## 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 semi-quantitative 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  
Figure 1: The general procedure of the NanoTrans-gel synthesis. EDC:HCl and BSA encapsulate the plasmid pEGFP-c3 forming nanogels by a crosslinking reaction followed by a solvent exchange to an aqueous milieu. The plasmid pEGFP-c3, coding for the fluorescent protein EGFP, contains two specific binding sites for primers enclosing a region of 118 base pairs (PCR product, highlighted in red). For transfection experiments, the cultivated cell lines A549 and MCF7 are incubated with the plasmid-containing nanogels as well as Lipofectamine as a reference in a 96-well plate, and the gene expression of the fluorescent protein EGFP is analyzed by flow cytometry. 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 [PERSON] 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).