Fig 1.
Synthesis and characterization of dendrimer-cysteamine nano-formulation.
(A) The schematic shows the reaction for the synthesis of PAMAM generation 4 (G4) dendrimers with terminal end decorated with a cysteamine-analogue. (B) Illustration showing the chemical structure of cysteamine. (C) Native PAGE gel analysis showing the characterization of our nano-formulation, where ‘band B’ represents the dendrimer alone (or dendrimer with one sulfhydryl group) and the slower migrating ‘band A’ depicts dendrimers with a few–SH groups. The different lanes represent the following: Lane 1 –ladder (G2-G5), Lane 2 –G5; Lane 3 –Untreated G4; Lane 4 –Nanoconjugate obtained after synthesis; Lane 5 –the synthesis reaction performed for 24 hrs; Lane 6–7 –the conjugate stored for 2 months showing that there is no cross linking of the conjugates by difulfide formation. The data shows the successful synthesis of a novel PAMAM-DENCYS nano-formulation for evaluation of its efficacy in CF. (D) Transmission electron microscopy (TEM) images were captured to determine the dispersion and size of the empty (PAMAM-DEN) and cysteamine-conjugated dendrimers PAMAM-DENCYS (Scale bar: 20 nm). Data shows the clear dispersion of PAMAM-DEN/PAMAM-DENCYS, without any significant aggregation (red arrows showing an example of aggregation). (E) Next, QUARTZ PCI TEM analysis software was used to quantify the average (mean ± SEM) diameter of the dendrimers (DEN/DENCYS), which was determined to be ~4nm (NS = not significant).
Fig 2.
PAMAM-DENCYS restores ΔF508-CFTR induced autophagy-impairment in CF cells.
(A) The IB3-1 cells were co-transfected with RFP-(Ub) Ubiquitin and GFP-(LC3), the autophagy-protein light chain-3 plasmid constructs, and after 24 hrs of transfection, cells were treated with PAMAM-DENCYS (500μM) and control (PAMAM-DEN). The fluorescence microscopy images were used to evaluate the efficacy of the nano-formulation to restore the intrinsic impaired-autophagy in CF cells. Administration of PAMAM-DENCYS significantly diminishes the LC3-GFP and Ub-RFP co-localization as compared to control untreated cells. The circles are shown to indicate the location and morphology of the fluorescent cells (red circles) in the respective brightfield image (black circles) to clarify that they are from the same field of view. (B) The data (n = 3, **p<0.01), suggests that PAMAM-DENCYS could restore autophagy-impairment in CF cells. Scale bar: 100 μm.
Fig 3.
PAMAM-DENCYS induces trafficking of ΔF508-CFTR to plasma membrane in CF cells.
(A, left panel; B) Immunoblotting of total protein extracts from CFBE41o- cells treated with cysteamine (500μM) show significantly (p<0.05) higher protein levels of membrane CFTR (C-form) as compared with untreated control group. (A, right panel; C) Immunoblotting of total protein extracts from CFBE41o- cells treated with PAMAM-DEN or PAMAM-DENCYS shows a significant increase in ‘C form’ (p<0.05) in PAMAM-DENCYS treated cells. (D) A comparative densitometry analysis of untreated control, PAMAM-DEN and PAMAM-DENCYS is shown, although it is more appropriate to compare control vs. cysteamine (B) and PAMAM-DEN vs. PAMAM-DENCYS (C). Data represent mean ± SEM of triplicate samples. (E) Flow cytometry of non-permeabilized IB3-1 cells treated with PAMAM-DENCYS show significant (p<0.05) increase in membrane CFTR protein levels as compared to PAMAM-DEN controls. Data represent mean ± SEM of triplicate samples. (F) Immunofluorescence microscopy of HEK-293 cells transfected with ΔF508-CFTR and treated with PAMAM-DENCYS (500μM, 12 hrs) show increased plasma membrane (PM, yellow arrows) trafficking of CFTR with decreased peri-nuclear aggresome-bodies (AB, red arrows) as compared to controls. To clarify the localization of CFTR, ‘N’ is shown as the nucleus and red arrows show the peri-nuclear aggresome-accumulation of ΔF508-CFTR, while yellow arrows show the membrane-localization of ΔF508-CFTR in PAMAM-DENCYS treated cells. High-magnification single cell images are shown as insets. Representative image of triplicate samples is shown. Scale bar: 50 μm.
Fig 4.
PAMAM-DENCYS decreases P. aeruginosa infection in CF cells.
(A, B) IB3-1 cells were seeded on a 6-well plate and treated with PAMAM-DENCYS for 12 hours followed by Pseudomonas aeruginosa-GFP (PA01-GFP) infection at MOI of 10 (A) and 1 (B) for 180 minutes. Cells were washed thoroughly with PBS (1x) to remove all extracellular bacteria. Bio-Rad ZOE™ Fluorescent Cell Imager was used to capture images. Representative bright field (left) and fluorescent images (right) show cell number and the number of intracellular bacteria respectively. (C) We quantified the number of fluorescent bacteria and found that PAMAM-DENCYS significantly decreased the PaO1 counts at both MOI of 1 and 10 (**p<0.01), verifying the efficacy of PAMAM-DENCYS as an autophagy-inducing antibacterial nano-formulation. (D) IB3-1 cells were transfected with WT-CFTR and infected with PA01 at MOI of 1 as a positive control. (E) In a parallel experiment, IB3-1 cells were infected with PA01-GFP (MOI of 1) for 180 minutes. Post infection, the media was removed and the cells were washed with PBS (1x) and used to quantify the number of intracellular bacteria using flow cytometry based analysis. Analysis of the flow cytometry data shows a significant (*p<0.05) decrease in the number of intracellular bacteria, and provide further evidence substantiating autophagy-mediated bacterial clearance by PAMAM-DENCYS formulation.
Fig 5.
PAMAM-DENCYS possesses direct bactericidal and mucolytic properties.
(A) Pseudomonas aeruginosa PaO1 bacteria were incubated in LB broth with either control (PBS), PAMAM-DEN, cysteamine (500 μM) and PAMAM-DENCYS (500 μM) and the standard growth of bacteria was monitored from 0 to 18 hours by measuring the OD at 625 nm, to assess bacterial growth. We found that PAMAM-DENCYS significantly (**p<0.01) restricts Pa growth as compared to control, PAMAM-DEN or cysteamine treatment groups, with significant differences at 15 and 18 hour time points (**p<0.01). The data suggest that PAMAM-DENCYS possess direct anti-bacterial activity, which may be one of the several potential benefits of this nano-formulation. The graph represents mean ± SEM, n = 3. (B) The mucolytic activity of PAMAM-DENCYS was quantified by incubating mucin (5% w/vol) solution with control (PBS), PAMAM-DEN, cysteamine (500 μM) and PAMAM-DENCYS (500 μM) and measuring their flow rate (velocity, mm/sec) through a 1 ml serological pipette. The data indicates that both cysteamine and PAMAM-DENCYS have direct mucolytic activity although PAMAM-DENCYS shows significantly (*p<0.5) better efficacy than cysteamine. Data represents mean ± SEM, n = 3.