The application of EDTA in drug delivery systems: doxorubicin liposomes loaded via NH₄EDTA gradient

Figures & data

Table 1 The dosage of DOX for pharmacodynamic test

Group	DOX solution	NH4EDTA-L			(NH4)2SO4-L
		Low	Medium	High
Dosage (mg/kg)	5	2.5	5	10	5

Abbreviations: DOX, doxorubicin; EDTA, ethylenediaminetetraacetic acid; L, doxorubicin-loaded liposomes.

Figure 1 A comparison of the solubilities of DOX in different salt solutions as a function of pH. The solubilities of DOX were quantified after a 24-hour incubation of 2 mg DOX within 1 mL of 200 mM salt solutions of different pH at 25°C.

Note: **P<0.01 versus (NH₄)₂SO₄ group.

Abbreviations: DOX, doxorubicin; EDTA, ethylenediaminetetraacetic acid.

Figure 2 Photographs of mixtures of DOX with (1) (NH₄)₂SO₄ and (2) NH₄EDTA at 25°C and pH 4 (A) before centrifugation, (B) after centrifugation, and (C) diluted with isochoric distilled water.

Abbreviations: DOX, doxorubicin; EDTA, ethylenediaminetetraacetic acid.

Figure 3 A comparison of the release of DOX encapsulated in liposomes in vitro via different ion gradients: 200 mM NH₄EDTA solution and 200 mM (NH₄)₂SO₄ solutions. Drug release profiles were analyzed in conventional PBS buffer (pH 7.4) and a special PBS buffer with ammonium chloride (80 mM), histidine (10 mM), penicillin (100 μg/mL), and streptomycin (100 μg/mL) (pH 7.4).

Note: **P<0.01 versus (NH₄)₂SO₄-L in PBS buffer with ammonium chloride.

Abbreviations: DOX, doxorubicin; EDTA, ethylenediaminetetraacetic acid; L, doxorubicin-loaded liposomes; PBS, phosphate-buffered saline.

Table 2 Long-term stability of HSPC/CH/mPEG₂₀₀₀-DSPE (3/1/1, w/w/w) liposomes loaded with doxorubicin (drug/lipid ratio 1:10, w/w) via 200 mM NH₄EDTA and (NH₄)₂SO₄ gradient stored at 4°C±2°C

Day	NH4EDTA-L			(NH4)2SO4-L
	Appearance	Particle size ± SD (nm; n =3)	Entrapment efficiency ± SD (%; n=3)	Appearance	Particle size ± SD (nm; n=3)	Entrapment efficiency ± SD (%; n=3)
0	+	79.4±1.87	95.54±0.59	+	76.9±2.15	95.07±0.78
15	+	80.9±2.83	97.29±1.16	+	82.5±8.00	95.92±0.74
30	+	87.1±3.25	96.02±1.35	+	87.4±5.48	95.52±0.78
60	+	93.1±4.85	95.60±1.04	↓	–	–
90	+	106.2±3.55	95.32±1.05	↓	–	–

Notes: + represents cherry red liposomes, clear, without precipitation, phase separation, and bacterium; ↓ represents cherry red liposomes, precipitation, or bacterium; and – represents no data.

Abbreviations: CH, cholesterol; EDTA, ethylenediaminetetraacetic acid; HSPC, hydrogenated soy phosphatidylcholine; L, doxorubicin-loaded liposomes; mPEG₂₀₀₀-DSPE, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-(methoxy[polyethylene glycol]-2000); SD, standard deviation; w/w, weight/weight.

Figure 4 (A) Tumor inhibition effects of different DOX formulations. The drug doses were 5.0 mg/kg for DOX solution and (NH₄)₂SO₄-L, and 2.5, 5.0, 10.0 mg/kg for the low, middle, and high concentration of NH₄EDTA-L, respectively. (B) Damage of different formulations on immune organs of H₂₂-bearing mice.

Notes: The data represent mean ± standard deviation. *P<0.05 versus NS-treated mice; **P<0.01 versus NS-treated mice; ^#P<0.05 versus DOX solution-treated mice.

Abbreviations: DOX, doxorubicin; EDTA, ethylenediaminetetraacetic acid; L, doxorubicin-loaded liposomes; NS, normal saline.

Figure 5 Tissue distributions of DOX in mice after intravenous administration of (A) 5 mg/kg DOX solution, NH₄EDTA-L, and (NH₄)₂SO₄-L and (B) 5.0 mg/kg DOX solution and different doses of NH₄EDTA-L (2.5, 5.0, and 10.0 mg/kg).

Notes: The data represent mean ± standard deviation (n=10). Differences between the groups versus DOX solution are statistically significant at the P<0.01 level.

Abbreviations: DOX, doxorubicin; EDTA, ethylenediaminetetraacetic acid; L, doxorubicin-loaded liposomes.

Figure 6 Typical images of pathological classification of myocardium injury of different DOX formulations: (A) NS; (B) DOX solution; (C) NH₄EDTA-L (low-dose); (D) NH₄EDTA-L (medium-dose); (E) NH₄EDTA-L (high-dose); and (F) (NH₄)₂SO₄-L.

Notes: Photographs were obtained under a fluorescence microscope (IX71; Olympus Corporation, Tokyo, Japan) using a 40× objective. The black circles point out the damaged sites of myocardium fragmentation.

Abbreviations: DOX, doxorubicin; EDTA, ethylenediaminetetraacetic acid; L, doxorubicin-loaded liposomes; NS, normal saline.