3.3.3. *In Silico* Binding Affinity of ssiRNAs with G5 TEA-Core Dendrimer Nanovectors

Before embarking in further time- and resource-consuming experimental investigations, we performed MD simulations to predict and understand if and how the different ssiRNA overhangs could impact G5 TEA-core mediated delivery. The *in silico* investigation started by verifying our own hypothesis, according to which the protruding, flexible overhangs could promote a better interaction and stronger binding of monomeric ssiRNAs to their dendrimeric nanocarriers via molecular dynamics simulations. The MD results are summarized in Figure 11a (see Table A1 in Appendix A for full MD results), while some exemplificative images extracted from the corresponding equilibrated MD trajectories are shown in Figure 12.

**Figure 11.** Total effective free energy (ΔGbind,eff = ΔHbind,eff–TΔSbind,eff), enthalpic (ΔHbind,eff), and entropic (–TΔSbind,eff) components for the binding of (**a**) ssiRNAs featuring complementary and non-complementary overhangs of different length and (**b**) dimeric ssiRNAs with the G5 TEA-core PAMAM dendrimer. Neff is the number of effective dendrimer positive charges involved in nucleic acid binding (see Tables A1 and A2 in Appendix A and text for more details). (**b**) Redrawn from [57], with permission of the American Chemical Society.

The computer simulations reveal that both the nature of the overhangs and their length influence the interaction of the relevant ssiRNAs with the G5 dendrimer nanocarrier. For the first aspect, the first three columns in Table A1 show that ssiRNAs with An/An overhangs are characterized by the most favorable free energy of binding values (ΔGbind = −409.9 kcal/mol for A5/A5 and −447.9 kcal/mol for A7/A7), followed by the ssiRNAs bearing complementary overhangs (ΔGbind = -387.4 kcal/mol for A5/T5 and -422.9 kcal/mol for A7/T7), and, last, by the ssiRNAs with Tn/Tn overhangs, which are characterized by the lowest nanovector affinity (ΔGbind = −316.8 kcal/mol for T2/T2, −344.8 kcal/mol for T5/T5, and −402.2 kcal/mol for T7/T7, respectively). Concerning the second aspect, these data clearly indicate that the presence of longer overhangs enhances nanovector/nucleic acid binding, the ssiRNA with the shorted overhangs T2/T2 being the one with the lowest ΔGbind value in the entire series.

**Figure 12.** Examples of equilibrated molecular dynamics (MD) snapshots of G5 TEA-core dendrimer in complex with A5/A5 (**a**), T5/T5 (**b**), A5/T5 (**c**), A7/A7 (**d**), T7/T7 (**e**), and A7/T7 (**f**) ssiRNAs at pH 7.4 and in the presence of 0.15 M NaCl. In all panels, the dendrimer is shown as cornflower blue sticks, and the terminal charged amine groups are highlighted as sticks-and-balls. The ssiRNA is portrayed as an orange ribbon, with the two overhangs (An) and (Tn) colored in red and navy blue, respectively. Some Cl<sup>−</sup> and Na<sup>+</sup> ions and counterions are shown as light green and light pink spheres, respectively. Water molecules are not shown for clarity. Redrawn from [57], with permission of the American Chemical Society.

Further interesting data were obtained by calculating the effective free energy of binding (ΔGbind,eff), i.e., the specific energetic contribution to ssiRNA/nanocarrier complex formation afforded only by those dendrimer branches in constant and productive interaction with its nucleic acid cargo. An analysis of each ssiRNA/dendrimer nanoassembly MD simulation allowed us to precisely identify and quantify these dendrimer residues (Neff); next, a per residue decomposition of the total binding free energy (see Supporting Information for details) led to the corresponding values of ΔGbind,eff (Figure 11a and Table A1). The first notable result regards Neff: Indeed, not only the smallest number of dendrimer branches involved in nanovector binding (38) pertains to the nucleic acid fragment with the shortest

overhangs (T2/T2), but also the values of Neff follow a clear increasing trend from Tn/Tn to An/An to An/Tn ssiRNAs. Concomitantly, the corresponding ΔGbind,eff values become more favorable (i.e., more negative) in the same order.

