The comparison of protein-entrapped liposomes and lipoparticles: preparation, characterization, and efficacy of cellular uptake

Figures & data

Table 1 The stability effect of different lipid molar ratios and L/E/W ratio formulas following the ethanol injection extrusion method

No	Lipid molar ratioa	L/E/W ratiob	Particle size (nm)			Zeta potential (mV)
			0 (day) mean ± SD (CV%)c	7 (day) mean ± SD (CV%)	28 (day) mean ± SD (CV%)	0 (day) mean ± SD (CV%)	7 (day) mean ± SD (CV%)	28 (day) mean ± SD (CV%)
A	59/36/5/0	75/1/6	121 ± 5 (4)	121 ± 7 (6)	112 ± 4 (4)	−42.8 ± 0.7 (1.6)	−35.2 ± 1.2 (3.4)***	−20.1 ± 0.7 (3.6)***
B	39/57/4/0	75/1/6	131 ± 3 (3)	110 ± 6 (5)**	114 ± 10 (9)*	−30.5 ± 2.3 (7.5)	−27.2 ± 1.3 (4.8)*	−55.5 ± 8.0 (14.3)***
C	59/36/5/0	50/1/6	138 ± 5 (4)	131 ± 6 (5)	120 ± 16 (13)	−30.6 ± 3.7 (12)	−18.1 ± 0.4 (2.4)***	−11.3 ± 2.1 (18.8)***
D	39/57/4/0	50/1/6	157 ± 23 (14)	162 ± 6 (4)	110 ± 16 (14)	−34.2 ± 1.2 (3.4)	−26.0 ± 1.4 (5.2)***	−17.2 ± 0.6 (3.4)***
E	59/36/5/0	75/1/9	124 ± 8 (6)	117 ± 14 (12)	122 ± 1 (1)	−31.8 ± 2.1 (6.7)	−31.8 ± 2.1 (6.7)	−37.5 ± 2.1 (5.6)
F	39/57/4/0	75/1/9	119 ± 11 (9)	126 ± 6 (5)	128 ± 5 (4)	−29.7 ± 0.5 (1.6)	−27.7 ± 1.2 (4.4)**	−28.3 ± 0.7 (2.4)***
E5	54/36/5/5	75/1/9	140 ± 5 (4)	137 ± 3 (2)	121 ± 3 (2)**	nd	nd	nd
E15	44/36/5/15	75/1/9	115 ± 5 (4)	118 ± 4 (3)	119 ± 11 (9)	−35.6 ± 4.9 (13.8)	−35.5 ± 4.0 (11.3)	−35.8 ± 1.4 (3.9)
E30	29/36/5/30	75/1/9	120 ± 8 (7)	129 ± 7 (5)	150 ± 14 (9)*	nd	nd	nd

a Notes: Indicates lipid molar ratio of DSPC (distearoylphosphatidylcholine)/cholesterol/DSPE-PEG₂₀₀₀ (distearoylphosphoethanolamine-PEG2000)/DHA (docosahexaenoic acid);

b indicates (lipid [mg]/ethanol [mL]/dd-water [mL]);

c mean ± standard deviation (% coefficient of variation);

* P <0.05;

** P <0.01;

*** P <0.001.

Abbreviations: L/E/W, lipid content/ethanol/water; SD, standard deviatio; nd, no detection.

Figure 1 The mean particle size (○) and zeta potential (●) stability tests for liposome suspensions as a function of time (stored at 4°C). (A) Formula No. E and (B) formula No. F. (C) FITC-BSA-loaded lipoparticles. (D) Comparison of FITC–BSA-loaded liposomes and lipoparticles.

Note: *Compared to 0 day, P < 0.001 (paired t-test) (n = 3).

Abbreviation: FITC-BSA, fluorescein isothiocyanate-conjugated bovine serum albumin.

Table 2 The characteristics of FITC-BSA-loaded liposomes and lipoparticles

Formula	Particle size(nm)					Zeta potential (mV)		Yield (%)d		EE(%)e
	Before lyoa		After lyob		Dc %
	Meanf	CV%g	Mean	CV%		Mean	CV%	Mean	CV%	Mean	CV%
Blank liposome-I	219 ± 3	2	281 ± 19*	7	25	−35.5 ± 3.9	11.1	90.0 ± 5.3	6	–
Blank liposome-IE	125 ± 2	19	177 ± 21*	12	41	−12.4 ± 1.5	12.0	75.6 ± 0.7	14	–
FITC-BSA-liposome-I	202 ± 5	24	218 ± 10	4	8	−17.26 ± 2.9	16.5	69.4 ± 4.7	7	19.0 ± 4.0	20.9
FITC-BSA-liposome-IE	126 ± 5	13	186 ± 24*	13	47	−13.7 ± 1.4	10.2	77.4 ± 3.2	4	51.4 ± 6.1	11.8
FITC-BSA-lipoparticle-I	215 ± 2	31	252 ± 25	10	17	3.2 ± 0.4	13.9	39.0 ± 9.9	51	48.4 ± 9.7	40.7
FITC-BSA-lipoparticle-IE	134 ± 9	19	148 ± 10	7	10	13.0 ± 1.6	12.3	72.4 ± 5.7	22	59.6 ± 0.4	0.7

a,b Notes: Liposomes particle size before/after lyophilization;

c difference in liposome particle size before and after lyophilization,

$\text{D}\%=\frac{\mathrm{\Delta }({\text{lyo}}_{\text{befor}}-{\text{lyo}}_{\text{after}})}{{\text{lyo}}_{\text{befor}}}\times 100\%;$

d

$\text{Yield}\%=\frac{{\text{MASS}}_{\text{lipo}}}{{\text{MASS}}_{\text{initial}}}\times 100\%;$

e

$\text{EE}\%=\frac{(\text{FITC}-{\text{BSA}}_{\text{enca}})}{\text{(FITC}-{\text{BSA}}_{\text{total}})}\times 100\%,$

where MASS_lipo is the mass of the lyophilized liposome powder, MASS_initial is the mass of the total materials, FITC-BSA_enca is the amount of adsorbed FITC-BSA measured in the liposomes; FITC-BSA_total is the initial amount of FITC-BSA, lyo_before is the particle size before lyophilization, lyo_after is the particle size after lyophilization;

f mean ± standard deviation, n = 3;

g % coefficient of variation = SD/mean × 100%.

