Formulation, characterization and tissue distribution of a novel pH-sensitive long-circulating liposome-based theranostic suitable for molecular imaging and drug delivery

Figures & data

Table 1 The chemical components and molar ratios of liposomes in this study

Liposome	Lipid components	Molar ratio	Loaded with calcein
4%PEG-HS-Chol	HSPC/Chol/DSPE-PEG2000/DSPE-DTPA	57:38:4:1	No
1%PEG-HS-CHEM	HSPC/CHEM/DSPE-PEG2000/DSPE-DTPA	57:41:1:1	No
2%PEG-HS-CHEM	HSPC/CHEM/DSPE-PEG2000/DSPE-DTPA	57:40:2:1	No
4%PEG-HS-CHEM	HSPC/CHEM/DSPE-PEG2000/DSPE-DTPA	57:38:4:1	No
4%PEG-HS-Chol-Cal	HSPC/Chol/DSPE-PEG2000/DSPE-DTPA	57:38:4:1	Yes
1%PEG-HS-CHEM-Cal	HSPC/CHEM/DSPE-PEG2000/DSPE-DTPA	57:41:1:1	Yes
2%PEG-HS-CHEM-Cal	HSPC/CHEM/DSPE-PEG2000/DSPE-DTPA	57:40:2:1	Yes
4%PEG-HS-CHEM-Cal	HSPC/CHEM/DSPE-PEG2000/DSPE-DTPA	57:38:4:1	Yes

Abbreviations: PEG, polyethylene glycol; HSPC, hydrogenated soy phosphatidylcholine; Chol, cholesterol; DSPE-PEG₂₀₀₀, PEG₂₀₀₀ conjugated distearoyl phosphatidylethanolamine; DSPE-DTPA, diethylenetriaminepentaacetic acid-derivatized DSPE; CHEM, cholesteryl hemisuccinate; Cal, calcein.

Table 2 Size, polydispersity, zeta potential, calcein encapsulation and lipid recovery of liposomes

Sample ID	Mean diameter (nm)	Polydispersity	Zeta potential (mV)	Calcein encapsulation (mg/µmol lipid)	Recovery of phosphatidylcholine (%)	Recovery of Chol/CHEM (%)
4%PEG-HS-Chol	90.55±1.33	0.103±0.014	−13.3±0.1	n/a	95.70±7.83	96.58±4.63
1%PEG-HS-CHEM	88.06±1.10	0.124±0.012	−18.1±0.7	n/a	86.69±4.76	98.33±3.95
2%PEG-HS-CHEM	98.26±1.05	0.120±0.013	−14.6±0.7	n/a	89.73±2.34	98.22±3.13
4%PEG-HS-CHEM	98.43±0.65	0.109±0.021	−12.7±0.6	n/a	93.23±4.39	97.05±4.01
4%PEG-HS-Chol-Cal	96.63±0.89	0.155±0.008	−13.4±0.4	0.100±0.026	85.07±2.03	91.73±1.22
1%PEG-HS-CHEM-Cal	91.17±0.68	0.142±0.001	−21.9±1.1	0.132±0.027	87.53±8.24	97.71±0.63
2%PEG-HS-CHEM-Cal	101.63±0.64	0.085±0.028	−15.6±0.7	0.093±0.003	87.91±5.17	92.32±2.53
4%PEG-HS-CHEM-Cal	95.18±0.59	0.143±0.022	−13.6±0.6	0.099±0.013	92.38±8.98	96.22±1.26

Note: Three measurements were taken with each sample and the final values were expressed as mean ± standard deviation.

Abbreviations: Chol, cholesterol; CHEM, cholesteryl hemisuccinate; PEG, polyethylene glycol; HS, hydrogenated soy; n/a, not applicable; Cal, calcein.

Figure 1 Percentage release of calcein from the four liposomes under acidic and physiological pHs.