The deconvolution of ΔGbind,eff in its enthalpic (ΔHbind,eff) and entropic (−TΔSbind,eff) components (Figure 11a and Table A1) reveals that the nanovector/siRNA interaction is prevalently enthalpic in nature, although entropic effects linked to the released of ions, counterions, and water molecules in the bulk solvent upon complex formation also contribute in modulating the individual intermolecular affinities. Thus, taking the ssiRNAs series bearing five-nucleotide long overhangs as an example, it is easily seen that the A5/A5 ssiRNA has both the most favorable enthalpic contribution (ΔHbind,eff = −592.0 kcal/mol) and the least unfavorable entropic term (−TΔSbind,eff = 227.2 kcal/mol) in the homologous series. The best results for the A5/A5 ssiRNA/G5 TEA core PAMAM complex can be rationalized, from the enthalpic viewpoint, by taking into account the high number of favorable electrostatic interactions, supported by the greatest value of Neff for this homologous series (46), along with other non-bonded, stabilizing contacts between the nanovector and the nucleic acid, including its overhangs (see Figure 12a). From the entropic perspective, the more rigid nature and the enhanced clamping propensity of the A-based overhangs with respect to the T-based ones translate into more permanent and effective contacts between the full nucleic acid fragments (overhangs included) and the positively charged dendrimer terminal groups (Figure 12a). The least performing nanoassembly in this series, i.e., the one involving the T5/T5 ssiRNA, is characterized by the smaller value of Neff (41), the lowest enthalpic variation (ΔHbind,eff = −503.1 kcal/mol) and the most unfavorable entropic component (−TΔSbind,eff = 241.1 kcal/mol). This latter term is quickly understood, considering the remarkable flexibility of the Tn protruding overhangs, which fluctuate in the solvent for most of the time of the corresponding MD trajectory (Figure 12b). When interacting with the nanovector terminal groups, these overhang molecular movements are frozen, resulting in a considerable loss of degrees of freedom and, hence, entropic penalty. As a further effect, the corresponding ssiRNA/nanocarrier opposite-charge contacts are suboptimal, and this directly reflects in a decrease of ΔHbind,eff. The ssiRNA with complementary overhangs A5/T5 exhibits an intermediate behavior (−ΔHbind,eff = −554.9 kcal/mol and −TΔSbind,eff = 233.4 kcal/mol), stemming from a compensatory effect between the rigid An arm (with high binding tendency) and the springy Tn arm (endowed with less efficient dendrimer clasping propensity), as illustrated in Figure 12c. An utterly analogous situation—governed by the same molecular factors described above—is observed for the homologous ssiRNA series bearing longer overhangs, for which the affinity towards the G5 TEA-core dendrimer increases in the order A7/A7 > A7/T7 > T7/T7 (Figures 11 and 12d–f, Table A1). These results allowed us to draw some general considerations about the effect of nature and lengths of the ssiRNA overhangs on their interaction with the G5 TEA-core dendrimer, as follows. First, longer overhangs are more beneficial to nanovector/ssiRNA interactions than shorter ones by virtue of the higher number of dendrimer residues (Neff) in permanent and efficient contact with the nucleic acid fragment. In addition, for a given length of non-complementary overhangs, the more flexible nature of the Tn sequence is detrimental to nanoassembly formation with respect to the alternative An strand, since the relevant less-optimized nanoparticle structure and the larger entropic penalty paid upon dendrimer/ssiRNA complex formation result in a lower affinity of the nucleic acid fragment for its nanovector.

The next part of the *in silico* investigation was devoted to verify the second hypothesis, according to which ssiRNAs could self-assemble into gene-like structures via the formation of hybrid bridges between the complementary overhang sequences and, in doing so, enhance their affinity for nanovectors. In his original work [54], Behr already showed that this oligomerization or concatenation process enhanced cooperative and multivalent PEI/A8/T8 ssiRNA interactions, thereby leading to better delivery efficiency. Most importantly, however, since no concatemers were detected in the absence of nanovectors (i.e., PEI or G5 TEA-core PAMAM dendrimers), we further reasoned that the nanocarriers themselves must play an active role in directing encounters between individual ssiRNA/nanovector complexes, thus promoting complementary overhang concatemerization. To assess these concepts,

