Dendron-functionalised hyperbranched bis-MPA polyesters as efficient non-viral vectors for gene therapy in different cell lines

Gene therapy has become a relevant tool in the biomedical field to treat or even prevent some diseases. The effective delivery of genetic material into the cell remains a crucial step to succeed in this purpose. In the search for efficient non-viral vectors, a series of amino-terminated dendronized hyperbranched polymers (DHPs) of different generations based either on bis-MPA or bis-GMPA have been designed. All of them have demonstrated an accurate ability to complex two types of genetic materials, a plasmid DNA and a siGFP, yielding dendriplexes. Moreover, some of them have proved to be able to deliver the genetic material inside the cells, resulting in the effective accomplishment of the desired genetic modification and improving the activity of some commercial transfection reagents. Different cell lines, including cancer and mesenchymal stem cells, have been studied here to evaluate the ability of DHPs as vectors for transfection. Treatments based on mesenchymal stem cells are gaining importance due to their pluripotency. Thus, it is of special relevance to introduce a genetic modification into a mesenchymal cell line as it allows it to act over a wide spectrum of tissues after inducing cellular differentiation.


Procedure II) Azide-alkyne cycloaddition reaction (CuAAC)
Scheme S3. Synthetic procedure for the CuAAC reaction (procedure II). As detailed in the manuscript (DHPs characterization section) and in the supporting information (Table S1), a small average number of residual groups, namely R = OH and/or R = COCH2CH2C≡C, can be present in the final DHP depending on the generation of the HP core and the nature of the dendron, either bis-MPA or bis-GMPA.

S2. DHPs labelled with a fluorophore
In order to obtain traceable molecules that allow their visualisation during the cellular internalisation process, the DHPs were labelled with the fluorophore rhodamine B (RhB) in an amount estimated to cover around 1% of the terminal functional groups of each DHP. The covalent bonding took place between the carboxylic acid of the RhB and the terminal amino groups of the DHPs (Scheme S2). Firstly, the carboxylic acid of RhB was activated by reaction with 1,1'-carbonyldiimidazole (CDI) in anhydrous dimethylformamide (DMF). Then, the mixture was allowed to react with the terminal amino groups of the DHP dissolved in the same solvent for 24 h at 40 °C. 1,2 The amount of RhB added was selected to obtain a final functionalisation of around 1% of the amino terminal groups of each pseudodendrimer. Purification was developed by three consecutive precipitations into cold ether and further recovery by centrifugation, pouring the supernatant and keeping the pellet obtained. Lastly, products were freeze-dried to obtain the final product as a pink solid.
Scheme S5. Synthetic procedure for the covalent labelling of the DHP-MPA with rhodamine B. As detailed in the manuscript (DHPs characterization section) and in the supporting information (Table S1), a small average number of residual groups, namely R = OH and/or R = COCH2CH2C≡C, can be present in the final DHP depending on the generation of the HP core and the nature of the dendron, either bis-MPA or bis-GMPA.
The correct labelling with RhB was assessed by 1 H NMR characterisation ( Figure S4). The aromatic protons of the fluorophore could be found in the region between 8.5 and 6.2 ppm in the 1 H NMR spectra. The methyl groups in β position from the N in the RhB are included in the up-field signal (around 1.29-1.25 ppm) together with the rest of the methyl groups of the pseudodendrimer. The number of RhB molecules in average per DHP was roughly determined by comparing the integration of the signal of the proton of the triazole at 7.8 ppm with the  RhB aromatic H Table S2. Hydrodynamic diameters (in nm) of the empty DHPs and the dendriplexes DHP/genetic material at different N/P ratios, measured by DLS in number. Data are represented as mean ± SD (n=3).  Table S3. Investigated ratios in w:w of the complexes formed by the DHPs and the pGFP. Table S4. Investigated ratios in w:w of the complexes formed by the DHPs and the siGFP.

S4. In vitro pGFP transfection
On the one hand, the effect of the incubation time of the dendriplexes with cells was evaluated for the complexes between DHP(G4)-MPA and pGFP at the N/P ratio 500. Namely, 4, 8 or 24 h of incubation in HeLa were assayed and the results were evaluated by fluorescence increase (transfection efficiency) and cell viability, comparing with the commercial reagent Lipofectamine 3000 ( Figure S6a). The highest fluorescence levels (8.7 a.u.) were found after 8 h of incubation with the dendriplexes, although the viability of the host cells was affected (60 % of viability). This cytotoxic effect was overcome after 24 h of incubation, when the transfection effectivity remained at high levels (7.3 a.u.) and the viability above 80 %. We hypothesise that this extratime may allow the culture to recover after the pGFP internalisation and then, the number of viable cells increases compared to 8 h. However, the commercial transfectant was not adequate as reference under these experimental conditions, since its cytotoxic activity provokes an almost complete elimination of cellular viability. The fluorescence levels for DHP(G4)-MPA after 4 h of incubation (5.2 a.u) were comparable to those given by Lipofectamine (6.0 a.u), and the 22 % of viability for the commercial reagent still allows comparisons between both gene delivery vectors at these conditions.
On the other hand, the amount of pGFP per well was varied between 0.2 and 0.4 µg following previous studies 14 . In Figure S6b, data for dendriplexes with DHP(G4)-MPA and pGFP at different N/P ratios (25, 50, 100, 250 and 500) with the different amounts of pGFP are shown. Despite the highest transfection efficiencies were reached with 0.4 µg of pGFP, more specifically at the ratio N/P 250, the low viability exhibited for these complexes (around 50 %) discard these conditions for the later transfection assays. The amount of 0.2 µg of pGFP allowed relevant fluorescence levels when increasing the N/P ratio to 500 while the cell viability was maintained above 86 %. . Transfection experiments with siGFP in a) HeLa-GFP and b) mMSCs-GFP cell lines. All the DHPs of the bis-MPA and bis-GMPA series were assayed as vectors at N/P ratios 150, 300, 500 and 750. Controls without treatment, naked siGFP, with Superfect and with Lipofectamine 3000 are also included for each cell lines. Black bars show transfection efficiency and red points indicate cell viability. Error bars indicate the standard deviation (SD) (n= 3). Statistical analysis was performed by one-way ANOVA; ns, p > 0.05; ***, p < 0.001; ****, p < 0.0001.