Notes: Calcein-encapsulated liposomes 4%PEG-HS-CHEM-Cal (A), 1%PEG-HS-CHEM-Cal (B), 2%PEG-HS-CHEM-Cal (C) and 4%PEG-HS-Chol-Cal (D) were tested in buffers of pH 4.5, 5.5 and 7.2 at 37°C. Measurements (n=3) were taken at 30 (initial time point), 60, 90, 120 and 180 min, respectively, into incubation. The release of calcein at each time point is expressed as a percentage of total release (mean% ± SD). Release at 30 min was compared to that of all other time points, and the statistical significance is expressed as follows: ns, P>0.05; *P<0.05; **P<0.001; ***P<0.0001.

Abbreviations: PEG, polyethylene glycol; HS, hydrogenated soy; CHEM, cholesteryl hemisuccinate; Cal, calcein; Chol, cholesterol; ns, not significant; SD, standard deviation.

Figure 2 Radiolabeling stability of the four ¹¹¹In-labeled liposomes under varied serum concentrations and PBS at different time points.

Notes: ¹¹¹In-labeled 4%PEG-HS-CHEM (A), 1%PEG-HS-CHEM (B), 2%PEG-HS-CHEM (C) and 4%PEG-HS-Chol (D) were incubated in human serum at two different ratios (2 and 0.5 μmol lipid/mL), as well as in PBS (control) at 37°C for evaluation of stability in serum. The stability of each liposome formula is expressed as a percentage of the radiochemical purity of the labeled liposomes (mean% ± SD). Statistical significance between individual serum challenge versus control condition is expressed as follows: ns, P>0.05; *P<0.05; **P<0.001.

Abbreviations: PBS, phosphate-buffered saline; PEG, polyethylene glycol; HS, hydrogenated soy; CHEM, cholesteryl hemisuccinate; Chol, cholesterol; ns, not significant; SD, standard deviation.

Table 3 Biodistribution of ¹¹¹In-liposomal nanoparticles in healthy Sprague Dawley rats

Tissues	Biodistribution of ^111In-labeled liposome in rats (%ID/g)
	4%PEG-HS-Chol			4%PEG-HS-CHEM
	2 h	24 h	48 h	2 h	24 h	48 h
Urine	0.264%±0.079%	0.263%±0.072%	0.237%±0.049%	0.215%±0.121%	0.133%±0.010%	0.138%±0.032%
Liver	0.446%±0.028%	0.735%±0.079%	0.633%±0.027%	0.499%±0.080%	0.574%±0.019%	0.422%±0.091%
Femur	0.163%±0.008%	0.213%±0.038%	0.174%±0.037%	0.102%±0.022%	0.112%±0.028%	0.096%±0.014%
Muscle	0.036%±0.011%	0.040%±0.004%	0.038%±0.003%	0.027%±0.006%	0.018%±0.004%	0.015%±0.003%
Spleen	1.747%±0.271%	4.996%±0.508%	4.918%±0.796%	2.602%±0.947%	4.915%±0.432%	4.677%±1.458%
Blood	2.401%±0.161%	0.818%±0.020%	0.275%±0.043%	1.531%±0.281%	0.450%±0.077%	0.132%±0.023%
Brain	0.054%±0.002%	0.024%±0.002%	0.013%±0.003%	0.040%±0.013%	0.013%±0.003%	0.005%±0.000%
Intestine	0.157%±0.014%	0.271%±0.035%	0.244%±0.011%	0.105%±0.034%	0.168%±0.019%	0.142%±0.020%
Kidney	0.312%±0.045%	0.444%±0.065%	0.418%±0.024%	0.188%±0.067%	0.210%±0.016%	0.214%±0.026%
Heart	0.451%±0.057%	0.360%±0.058%	0.367%±0.195%	0.252%±0.022%	0.140%±0.009%	0.112%±0.026%
Lung	0.610%±0.015%	0.452%±0.089%	0.310%±0.049%	0.461%±0.093%	0.187%±0.014%	0.142%±0.009%
Stomach	0.027%±0.006%	0.035%±0.011%	0.049%±0.009%	0.017%±0.002%	0.027%±0.006%	0.037%±0.019%
Thyroid	0.074%±0.009%	0.100%±0.017%	0.089%±0.006%	0.079%±0.049%	0.041%±0.005%	0.040%±0.006%
Testis	0.059%±0.007%	0.078%±0.008%	0.078%±0.013%	0.042%±0.010%	0.036%±0.005%	0.033%±0.001%