we performed further MD simulations on G5 TEA-core PAMAMs in complex with two dimeric ssiRNAs, (A5/T5)2 and (A7/T7)2 (see Figure 13a,b). The computational results are graphically reported in Figure 11b (and numerically listed in Table A2). When comparing these data with those relative to the monomeric ssiRNAs (i.e., A5/T5 and A7/T7, Figure 11a and Table A1), some important information can be immediately appreciated. First, the number of nanovector-charged branches in productive contact with the nucleic acid are larger than twice the sum of the value predicted for the analogous monomeric nanoassemblies (i.e., Neff = 96 for (A5/T5)2 and (2 × 44) = 88 for A5/T5, and Neff = 107 for (A7/T7)2 and (2 × 47) = 94 for A7/T7, respectively, Table A3). This enhancement of stabilizing intermolecular contacts for the concatenated systems can be ascribed to the presence of the extra double-stranded portion of the hybridized ssiRNAs (Figure 13a,b), which, being more rigid and globally more negatively charged than the single stranded overhangs, induces a further conformational adaptation of the dendrimer terminal units to accommodate a larger number of favorable electrostatic interactions.

**Figure 13.** Equilibrated MD snapshots of the (A5/T5)2 (**a**) and (A7/T7)2 (**b**) dimeric ssiRNAs in complex with the G5 TEA-core dendrimer pH 7.4 and in the presence of 0.15 M NaCl. Molecule representations and color scheme as in Figure 12. The double-stranded portion of the concatenated (hybridized) ssiRNAs is highlighted in purple. (**c**) and (**d**) Experimental binding of ssiRNAs bearing complementary and non-complementary overhangs with the G5 TEA-core dendrimer by ethidium bromide (EB) displacement assay. Color legend: (**c**) Light blue, T5/T5 ssiRNA; orange, A5/A5 ssiRNA; light purple, A5/T5 ssiRNA; dark blue; (**d**) T7/T7 ssiRNA; red, A7/A7 ssiRNA; light green, A7/T7 ssiRNA. Adapted from [57], with permission of the American Chemical Society.

The synergistic effect of ssiRNA concatemerization is also evident in the corresponding binding thermodynamics. Indeed, both ΔGbind,eff and ΔHbind,eff for the hybridized ssiRNAs are more favorable than two times the corresponding values for the monomeric ssiRNAs, while the decrease in the entropic contributions (–TΔSbind,eff) for the dimeric ssiRNAs is less disfavoring for the former systems with respect to twice the values for the latter ones, as summarized in Table A3.

All these data indeed provide a computational support to the idea that dimeric ssiRNAs generated by nucleic acid fragments bearing complementary overhangs which might hybridize into a central (An/Tn)2 double-stranded portion result in a synergistic binding with the G5 TEA-core dendrimer nanovector with respect to siRNAs characterized by both short and/or non-complementary overhangs. Pleasingly, these theoretical predictions were confirmed by ethidium bromide (EB) displacement fluorescence spectroscopy assays (Figure 13c,d), according to which the experimental binding affinity of the different ssiRNAs for the dendrimer follows exactly the same order anticipated by simulations, that is: An/Tn > An/An > Tn/Tn.

The final step of the *in silico* study concerned another fundamental aspect in nanovector-assisted effective siRNA delivery and gene silencing—the disassembly of the nanocomplexes in the cellular cytoplasm to make the nucleic acid cargo available to the RNAi machinery. To the purpose, advanced computational techniques based on steered molecular dynamics (SMD) simulations were applied to the four complexes formed by ssiRNAs bearing non-complementary overhangs, as well as by the standard (T2/T2) siRNA and the G5 TEA-core dendrimer (see Supplementary Materials)). Briefly, during SMD runs, each siRNA molecule was drifted away from its nanocarrier using a constant pulling speed, and the behavior of the force require to break the corresponding complexes was recorded as a function of time. The results from SMD simulations are shown in Figure 14a, from which it is seen that the peak force that needs to be exerted to dissociate the nucleic acids fragments from their nanocarrier increases in the order: 730 pN for T2/T2, 753 pN for T5/T5, 794 pN for A5/A5, 824 pN for T7/T7, and 862 pN for A7/A7. If the nanovector/ssiRNA disassembly force is plotted against the corresponding effective formation free energy (ΔGbinf,eff) value (Figure 11a and Table A1), a linear relationship is obtained (Figure 14b, R2 = 0.95), indicating that the tighter the ssiRNA/dendrimer binding, the stronger the force required to disassemble the corresponding complex. In other words, a very high affinity between nanocarrier and cargo, although useful for protection and transport, will ultimately be detrimental to the final step, i.e., efficient release; accordingly, the ideal system must represent the best compromise among these counteracting effects.

