Supplementary Figures

Supplementary Figure 1. Screening of the EGFP gene transfection efficacy of

fluorinated dendrimers in HEK293 cells. HEK293 cells were seeded in the 24-well

plates for 24 h before in vitro gene transfection. The gene transfection experiments for

the fluorinated dendrimers are conducted at their optimized N/P ratios. The positive

EGFP cells and mean fluorescence intensity were analyzed by flow cytometry after

the cells were incubated with the polyplexes for 24 h (Figure 1b). The N/P ratios for

G5-F326, G5-F344, G5-F536, G5-F548, G5-F749, G5-F768, G5-F932, and G5-F954 are

14:1, 14:1, 8:1, 11:1, 7:1, 2:1, 8:1, 8:1, respectively. Error bars represent the standard

error (n=3). Diamonds represent mean fluorescence intensity.

Supplementary Figure 2. Agarose gel electrophoresis assay of G5, G5-F749, and

G5-F768 polyplexes with EGFP plasmid at different N/P ratios. Lane 1 represents

DNA marker. Lane 2 represents naked plasmid DNA (pDNA). Lanes 3-7 represent

G5/DNA polyplexes at N/P ratios of 0.5:1, 0.75:1, 1:1, 2:1, and 4:1, respectively.

Lanes 8-12 represent G5-F749/DNA polyplexes at N/P ratios of 0.5:1, 0.75:1, 1:1, 2:1,

and 4:1, respectively. Lanes 13-17 represent G5-F768/DNA polyplexes at N/P ratios of

0.5:1, 0.75:1, 1:1, 2:1, and 4:1, respectively.

Supplementary Figure 3. Hydrodynamic sizes of G5-F749/DNA and G5-F768/DNA

polyplexes at different N/P ratios determined by dynamic light scattering.

G5-F749/DNA and G5-F768/DNA polyplexes were formed at a final DNA

concentration of 1.6 µg/mL. The N/P ratio ranges from 0:1 to 8:1. All the samples

were examined at 25 oC. Error bars represent the standard error (n=3).

Supplementary Figure 4. Zeta potentials of G5-F749/DNA and G5-F768/DNA

polyplexes at different N/P ratios. G5-F749/DNA and G5-F768/DNA polyplexes were

formed at a final DNA concentration of 1.6 µg/mL. The N/P ratio ranges from 0:1 to

8:1. All the samples were examined at 25 oC. Error bars represent the standard error

(n=3).

Supplementary Figure 5. EGFP expression of G5-F768/DNA polyplexes in HeLa

cells for 48 h. DNA amounts in the polyplexes range from 0.1 to 3.0 μg. The N/P ratio

for the G5-F768/DNA polyplexes is fixed at 2.5:1. Scale bar: 200 µm. Diamonds

represent mean fluorescence intensity, n=1.

Supplementary Figure 6. Transfection efficacy of fluorinated dendrimers in COS-7

cells after 48 h. EGFP gene transfection efficacies of G5-F749 and G5-F768 were

compared with those of G5 and Lipofectamine 2000. Error bars represent the standard

error (n=3). Squares represent mean fluorescence intensity Scale bar: 200 µm.

Supplementary Figure 7. Transfection efficacy of fluorinated dendrimers in CHO

cells after 48 h. EGFP gene transfection efficacies of G5-F749 and G5-F768 were

compared with those of G5 and Lipofectamine 2000. Error bars represent the standard

error (n=3). Diamonds represent mean fluorescence intensity Scale bar: 200 µm.

Supplementary Figure 8. Transfection efficacy of fluorinated dendrimers in NIH3T3

cells after 48 h. EGFP gene transfection efficacies of G5-F749 and G5-F768 were

compared with those of G5 and Lipofectamine 2000. Error bars represent the standard

error (n=3). Diamonds represent mean fluorescence intensity Scale bar: 400 µm.

Supplementary Figure 9. Transfection efficacy and characterization of

G5-F768/DNA polyplexes at extremely low N/P ratios. The gene transfection

experiments were conducted on HEK293 cells and HeLa cells at extremely low N/P

ratios of 0.5:1, 0.75:1, and 1:1, respectively. Error bars represent the standard error

(n=3). Diamonds represent mean fluorescence intensity Scale bar: 200 µm.