Notes: Eighteen animals were injected with ¹¹¹In-4%PEG-HS-Chol or ¹¹¹In-4%PEG-HS-CHEM. Blood samples were taken through tail vein at 2, 24 and 48 h post injections before animals were sacrificed for tissue counting and weighing. Values were expressed as averaged percent injected dose/gram of tissue (%ID/g) (mean ± standard deviation, n=3).

Abbreviations: PEG, polyethylene glycol; HS, hydrogenated soy; Chol, cholesterol; CHEM, cholesteryl hemisuccinate.

Table 4 Kinetic parameters of 4%PEG-HS-Chol and 4%PEG-HS-CHEM

Sample ID	Fast (α) component		Slow (β) component
	t1/2 (h) (95% CI)	Fraction (%)	t1/2 (h) (95% CI)	Fraction (%)
4%PEG-HS-Chol	0.91 (0.55–2.59)	43.26±4.84	15.26 (10.74–26.39)	56.74±4.94
4%PEG-HS-CHEM	0.55 (0.35–1.23)	55.23±4.71	13.65 (8.34–37.56)	44.77±4.71

Notes: A two-compartment exponential decay model was fitted over the % injected liposome-associated activity to estimate the clearance half-life (t_1/2 in hours, with confidence interval) and fraction % of removed liposome content from blood, in both the fast and slow phase.

Abbreviations: PEG, polyethylene glycol; HS, hydrogenated soy; Chol, cholesterol; CHEM, cholesteryl hemisuccinate; CI, confidence interval.

Figure 3 Tissue uptake of ¹¹¹In-labeled 4%PEG-HS-Chol (A) and 4%PEG-HS-CHEM (B) in healthy rats.

Notes: A group of three animals were sacrificed at 2, 24 and 48 h after the injection of each type of radiolabeled liposome. Tissues (bladder, liver, femur, muscle, spleen, blood, brain, intestine, kidney, heart, lung, stomach, thyroid and testis) were extracted for gamma counting and weight measurement. The uptake values are expressed as the percentage of injected dose per gram (%ID/g ± SD).

Abbreviations: PEG, polyethylene glycol; HS, hydrogenated soy; Chol, cholesterol; CHEM, cholesteryl hemisuccinate; SD, standard deviation.

Figure 4 Accumulation of ¹¹¹In-labeled 4%PEG-HS-Chol and 4%PEG-HS-CHEM.

Notes: The majority of injected liposomes (expressed as percentage of injected dose %ID ± SD, n=3) were found in liver (A), intestine (B), spleen (C) and kidney (D). Statistical significance between the two formulas is expressed as follows: *P<0.05; **P<0.001.

Abbreviations: PEG, polyethylene glycol; HS, hydrogenated soy; Chol, cholesterol; CHEM, cholesteryl hemisuccinate; SD, standard deviation.

Figure 5 Blood clearance of ¹¹¹In-labeled 4%PEG-HS-Chol and 4%PEG-HS-CHEM.

Notes: The percentage of injected liposome (%ID ± SD, n=5 at 2, 24 and 48 h, n=2 for the rest) in blood was calculated and plotted against time. A two-compartment elimination curve was used to model the blood activity over time for both formulas: 4%PEG-HS-Chol, y=43.26e^−0.6732x+56.74e^−0.04492x, r²=0.993; 4%PEG-HS-CHEM, y=55. 23e^−1.190x+44.77e^−0.05057x, r²=0.991. Statistical significance between the two formulas is expressed as follows: *P<0.05; **P<0.001.

Abbreviations: PEG, polyethylene glycol; HS, hydrogenated soy; Chol, cholesterol; CHEM, cholesteryl hemisuccinate; SD, standard deviation.