**Figure 14.** (**a**) Profiles of the average force required to unbind ssiRNAs from their G5 TEA-core dendrimer nanovectors as obtained from steered molecular dynamics (SMD) simulations. Color legend: Dark blue, (T7/T7) ssiRNA; light blue, (T5/T5) ssiRNA; gray, (T2/T2) (i.e., non-sticky) siRNA; red, (A7/A7) ssiRNA; orange, (A5/A5) ssiRNA. (**b**) Relationship between the SMD peak force and the corresponding effective free energy of binding ΔGbind,eff for the corresponding ssiRNA and the G5 dendrimers. Redrawn from [57], with permission of the American Chemical Society.

#### 3.3.4. In Vitro Delivery of ssiRNAs with G5 TEA-Core Dendrimer Nanovectors

The uptake of ssiRNA/G5 TEA-core PAMAM dendriplexes by PC-3 cells was first verified using live-cell confocal microscopy that confirmed both efficient internalization and cytoplasmic localization of the nucleic acid-loaded nanocarriers. Since the mechanism presiding cellular uptake of nanoparticles can involve several pathways, including macropinocytosis, clathrin-mediated endocytosis, and caveolae-mediated endocytosis [58], the mechanism of uptake was investigated using specific inhibitors and biomarkers of various endocytic pathways. As an example, Figure 15a shows that a meaningful reduction of G5 TEA core dendrimer/A5/T5 ssiRNA complexes was achieved only in the presence of the macropinocytosis inhibitor cytochalasin D, while only very weak effect was obtained in the presence of the two alternative inhibitors, that is genistein (an inhibitor of caveolae-mediated endocytosis) and chlorpromazine, a clathrin-mediated uptake specific blocker. In addition, a robust colocalization of the Alexa 647-labelled nanoparticles with dextran (a prototypic macropinocytosis biomarker) was observed, while minor-to-moderate colocalization was evidenced using either the transferrin of cholera toxin B (biomarkers for clathrin- and caveolae-mediated endocytosis) (Figure 15b).

**Figure 15.** (**a**) Effect of cytochalasin D (a macropinocytosis inhibitor, red bars), genistein (a caveolae-mediated endocytosis inhibitor, light blue bars), and chlorpromazine (a clathrin-mediated endocytosis inhibitor, gray bars) on the uptake of Alexa 647-labelled A5/T5 ssiRNA/G5 TEA-core dendrimer nanoparticles by PC-3 cells. Values are normalized to Alexa 647-labeled ssiRNA/G5 TEA-core dendrimer nanoparticles uptake in the absence of any inhibitor. (**b**) Colocalization of the Alexa 647-labelled A5/T5 ssiRNA/G5 TEA-core dendrimer nanoparticles with different endocytosis biomarkers: Top panel, dextran (macropinocytosis biomarker); middle panel, cholera toxin B (caveolae-mediated endocytosis biomarker); bottom panel, transferrin (clathrin-mediated endocytosis biomarker).

Based on all results discussed above, the ssiRNAs An/Tn, An/An, and Tn/Tn (*n* = 5 or 7) were selected for further in vitro experiments. Taking again the Hsp27 as the target gene in different cancer cell lines, the efficiency and specificity of the gene silencing effect were evaluated both at the mRNA and protein levels [57]. Figure 16 illustrates some of the results obtained in these tests.

As seen in this figure, the G5 TEA-core dendrimer/non-sticky siRNA (A2/T2) assembly confirmed its inability to elicit gene silencing in all cell lines, while, in agreement with computational predictions, a substantial effect (approximately 90%) was achieved with ssiRNAs bearing complementary overhangs (i.e., An/Tn). Moreover, again in line with *in silico* results, ssiRNAs ending with non-complementary sequences can induce up to 70% gene silencing, as also seen in Figure 16. Specifically, for these systems, as revealed by simulations, if, on the one side, a higher overhang rigidity leads to stronger nanovector/ssiRNA interaction, on the other side, longer and more flexible overhangs are more beneficial for the subsequent nucleic acid delivery. The results from all *in silico* investigations described above unambiguously supported the conclusion that, among all ssiRNAs with non-complementary overhangs we synthesized to empower low-generation TEA-core PAMAMs with effective delivery capacity, those bearing A7/T7 terminals represent the best compromise in terms of both nanovector binding and unbinding ability.