Supplementary Figure 10. Cytotoxicities of G5-F749 and G5-F768 on the cells.

HEK293 cells (a) and HeLa cells (b) were incubated with fluorinated dendrimers for

24 h and 48 h, respectively.. G5 and Lipofectamine 2000 were used as controls. The

concentrations of the polymers were equal to those in the gene transfection

experiments at different N/P ratios. The concentration of Lipofectamine 2000 was

chosen according to the optimized condition in the EGFP gene transfection

experiments (0.8 μg DNA/2 μL Lipofectamine 2000). Error bars represent the

standard error (n=5).

Supplementary Figure 11. Stability of the fluorinated dendrimer G5-F768 during

storage. EGFP gene transfection efficacy of G5-F768 in HeLa cells after the samples

were stored at -20 oC for three (a), five (b), and eight (c) months, respectively. The

transfection efficacy of a sample further stored at 4 oC for one month is shown in (d).

Error bars represent the standard error (n=3). Scale bar: 400 µm. G5-F768 maintained

efficient transfection efficacy on HeLa cells during storage.

Supplementary Figure 12. Synthesis and characterization of butyric acid modified

G5 PAMAM dendrimer. The synthesis route is the same with the one used in the

synthesis of fluorinated dendrimers (a). The product was characterized using 1H NMR

spectrum (b) in D2O. According to the 1H NMR spectrum, an average number of 69

butyric acid groups were conjugated to each G5 dendrimer. The product was termed

as G5-H769.

Supplementary Figure 13. Comparison of G5-H769, G5 PAMAM, and G5-F768 on

EGFP gene expression on HEK293 and HeLa cells. The N/P ratios for G5-H769/DNA

polyplexes are chosen at 2:1, 4:1, 6:1, and 8:1, respectively. The N/P ratios for

G5-F768/DNA and G5/DNA polyplexes are 2:1 and 8:1, respectively. Scale bar: 400

µm.

Supplementary Figure 14. EGFP expressions of G5/DNA polyplexes in HeLa cells

in the absence and presence of different amounts of perfluorobutyric acid. The N/P

ratio for G5/DNA polyplex in the absence of perfluorobutyric acid is chosen at its

optimized condition (8:1). The compositions of G5/perfluorobutyric acid/DNA

(G5+F768) polyplexes are equal to those of G5-F768/DNA polyplexes at N/P ratios of

2:1, 2.5:1, and 3:1, respectively. The addition of perfluorobutyric acid does not

enhance the gene transfection efficacy of G5 dendrimer on HeLa cells. Scale bar: 400

µm.

Supplementary Figure 15. Endosomal escape abilities of G5-F768/DNA and G5

PAMAM/DNA polyplexes. HeLa cells were pretreated with bafilomycin A1 or

sucrose, followed by the incubation of G5-F768/DNA and G5 PAMAM/DNA

polyplexes for 48 h. The EGFP expressions were determined by flow cytometry and

the data were expressed as means±S.D. of triple parallel samples. **p < 0.01 by

students’ t-test. Diamonds represent EGFP positive cells.

Supplementary Figure 16. Confocal images of HeLa cells treated with

G5-F749/DNA polyplexes for 2 and 4 h, respectively. DNA was labeled with YOYO-1,

the endosomes were stained with LysoTracker® Red, and the nuclei were stained with

DAPI. The arrows indicate co-localization of YOYO-1 labeled DNA and

LysoTracker® Red stained endosomes in HeLa cells. Scale bar: 20 µm.

Supplementary Figure 17. DNA release from the polyplexes in the presence of

different concentrations of heparin. G5/DNA polyplexes were used as controls.

G5/DNA polyplexes were prepared at an N/P ratio of 8:1, which is proved as the

optimized condition in the gene transfection experiments. G5-F768/DNA polyplexes

were prepared at N/P ratio of 2:1 (optimized N/P ratio) or 8:1 (N/P ratio equal to that

of G5/DNA polyplex). Heparin molecules (0-176 μM) were added to the polyplex

solutions as competitors of DNA. It is clear that G5-F768 is easier to release its bound

DNA in the presence of heparin than G5 dendrimer no matter the N/P ratio of the

G5-F768/DNA polyplex is 2:1 or 8:1.