* Compared to before lyophilization, P < 0.05 (paired t-test).

Abbreviation: FITC-BSA, fluorescein isothiocyanate-conjugated bovine serum albumin.

Figure 2 TEM image (left) and FE-SEM image (right) with 1% uranyl acetate negative staining. (A) FITC-BSA-loaded liposomes and (B) FITC-BSA-loaded lipoparticles. (C) FITC-BSA-loaded liposomes and (D) FITC-BSA-loaded lipoparticles. The scale bar is 100 nm. (E) Particle size distribution of FITC-BSA-loaded lipoparticles containing a protamine/FITC-BSA core.

Abbreviations: TEM, transmission electron microscope; FE-SEM, field-emission scanning electron microscope; FITC-BSA, fluorescein isothiocyanate-conjugated bovine serum albumin.

Figure 3 The remaining amount of FITC-BSA from the release profile of FITC-BSA-loaded liposomes and lipoparticles in PBS (pH 7.4) at 37°C. (A and C) Before lyophilization and (B and D) after lyophilization. FITC-BSA-loaded liposomes (⋄) and FITC–BSA-loaded lipoparticles containing a protamine/FITC-BSA 20/1 core (○) (n = 3).

Abbreviations: FITC-BSA, fluorescein isothiocyanate-conjugated bovine serum albumin; PBS, phosphate buffered saline.

Table 3 Retention parameters for FITC-BSA-loaded particles obtained after fitting in vitrodrug release data (0.5–24 hours) to three different mathematical models for FITC-BSA retention kinetics

Formulation	Mathematical modelsa for retention kinetics
	Zero order	First order	Higuchi model
Before lyophilization
FITC-BSA-liposome	k′0 = −0.0319	k′1 = −0.0556	k′h = −0.1953
	R^2 = 0.7838	R^2 = 0.6688	R^2 = 0.9307
FITC-BSA lipoparticle	k′0 = −0.0314	k′1 = −0.0882	k′h = −0.1879
	R^2 = 0.8543	R^2 = 0.5940	R^2 = 0.9669
After lyophilization
FITC-BSA-liposome	k′0 = −0.0377	k′1 = −0.0735	k′h = −0.2332
	R^2 = 0.7580	R^2 = 0.5584	R^2 = 0.9154
FITC-BSA lipoparticle	k′0 = −0.0374	k′1 = −0.0965	k′h = −0.2172
	R^2 = 0.9306	R^2 = 0.7183	R^2 = 0.9922

a Notes: Zero order, M_t/M_⊥ = k′₀t; first order, M_t/M_⊥ = 1 − exp(−k′₁ t); higuchi model, M_t/M_⊥ = k′_h t^1/2, where M_t/M_⊥ is the fraction of the drug retained at time t and k′₀, k′₁, and k′_h, represent the zero-order release constant, first-order retention constant, and Higuchi constant, respectively.

Abbreviation: FITC-BSA, fluorescein isothiocyanate-conjugated bovine serum albumin.

Figure 4 The 50% retention time (t_50%) of FITC-BSA (before [white] and after [black] lyophilization) of the various formulations.

Notes: The results were calculated using the Higuchi equation. *P < 0.01.

Abbreviation: FITC-BSA, fluorescein isothiocyanate-conjugated bovine serum albumin.

Figure 5 Protein stability evaluation by SDS-PAGE (M, molecular weight markers; 1, BSA; 2, FITC-BSA; 3, FITC-BSA particle; 4, FITC-BSA liposome; 5, FITC-BSA lipoparticle-I; 6, FITC-BSA lipoparticle-IE; 7, Freeze-dried FITC-BSA lipoparticle-IE; 8, FITC-BSA lipoparticle-IE in 13.7 mM PBS for 4 hours; and 9, FITC-BSA lipoparticle-IE in 13.7 mM PBS for 8 hours).

Abbreviations: SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; FITC-BSA, fluorescein isothiocyanate-conjugated bovine serum albumin.

Figure 6 Cytotoxicity profile containing a protamine/FITC-BSA core (□) after a 72-hour incubation with Caco-2 cell, as determined by WST-1 assay. Percent viability of fibroblasts is expressed relative to control cells (n = 6).

Abbreviation: FITC-BSA, fluorescein isothiocyanate-conjugated bovine serum albumin.

Figure 7 Caco-2 cell uptake profiles of FITC-BSA-loaded lipoparticles (blank) and liposomes (black). Data represents mean ± S D, n = 3. (A) Forward and side light scatter was used to gate the desired scattering events (normal cells) from dead cells and cell debris. (B) Uptake of FITC-BSA-loaded PEGylated liposomes and lipoparticles into Caco-2 cells. Cells were analyzed with 488 nm excitation and 522 nm band-pass filters in the emission path. (C) Fluorescence intensity of FITC-BSA-loaded liposome and lipoparticle preparations in normal cells expressed as the intensity ratio by dividing by the untreated control.

Notes: Data are expressed as mean ± SD, n = 3 (student’s t-test; ** P < 0.01).

Abbreviations: FITC-BSA, fluorescein isothiocyanate-conjugated bovine serum albumin; SD, standard deviation.