Before moving the A7/T7 ssiRNA/G5 TEA-core dendrimer complexes to in vivo tests, we further investigated their anticancer effects resulting from Hsp27 silencing in PC-3 cells. Giving the previous results obtained with the delivery of siRNA by the TEA-core dendrimer of Generation 6 and 7 discussed above, we expected this smaller PAMAM to be devoid of toxicity and its complexes with the Hsp27-targeting ssiRNAs to significantly suppress cell growth via a caspase-induced apoptosis. Indeed, as shown in Figure 17a, a notable inhibition of cell proliferation was detected after delivering

A7/T7 ssiRNA with the G5 TEA-core nanovector with respect to non-treated cells, cells treated with the G5 dendrimer alone, or with a scrambled (non-silencing) ssiRNA.

**Figure 16.** Hsp27 gene silencing upon delivery of different ssiRNAs (50 nM) mediated by G5 TEA-core PAMAM (N/P = 10) to PC-3 cells (**a**), MDA-MB-231 cells (**b**), and MCF-7 cells (**c**). Checked bars: Data for ssiRNA with n = 5; solid bars: Data for ssiRNA with n = 7. In these experiments, vinculin was used as reference, and non-treated cells were used for control (green solid bar). Data for non-sticky siRNA (A2/T2) are also shown for comparison (red solid bar). MDA-MB-231 and MCF-7 are two different breast cancer cell lines. Redrawn from [57], with permission of the American Chemical Society.

**Figure 17.** (**a**) Cell proliferation, (**b**) apoptosis, and (**c**) caspase 3/7 activity in PC-3 cells treated with A7/T7 ssiRNAs (50 nM) delivered by G5 TEA-core PAMAM (N/P = 10). Non-treated cells, the G5 dendrimer alone and a scrambled (non-silencing) ssiRNA sequence were used for control. Data in panels (**a**) and (**c**) were measured as described in Figure 6. The apoptotic index was measured with fluorescence-activated cell sorting (FACS) flow cytometry by the annexin V assay four days after treatment. Redrawn from [56], with permission of the American Chemical Society.

Concomitantly, fluorescence-activated cell sorting (FACS) flow cytometry revealed a considerable increase of annex V-positive apoptotic cells paralleled with the relevant activation of the apoptotic Caspases 3 and 7. Finally, no toxicity mediated by the ssiRNA/dendrimer complexes was observed via further MTT and lactate dehydrogenase assays, supporting the potential for in vivo experiments with the A7/T7 ssiRNA/G5 TEA-core dendrimer nanoparticles.

3.3.5. In Vivo Delivery of ssiRNAs with G5 TEA-Core Dendrimer Nanovectors

The final part of the study concerned the evaluation of in vivo gene silencing by the in vitro most efficient ssiRNA/nanovector system. Accordingly, a prostate cancer PC-3 xenografted mouse model was adopted, to which the A7/T7/G5 TEA core dendrimer nanoparticles were slowly administered via slow intratumoral injection. Treatment lasted one week, during which the mice survived well, showing no sign of induced toxicity or weight loss. After mice sacrifice, the expression of Hsp27 in the tumors was measured, as shown in Figure 18a,b. A significant downregulation of Hsp27 at both the mRNA and protein levels was observed, compared to all controls, confirming that the ssiRNA delivered by the dendrimer nanovector was able to elicit potent and specific RNAi also in vivo.

**Figure 18.** In vivo downregulation of Hsp27 at both mRNA (**a**) and protein (**b**) levels achieved after treating PC-3 cell xenografted nude mice with intratumoral injection of Hsp27 A7/T7 ssiRNA/G5 complex, buffer solution (control), the dendrimer G5 alone, the A7/T7 ssiRNA alone and a scrambled (non-silencing) ssiRNA sequence/G5 complex (all used as negative controls). (**c**) Evaluation of tumor cell proliferation via immunohistochemistry using Ki-67 staining after treatment with a scrambled ssiRNA sequence/G5 (left) and the Hsp27 A7/T7 ssiRNA/G5 complexes (right). Adapted from [56], with permission of the American Chemical Society.