Supplementary Figure 18. Stability of the fluorinated dendrimer G5-F768 stored for

eight months revealed by 19

F NMR. (a) free heptafluorobutyric acid (F7), (b) G5-F768

(G5-F7) (c) fluorinated G5 added with free heptafluorobutyric acid (G5-F7+free F7).

CF3CH2OH is added as an internal standard. Conjugation of F7 to G5 dendrimer leads

to a significant shift of peak (1) in F7 to peak (1’) in G5-F768. The extremely weak

intensity of peak (1) in the spectrum of G5-F768 (b) suggests excellent stability of

G5-F768.

0

20

40

60

80

100

G5+F768

Cell v

iab

ilit

y (

%)

N/P=2

N/P=2.5

N/P=3

G5-F768

Supplementary Figure 19. Cytotoxicities of G5-F768 and G5/heptafluorobutyric acid

mixture on HeLa cells for 48 h. The concentrations of G5-F768 were equal to those in

the gene transfection experiments at N/P ratios of 2:1, 2.5:1, and 3:1. For the

G5/heptafluorobutyric acid mixture, the amount of both G5 and heptafluorobutyric

acid are equal to those in G5-F768. Error bars represent the standard error (n=5).

Supplementary Figure 20. Gene transfection efficacies of fluorinated and

unmodified G4 PAMAM dendrimers. The in vitro EGFP transfection efficacies of

fluorinated G4 PAMAM dendrimer (G4-F736) and unmodified G4 PAMAM

dendrimer were measured on HeLa cells at their optimal N/P ratios (3:1 for G4-F736

and 8:1 for G4 PAMAM dendrimer, respectively) for 48 h. Positive EGFP cells (%)

were determined by flow cytometry. Error bars represent the standard error (n=3).

Scale bar: 200 µm.

Supplementary Figure 21. Gene transfection efficacies of fluorinated and

unmodified G5 polypropylenimine (PPI) dendrimers. Fluorinated G5 PPI dendrimer

(PPI-F716) was compared with unmodified G5 PPI dendrimer on in vitro EGFP

transfection efficacy on HeLa cells at their optimal N/P ratios (3:1 for PPI-F716 and

8:1 for G5 PPI dendrimer, respectively) for 48 h. Positive EGFP cells (%) were

determined by flow cytometry, n=1 Scale bar: 200 µm.

Supplementary Figure 22. Gene transfection efficacies of fluorinated and

unmodified bPEI25K. Fluorinated bPEI25K (bPEI-F773 and bPEI-F7109) were

compared with unmodified bPEI25K on in vitro EGFP gene transfection efficacy on

HEK293 cells (2:1, 4:1, and 8:1 for bPEI-F773, bPEI-F7109, and bPEI25K, respectively)

for 24 h. Positive EGFP cells (%) were determined by flow cytometry, n=1. Scale bar:

400 µm.

Supplementary Table

Supplementary Table 1. Characterization of G5 PAMAM and fluorinated G5

PAMAM dendrimers.

Polymer n n’ Mw

(Da)*

G5-F326 26.3241±0.9963 26 31322

G5-F344 44.1697±2.0704 44 33050

G5-F536 35.8809±0.9701 36 34082

G5-F548

G5-F749

G5-F768

G5-F932

G5-F954

48.0416±2.0115

48.8611±1.3225

67.9926±0.2499

31.8462±0.4627

54.1114±0.8249

48

49

68

32

54

35834

38430

42154

37850

44054

Note: n is the number of fluorinated groups conjugated to each G5 dendrimer

determined by the ninhydrin assay (average±S.D.). n’ is the approximate number of

the fluorinated groups conjugated to each G5 dendrimer according to the ninhydrin

assay, which is used to calculate the molecular weight (Mw) of the fluorinated

dendrimer. *molecular weights of the fluorinated G5 PAMAM dendrimers were

calculated according to the number of fluorinated groups conjugated and the

theoretical molecular weight of G5 PAMAM dendrimer (28826 Da).

