Fig. 2: Development of ratiometric nanoformulation for the co-delivery of PAL and HCQ. | Nature Communications

Fig. 2: Development of ratiometric nanoformulation for the co-delivery of PAL and HCQ.

From: Use of ratiometrically designed nanocarrier targeting CDK4/6 and autophagy pathways for effective pancreatic cancer treatment

Fig. 2Fig. 2

a Schemes depicting the structure of lipid-coated MSNP nanocarrier and the mechanism for simultaneous remote loading of PAL and HCQ using a trapping agent. TEA8SOS-laden particles were incubated with the solution of PAL and HCQ mixed at predefined molar ratios, allowing the amphipathic drugs to diffuse across the lipid bilayer for protonation by TEA8SOS. b Preparation of co-delivery nanocarriers with various PAL/HCQ ratios. c BrdU proliferation assay to calculate IC50 and CI for co-delivery nanoparticles. Each concentration was tested in triplicate (n = 3). Co-delivery formulation with PAL/HCQ feed molar ratio of 1:5 (loading ratio of 1:4.3) displayed the best level of synergy and has been chosen for the following study (red arrow). d Summary of physiochemical properties of co-delivery NP. CryoEM of co-delivery NP suggested intact lipid bilayer coating and uniform size distribution. Scale bar: 100 nm. e Crystal violet staining to visualize the growth-inhibitory effect in PANC-1 cells incubated with co-delivery NP for 72 h with equivalent concentration (PAL = 2.5 μM, HCQ = 10.8 μM). f Co-delivery NP significantly improved the anti-proliferative effect compared to PAL NP (p = 5.7 × 10−4). Six ROIs were analyzed for the Ki67 expression (n = 6). g Cell cycle analysis using flow cytometry (triplicate). Both PAL NP and co-delivery NP induced significant G1 cell cycle arrest compared to control with p values of 3.3 × 10−4 and 1.2 × 10−4, respectively. h Western blot to reflect the impact on the expression of phospho-Rb and total Rb. i Representative TEM characterization of autophagic vacuoles in PANC-1 cells. j GFP-RFP-LC3B reporter assay (n = 6 ROIs) to visualize and compare the intracellular level of autophagosomes (GFP and RFP puncta) and autolysosomes (RFP-only puncta). Scale bar: 20 μm. Quantification of GFP-RFP-LC3B is also shown in Supplementary Fig. 7D. k ROS production probed by CellROX staining suggested that the autophagy blockade by co-delivery NP was ROS mediated. One of the two independent repetitions with similar results is shown here for b–k. Data represent mean ± SD; statistical difference was evaluated by one-way ANOVA followed by Tukey’s post hoc test (*p < 0.05). Source data are provided as a Source Data file.

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