Peptide dendrimers as “lead compounds” for the treatment of chronic lung infections by Pseudomonas aeruginosa in cystic fibrosis patients: in vitro and in vivo studies

Figures & data

Figure 1 Peptide dendrimers dG3KL and dTNS18 tested in the present study.

Notes: (A) Chemical structure. (B) Physicochemical properties. C-termini are carboxamide CONH₂. ^aOne-letter codes for amino acids (l, leucine; k, lysine; o, ornithine; b, diaminobutyric acid; k, branching lysine and K [C₁₀], side-chain decanoylated lysine).
Abbreviation: MS, mass spectrometry (to assess molecular weight).

Table 1 In vitro antibacterial activity of dendrimer peptides compared to Tob against Pseudomonas aeruginosa strains from CF patients

Strain	dG3KL		dTNS18		Tob
	MIC	MBC	MIC	MBC	MIC	MBC
PaPhl3	128	128	16	16	1	2
PaPhl4	8	8	8	8	l	l
PaPh26	8	l6	8	8	8	l6
PaPh32	8	8	8	8	>128	>128

Notes: Values expressed as µg/mL.

Abbreviations: MIC, minimum inhibitory concentration; MBC, minimum bactericidal concentration; Tob, tobramycin; CF, cystic fibrosis.

Figure 2 Time–kill kinetics of dendrimers and Tob against Pseudomonas aeruginosa.

Notes: A standardized inoculum (1–2×10⁶ CFU/mL) of each P. aeruginosa strain (PaPh13, PaPh14, PaPh26, and PaPh32) was exposed to each peptide or Tob at MIC value. OD₅₅₀ was measured every 15 minutes over 24 hours. Each experiment was carried out in triplicate and repeated twice. In the case of PaPh32, Tob was not tested because of out-of-range MIC (>128 µg/mL).
Abbreviations: MIC, minimum inhibitory concentration; Tob, tobramycin.

Figure 3 Screening for biofilm formation by Pseudomonas aeruginosa.

Notes: Biofilm was allowed to form in 96-well microtiter in trypticase soy broth for 24 and 48 hours. Biofilm cellularity was evaluated by viable cell counts. Each experiment was carried out in triplicate and repeated three times, and results are shown as mean + SD. No statistically significant differences were observed between 24 hours and 48 hours by unpaired t-test. **P<0.01, ***P<0.001 vs other strains; ANOVA + Tukey’s multiple comparison posttest.

Figure 4 Effect of dendrimers and Tob on Pseudomonas aeruginosa-biofilm formation.

Notes: Biofilms were allowed to form (37°C, 24 hours) in each well of a 96-well microtiter plate in the presence of each peptide tested at several sub-MIC levels (1/2×, 1/4×, and 1/8×). Biofilm biomass was then measured by crystal violet assay. Effect against biofilm formation shown as variation in percentage of biofilm biomass measured after exposure compared to control (exposed to vehicle only). Each experiment was carried out in triplicate and repeated twice, and results are shown as mean + SD. Dotted lines show statistically significant variations (>±30% vs control; P<0.05, Fisher’s exact test) in biofilm formation.
Abbreviations: MIC, minimum inhibitory concentration; Tob, tobramycin.

Figure 5 Pseudomonas aeruginosa-biofilm dispersion caused by dendrimers and Tob.

Notes: Biofilm was allowed to form in a 96-well microtiter plate at 37°C for 24 hours, and then exposed for a further 24 hours to each peptide (dG3KL, dTNS18) or Tob at MIC and multiple MIC (2×, 4×, 8×, and 16×). Tob was not tested against PaPh32 strain because of out-of-range MIC (>128 µg/mL). Effect against preformed biofilm shown as variation in percentage of biofilm biomass measured after exposure compared to control (exposed to vehicle only). Each experiment was carried out in triplicate and repeated twice, and results are shown as mean + SD. Dotted lines show statistically significant variations compared to control (>±30% vs control; P<0.05, Fisher’s exact test).
Abbreviations: MIC, minimum inhibitory concentration; Tob, tobramycin.

Figure 6 In vitro activity of peptides and Tob against viability of preformed biofilm by Pseudomonas aeruginosa.

Notes: Biofilm was allowed to form in a 96-well microtiter plate at 37°C for 24 hours, and then exposed for a further 24 hours to each peptide (dG3KL, dTNS18), or Tob at 10 × MIC. Control biofilm was exposed to vehicle only (control). Results expressed as mean + SD (left) and proportion of dead and live cells after exposure (right). **P<0.01, ***P<0.001, ****P<0.0001 vs control; ^◦◦P<0.01, ^◦◦◦P<0.001 vs Tob; ANOVA + Tukey’s multiple comparison post-test.
Abbreviations: MIC, minimum inhibitory concentration; Tob, tobramycin.

Figure 7 In vivo toxicity assays.

Notes: Galleria mellonella larvae (n=20/group) were administered by intra-hemocoel injection with peptides (dG3KL and dTNS18) or Tob, each at doses equal to 10 × MIC (corresponding to 80, 160, and 80 µg/larva, respectively). Survival of larvae was monitored daily over 96 hours. Results shown as mean values from two independent determinations (n=20 each). No significant differences between curves found by Log-rank (Mantel–Cox) test.
Abbreviations: MIC, minimum inhibitory concentration; Tob, tobramycin.

Figure 8 In vivo Pseudomonas aeruginosa-killing assays.

Notes: Galleria mellonella larvae (n=20/group) were infected with different doses (10, 10², 10³, 10⁴, 10⁵, and 10⁶ cells) of PaPh13, PaPh14, PaPh26, and PaPh32 strains. Survival was then monitored daily over 96 hours. Results shown as mean + SD from two independent experiments (n=20 each). Control larvae administered PBS only. Dotted lines, LD₅₀.
Abbreviation: LD₅₀, lethal dose 50.

Figure 9 In vivo protection against systemic Pseudomonas aeruginosa (Pa) infection.

Notes: Galleria mellonella larvae (n=20/group) were infected by injection of the selected infectious dose of Pa. After 30 minutes, larvae were treated with peptides (dG3KL, dTNS18) or Tob at 10 × MIC. Control larvae were infected and then exposed to vehicle only (Pa + H₂O). Negative control consisted of uninfected and untreated larvae (PBS + H₂O). Survival was monitored daily over 96 hours. Results shown as percentage survival from two experiments (n=20 each). **P<0.01, ***P<0.001, ****P<0.0001 vs positive control; Log-rank (Mantel–Cox) test. Tob not tested against PaPh32 strain because of out-of-range MIC value (>128 µg/mL). Dotted lines, LD₅₀.
Abbreviations: MIC, minimum inhibitory concentration; Tob, tobramycin; LD₅₀, lethal dose 50.

Figure 10 Kinetics of Pseudomonas aeruginosa (Pa) growth in Galleria mellonella hemolymph.

Notes: G. mellonella (n=20/group) was infected by injection of an infectious dose of PaPh14 and PaPh26 Pa strains. After 30 minutes, larvae were treated with peptides (dG3KL, dTNS18) or Tob at 10 × MIC or vehicle only (H₂O; Ctrl). Following 4 and 12 hours of treatment, hemolymph was collected from each larva, pooled (as groups of five larvae), and underwent viable cell counts. Results shown as mean + SD from two experiments. *P<0.05; **P<0.01; ***P<0.001; unpaired t-test.
Abbreviations: MIC, minimum inhibitory concentration; Tob, tobramycin.