Supplementary Note

Supplementary Note 1

In this study, “extremely low N/P ratio” was used to describe fluorinated dendrimer

such as G5-F768 mediated gene transfection. Usually, the optimal N/P ratio for

lipid-based gene transfection reagents is relatively low (for example, DOGS with the

highest luciferase transfection efficacy at N/P ratio of 2.5:140

; DOTAP with an

optimal N/P ratio around 4:141

). However, for cationic polymers, the optimal N/P

ratio is relatively high. For example, bPEI25K has an optimal N/P ratio around 8:149

.

For PAMAM dendrimers, the optimal N/P ratio of G5 PAMAM dendrimer is also

around 8:1. Functional dendrimers such as amino acid (phenylalanine and leucine)

modified dendrimers50

and cross-linked dendrimers51

have optimal N/P ratios above

20:1.

In comparison, the fluorinated dendrimer such as G5-F768 has an optimal N/P ratio

around 2:1, and the material also works well at extremely low N/P ratios of 0.5:1 and

0.75:1 (~50% EGFP positive cells on both HEK293 and HeLa cells in Supplementary

Figure S9). Based on the discussions, we used “extremely low N/P ratio” to describe

G5-F768 mediated gene transfection in the title of the manuscript.

Supplementary Method

The ninhydrin assay. To determine the number of fluorinated groups on each G5

dendrimer, we use a ninhydrin assay to test the remaining number of primary amine

groups on the fluorinated G5 dendrimers. The G5 PAMAM dendrimer obtained from

Dendritech in this study was characterized by 13

C NMR52

and polyacrylamide gel

electrophoresis (PAGE)53

before the assay. Generally, 85 mg ninhydrin and 15 mg

hydrindantin were dissolved in 10 mL ethylene glycol-monomethyl ether. 100 µL of

the solution was mixed with 100 µL sodium acetate buffer (0.2 M, pH = 5.4). The

mixtures were added with different amounts of G5 PAMAM dendrimer or fluorinated

dendrimers and the volume of the solutions was fixed to 300 µL using distilled water.

The mixtures were incubated in boiling water for 10 min. After cooling to room

temperature, the mixtures were added with 300 µL ethanol/water (v/v~60:40) solution.

The absorbances of the samples were measured at 570 nm using a UV-Vis

spectroscopy. The calibration curve was made according to the dendrimer

concentration and the absorbance. The numbers of fluorinated groups modified on

each G5 dendrimer were determined by the calibration curve (Abs = 3.1142C – 0.0912,

R2 = 0.99905, Abs is the absorbance of the sample at 570 nm, C is the concentration

of primary amine groups, mM). Three repeats were conducted for each sample. The

resulting fluorinated dendrimers were termed as G5-F326, G5-F344, G5-F536, G5-F548,

G5-F749, G5-F768, G5-F932, and G5-F954, respectively, according to the ninhydrin

assay.

Supplementary References

49 Choi, J. S. et al. Enhanced transfection efficiency of PAMAM dendrimer by surface

modification with L-arginine. J. Control. Release 99, 445-456, (2004).

50 Kono, K., Akiyama, H., Takahashi, T., Takagishi, T. & Harada, A. Transfection activity of

polyamidoamine dendrimers having hydrophobic amino acid residues in the periphery.

Bioconjugate Chem. 16, 208-214, (2005).

51 Liu, H., Wang, H., Yang, W. & Cheng, Y. Disulfide cross-linked low generation dendrimers

with high gene transfection efficacy, low cytotoxicity, and low cost. J. Am. Chem. Soc. 134,

17680-17687, (2012).

52 Tomalia, D. A., Naylor, A. M. & Goddard III, W. A. Starburst Dendrimers: Molecular-Leve

Control of Size, Shape, Surface Chemistry, Topology, and Flexibility from Atoms to

Macroscopic Matter. Angew. Chem. Int. Ed. 29, 138-175 (1990).

53 Lesniak, W. G. et al. Synthesis and characterization of PAMAM dendrimer-based

multifunctional nanodevices for targeting alphavbeta3 integrins. Bioconjugate Chem. 18,

1148-1154, (2007).