In vitro evaluation of physicochemical-dependent effects of polymeric nanoparticles on their cellular uptake and co-localization using pulmonary calu-3 cell lines

Figures & data

Figure 1. Papp of [¹⁴C]-mannitol measured under different NPs exposures over time: (A) PG-2, (B) PG + 2, (C) PL-2, (D) PL + 2, (E) PG-5, (F) PG + 5, (G) PG-8, and (H) PG + 8 (Mean ± SD, n = 3).

Table 1. Characterization of polymeric of NPs; size, PDI, and zeta potential/charge of 8 different NPs (mean ± SD, n = 3).

NPs Type	Characterization
	Size (nm)	PDI	Zeta Potential (mV)
PG-2	210.11 ± 5.01	0.21 ± 0.02	−26.80 ± 0.90
PG + 2	205.20 ± 7.50	0.23 ± 0.03	+33.30 ± 1.60
PL-2	215.20 ± 3.01	0.20 ± 0.02	−27.70 ± 1.70
PL + 2	199.31 ± 5.50	0.20 ± 0.01	+32.40 ± 1.50
PG-5	524.31 ± 6.10	0.32 ± 0.04	−29.30 ± 2.50
PG + 5	513.41 ± 4.30	0.30 ± 0.05	+27.50 ± 3.00
PG-8	825.50 ± 4.50	0.34 ± 0.04	−28.40 ± 1.50
PG + 8	810.50 ± 10.50	0.36 ± 0.07	+28.90 ± 3.50

Figure 2. NPs internalization and the effects of different inhibitors (inhibitor mechanisms detailed in Materials section) on the internalization of (A) PG-2, (B) PG + 2, (C) PL-2, (D) PL + 2, (E) PG-5, (F) PG + 5, (G) PG-8, (H) PG + 8; where Inh1: Dynasore, Inh2: Genistein, Inh3: Cytochalasin D, Inh4: Nocodazole, Inh5: EIPA, Inh6: Methyl-β-cyclodextrin, Inh7: Chlorpromazine (• Dunnett test comparison to NC, •• p < 0.01, • • • p < 0.001, • • • • p < 0.0001) (Mean ± SD, n = 3).

Figure 3. Confocal microscopy images of Calu-3 cells where nuclei labeled by DAPI/Blue and cell membranes labeled by AF488/Green after incubation for 1 h with (0.5 mg/mL) NP-NRs/Red (A: all negatively-charged NPs and B: their positively-charged counterparts).

Figure 4. Confocal microscopy images of Calu-3 cells where nuclei labeled by DAPI/Blue and lysosomes labeled by LT-26/Green after incubation for 1 h with (0.5 mg/mL) NP-NRs/Red (A: all negatively-charged NPs and B: their positively-charged counterparts).

Table 2. Summary of the endocytosis transport inhibitors results (overview of all NPs and the different inhibitors used, inhibitor mechanisms detailed in materials section) (^-non-significant, ⁺⁺p < 0.01, ⁺⁺⁺p < 0.001, ⁺⁺⁺⁺p < 0.0001).

Mechanism Inhibited	PG-2	PG + 2	PL-2	PL + 2	PG-5	PG + 5	PG-8	PG + 8
Energy/active process (4 ^oC)	+ + + +	+ + + +	+ + + +	+ + + +	+ + + +	+ + + +	+ + + +	+ + + +
GTPase-dynamin (Inh1)	+ + + +	+ + +	+ + +	+ +	–	–	–	–
Tyrosine Kinase-Caveolin assembly (Inh2)	+ + + +	+ + +	+ + + +	+ +	+ + + +	+ +	–	–
Actin polymerization (Inh3)	–	+ + +	–	+ +	–	–	–	+ +
Microtubules arrangement (Inh4)	+ + + +	+ + +	–	–	+ +	+ +	–	–
Na^+/H^+ exchange marcopinocyctosis (Inh5)	–	–	–	–	–	–	–	–
Cholesterol depletion-Caveolin (Inh6)	+ + + +	–	–	–	–	–	–	–
Clathrin pits assembly (Inh7)	–	–	+ +	–	+ + + +	+ + + +	+ +	+ + +
Endocytosis Mechanism	Caveolin and macropinocytosis	Caveolin and macropinocytosis	Caveolin and Clathrin	Caveolin and macropinocytosis	Caveolin and Clathrin and	Clathrin and caveolin	Clathrin	Clathrin and macropinocytosis