Apoptotic caspase activation was also detected only in mice treated with the nanodelivered ssiRNA, and immunohistochemistry images obtained with Ki-67 antibody staining finally confirmed the remarkable inhibition of cell proliferation in the treated animals (Figure 18c,d).

#### **4. Conclusions**

In the last ten years, the number of studies involving dendrimers as safe, efficient and effective nanovectors for drug and nucleic acid delivery have increased exponentially. This is mainly due to the exquisite properties of these hyperbranched molecules which, by virtue of their nanoscale size, regularly repeating structure and functional surface groups, make them an ideal drug delivery platform. PAMAM dendrimers in particular bear primary amine groups on their periphery which, being positively charged at physiological pH (7.4), can aptly condense negatively charged nucleic acids for efficient gene or siRNA delivery. In addition, this class of dendrimers features tertiary amines in their interior which become protonated at endosomal pH (5.5), thereby promoting the so-called proton sponge effect and the subsequent release of their DNA/siRNA cargo in the cell cytoplasm.

During the same decade, our group has been particularly active in the field of design and optimization of PAMAM-based dendrimers for siRNA delivery. In particular, we designed, synthesized, and tested highly flexible triethanolamine-core PAMAM dendrimers which proved to be highly effective for siRNA delivery in cancer therapeutics both in vitro and in vivo, as discussed in this brief review. Based on its successful performance, the G5 TEA-core PAMAM dendrimer was scheduled to enter clinical trials for siRNA-based cancer therapy in 2014; unfortunately, however, due to the unavailability of GMP dendrimer material, the foreseen clinical trial was delayed and ultimately replaced by the use of Smarticles® for the delivery of siRNA therapeutics [59].

Since the GPM production of dendrimer is quite a challenging process, we decided to exploit the quintessence of nanotechnology, i.e., the controlled self-assembly of small, synthetically amenable building blocks to generate nanosystems for siRNA delivery. Accordingly, we designed, synthesized, and tested amphiphilic dendrons which, upon auto-organization into micelles, were able to mimic the covalent, high generation dendrimers in size, structure and function—in particular for siRNA delivery. These exciting self-assembled nanovectors will be the subject of the second part of this review work.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/1999-4923/11/7/351/s1, Table S1, Computational details.

**Funding:** This research was funded by the Italian Association for Cancer Research (AIRC), grant IG17413 to SP. The assistant professor position (RTDa) of SA is fully supported by the University of Trieste, in agreement with the actuation of the strategic planning financed by the Italian Ministry for University and Research (MIUR, triennial program 2016–2018) and the Regione Friuli Venezia Giulia (REFVG, strategic planning 2016-18), assigned to SP. This award is deeply acknowledged.

**Acknowledgments:** Authors wish to thank Ling Peng and her group for the longstanding, fruitful collaboration, the challenges in siRNA delivery nanovector design and optimization, the inspiring discussions and, above all, the personal friendship.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **Appendix A**

**Table A1.** Total and effective enthalpy (ΔHbind, ΔHbind,eff), entropy (-TΔSbind, -TΔSbind,eff), and free energy of binding (ΔGbind = ΔHbind−TΔSbind, ΔGbind,eff = ΔHbind,eff−TΔSbind,eff) for ssiRNAs featuring complementary and non-complementary overhangs of different length with G5 TEA-core PAMAM dendrimers. All values are in kcal/mol (standard deviation for all data in Table A1 is less that 3%). Neff is the number of positive dendrimer charges effectively involved in ssiRNA binding. Adapted from [57] with permission of the American Society of Chemistry.


**Table A2.** Total and effective enthalpy (ΔHbind, ΔHbind,eff), entropy (−TΔSbind, −TΔSbind,eff), and free energy of binding (ΔGbind = ΔHbind−TΔSbind, ΔGbind,eff = ΔHbind,eff−TΔSbind,eff) for the two dimeric ssiRNAs with G5 TEA-core PAMAM dendrimers. All values are in kcal/mol (standard deviation for all data in Table A2 is less that 3%). Neff is the number of positive dendrimer charges effectively involve in ssiRNA binding. Adapted from [57] with permission of the American Society of Chemistry.


**Table A3.** Synergistic effect of the dimeric ssiRNA concatemerization on their G5 TEA-core PAMAM dendrimer effective binding thermodynamics with respect to the corresponding monomeric ssiRNAs. Data from Tables A1 and A2. All values are in kcal/mol.


#### **References**


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*Review*
