Functional coating of liposomes using a folate– polymer conjugate to target folate receptors

Figures & data

Figure 1 Schematic illustration of F–PLL conjugate synthesis.

Abbreviations: EDC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; F-PLL, folatepoly(L-lysine).

Figure 2 ¹H NMR spectra of (A) PLL, (B) FA, and (C) F–PLL.

Abbreviations: FA, folic acid; F-PLL, folate-poly(L-lysine); ppm, parts per million.

Table 1 Physicochemical properties of prepared liposomal DXR

Liposome	Particle size (nm)	PDI	Zeta potential (mV)
Liposomal DXRa	115.3 ± 10.0	0.225 ± 0.014	−55.4 ± 5.7
F-PLL-coated liposomal DXRb	117.4 ± 2.2	0.180 ± 0.015	34.1 ± 1.9
PLL-coated liposomal DXRb	119.7 ± 2.1	0.192 ± 0.009	46.8 ± 1.3

Notes: Each value represents the mean ± SD;

a n = 4;

b n = 3.

Abbreviations: DXR, doxorubicin; F-PLL, folate-poly(L-lysine); PDI, polydispersity index.

Figure 3 Cellular association of F–PLL or low F–PLL-coated liposomes labeled with DiI in KB cells analyzed by flow cytometry after a 1-hour incubation.

Notes: The control indicates the autofluorescence of untreated cells. Each analysis was generated by counting 10⁴ cells.

Abbreviations: DiI, 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate; F-PLL, folate-poly(L-lysine).

Figure 4 Cellular association of PLL- or F–PLL-coated liposomal DXR in (A) KB cells and in (B) A549 cells incubated at 37°C, and in (C) KB cells incubated at 4°C for 3 hours with or without 5 mM FA analyzed by flow cytometry.

Note: Each value represents the mean ± SD (n = 3).

Abbreviations: DXR, doxorubicin; FA, folic acid; F-PLL, folate-poly(L-lysine).

Figure 5 Confocal laser scanning microscopy image of KB cells incubated with F–PLL-coated liposomal DXR for 3 hours at 37°C (A) without or (B) with 5 mM FA in culture medium.

Note: The scale bar represents 10 μm.

Abbreviations: DXR, doxorubicin; FA, folic acid; F-PLL, folate-poly(L-lysine).

Table 2 IC₅₀ of DXR solution, F–PLL- and PLL-coated liposomal DXR against KB cells or A549 cells at 48 hours

Formulation	IC50 (μM)
	KB cells	A549 cells
DXR solution	1.20	2.17
F–PLL-coated liposomal DXR	30.75	104.31
PLL-coated liposomal DXR	64.97	105.71

Abbreviations: DXR, doxorubicin; F-PLL, folate-poly(L-lysine).

Figure 6 Cytotoxicity of F–PLL-coated liposomal DXR or PLL-coated liposomal DXR against (A) KB cells and (B) A549 cells.

Notes: Cells were incubated with liposomes in folate-free medium for 2 hours, then in fresh medium without the drug for 48 hours at 37°C. Data indicate the mean ± SD (n = 6).

Abbreviations: DXR, doxorubicin; F-PLL, folate-poly(L-lysine).

Figure S1 UV spectra of F–PLL, FA, and PLL solutions.

Abbreviations: FA, folic acid; F-PLL, folate-poly(L-lysine).

Figure S2 Confocal laser scanning microscopy image of A549 cells incubated with F–PLL-coated liposomal DXR for 3 hours at 37°C (A) without or (B) with 5 mM FA in culture medium.

Note: The scale bar represents 10 μm.

Abbreviations: DXR, doxorubicin; FA, folic acid; F-PLL, folate-poly(L-lysine).