Folic acid-tethered Pep-1 peptide-conjugated liposomal nanocarrier for enhanced intracellular drug delivery to cancer cells: conformational characterization and in vitro cellular uptake evaluation

Figures & data

Figure 1 Schematic of synthesis of DSPE-PEG₂₀₀₀-folate.

Note: The compound was synthesized by DCC chemistry linking the amine group of DSPE-PEG₂₀₀₀-amine and the γ-carboxyl of folic acid.

Abbreviations: DSPE, distearoyl phosphatidyl ethanolamine; PEG₂₀₀₀, polyethylene glycol; DCC, dicyclohexylcarbodiimide.

Table 1 Conformational and physical characteristics of liposomal nanocarriers

	C-Lipo	P-Lipo	F-Lipo	FP-Lipo
				FP(100)-PB	FP(350)-PB	FP(100)-PEG	FP(350)-PEG
Composition
PC:Tween 80	90:10	89.5:10	89.5:10	89.5:10	89.5:10	89.5:10	89.5:10
DSPE-PEG2000-mal	–	–	–	–	–	0.15	0.5
PE-PB-mal	–	0.5	–	0.15	0.5	–	–
Pep-1 peptide	–	0.25	–	0.07	0.25	0.07	0.25
DSPE-PEG2000-folate	–	–	0.55	0.55	0.55	0.55	0.55
Conformational properties
Spacer of Pep-1 peptide	–	Phenylbutyryl	–	Phenylbutyryl	Phenylbutyryl	PEG2000	PEG2000
Spacer of folic acid	–	–	PEG2000	PEG2000	PEG2000	PEG2000	PEG2000
Pep-1 molecules/vesicle	–	338 ± 62	–	100 ± 26	348 ± 21	104 ± 11	337 ± 19
Folate molecules/vesicle	–	–	762 ± 52	737 ± 68	751 ± 33	778 ± 41	761 ± 20
Physical properties
Size (nm)	130.9 ± 4.5	132.4 ± 6.3	142.9 ± 4.8	136.7 ± 3.7	137.5 ± 2.7	143.0 ± 2.4	141.0 ± 4.6
Polydispersity index	0.220	0.117	0.125	0.288	0.274	0.261	0.259
Zeta potential (mV)	−10.3 ± 0.6	14.9 ± 3.8	−7.1 ± 0.3	4.2 ± 0.2	7.2 ± 3.6	3.8 ± 0.7	7.4 ± 1.7

Abbreviations: C-Lipo, conventional liposomes; P-Lipo, Pep-1 peptide–modified liposomes; F-Lipo, folate-modified liposomes; FP-Lipo, dual ligand–modified liposomes; FP(100)-PB, FP-Lipo with 100 Pep-1 peptide molecules and PE-PB-mal spacer; FP(350)-PB, FP-Lipo with 350 Pep-1 peptide molecules and PE-PB-mal spacer; FP(100)-PEG, FP-Lipo with 100 Pep-1 peptide molecules and DSPE-PEG₂₀₀₀-mal spacer; FP(350)-PEG, FP-Lipo with 350 Pep-1 peptide molecules and DSPE-PEG₂₀₀₀-mal spacer; PC, soya phosphatidylcholine; DSPE, distearoyl phosphatidyl ethanolamine; PE, phosphatidylethanolamine; PB, phenylbutyryl; PEG₂₀₀₀, polyethylene glycol; mal, maleimide.

Figure 2 Schematic illustration of the three steps used to prepare FP-Lipo: Step 1 maleimide derivatization of liposomes; Step 2 insertion of DSPE-PEG₂₀₀₀-folate to maleimide-derivatized liposomes; and Pep-1 peptide coupling; Step 3 purification of FP-Lipo systems.

Abbreviations: FP-Lipo, dual ligand–modified liposomes; DSPE, distearoyl phosphatidyl ethanolamine; PEG₂₀₀₀, polyethylene glycol; PC, soya phosphatidylcholine; mal, maleimide; MLV, multi-lamellar vesicle; SUV, small unilamellar vesicle.

Figure 3 Effect of the number of Pep-1 peptide per vesicle and type of spacer on cell uptake behavior of various FP-Lipo formulations.

Note: Composition of each formulation can be found in Table 1.

Abbreviations: FP-Lipo, dual ligand–modified liposomes; FP(100)-PB, FP-Lipo with 100 Pep-1 peptide molecules and PE-PB-mal spacer; FP(350)-PB, FP-Lipo with 350 Pep-1 peptide molecules and PE-PB-mal spacer; FP(100)-PEG, FP-Lipo with 100 Pep-1 peptide molecules and DSPE-PEG₂₀₀₀-mal spacer; FP(350)-PEG, FP-Lipo with 350 Pep-1 peptide molecules and DSPE-PEG₂₀₀₀-mal spacer; PE, phosphatidylethanolamine; PB, phenylbutyryl; mal, maleimide; DSPE, distearoyl phosphatidyl ethanolamine; PEG₂₀₀₀, polyethylene glycol.

Figure 4 The quantitative analysis of FITC-dextran translocated into the cells by various liposomal formulations.

Notes: Values represent mean ± SD (n = 3), and statistical analysis was performed using the Student’s t-test (*P < 0.05 versus paired group).

Abbreviations: FITC-dextran, fluorescein dextran isothiocyanate; FP-Lipo, dual ligand–modified liposomes indicating FP (100)-PEG-Lipo; C-Lipo, conventional liposomes; F-Lipo, folate-modified liposomes; P-Lipo, Pep-1 peptide–modified liposomes.

Figure 5 Fluorescence microscopy of HeLa and HaCaT cells incubated with various liposomal formulations containing FITC-dextran at 37°C and pH 7.4 for 4 hours.

Notes: green fluorescence represents cellular translocation of the probe-entrapped liposomal carrier. While peptide conjugation (P-Lipo) increased translocation into both cells with no difference, folate modification (F- and FP-Lipo) manifested the selectivity to FR-positive HeLa cells, resulting in the greatest uptake of FP-Lipo.

Abbreviations: FITC-dextran, fluorescein dextran isothiocyanate; P-Lipo, Pep-1 peptide–modified liposomes; F-Lipo, folate-modified liposomes; FP-Lipo, dual ligand–modified liposomes; C-Lipo, conventional liposomes.

Figure 6 Effect of FR saturation by free folic acid pretreatment on the cellular uptake of various liposomal formulations.

Notes: 1 mM of free folic acid was added to block Fr on HeLa cells. Values represent mean ± SD (n = 3), and statistical analysis was performed using the Student’s t-test (*P < 0.05 versus paired group).

Abbreviations: FR, folic acid receptor; SD, standard deviation; C-Lipo, conventional liposomes; F-Lipo, folate-modified liposomes; P-Lipo, Pep-1 peptide–modified liposomes; FP-Lipo, dual ligand–modified liposomes.

Figure 7 Cytotoxicities of empty liposomal nanocarriers in HaCaT cells by WST-1 assay under the same experimental conditions as in the cell uptake studies.

Notes: Cells were treated with C-Lipo, F-Lipo, P-Lipo, and FP-Lipo for 4 hours, while control cells were treated with double-distilled water (CTL). None of the formulations were cytotoxic to HaCaT cells in the concentrations used for treatment. Data are expressed as mean ± SD (n = 3).

Abbreviations: FITC-dextran, fluorescein dextran isothiocyanate; C-Lipo, conventional liposomes; F-Lipo, folate-modified liposomes; P-Lipo, Pep-1 peptide–modified liposomes; FP-Lipo, dual ligand–modified liposomes; CTL, double-distilled water (control); SD, standard deviation.