nucleic acids have to cross the cell membrane, escape from the lysosome, and then enter the nucleus thereby—most importantly—avoiding their degradation [54,55,56,57,58,59,60,61]. Without yet having strong experimental evidence for increased protection of the nucleic acid when it is on the way from the membrane to the expressed marker gene(s), the speculation appears to be reasonable that DNA molecules tightly cocooned in a protein-based hydrogel may be stored extremely safely against (enzymatic) degradation like in a molecular secure container. If degradation-prone inactivation of functional vectors in a hard-to-transfect cell line like A549 is a crucial parameter, in turn, a hydrogel-mediated stabilization may explain the drastic effect observed for this cell line. However, the particle uptake, the release of cargo DNA, and its fate until the expression of the transfected phenotypes require more detailed analyses, which will be part of a follow-up study also involving more cell lines and different cargo DNAs. Deeply appreciating the common perception that the complex physiological process of transfection requires optimized conditions for every potential target cell line [62,63], we dare to hope that the NanoTrans-gel concept may develop into a broadly applicable, novel, and valuable expansion of the portfolio of transfection technologies available in cell biology.

5. Conclusions

Following a very simple protocol, nanometer-sized BSA/EDC hydrogels can be fabricated in the presence of increasing amounts of passenger DNA. These NanoTrans-gels were shown to be functional for transfection of A549 and MCF7 human cell lines with high efficiencies exceeding those obtained with commercially available DOSPA/DOPE (Lipofectamine) lipid nanoparticles and without extensive further optimization of the transfection protocol. These results may represent an ideal starting point for our already preconceived in-depth characterization studies to evaluate the full application potential of the NanoTrans-gel concept with a comprehensive series of different human and mammalian cell lines, different types and concentrations of nucleic acids (RNA and DNA), and under variations of experimental transfection parameters (e. g. time, temperature, medium, and cell number).