Characterization and in vivo evaluation of novel lipid–chlorambucil nanospheres prepared using a mixture of emulsifiers for parenteral administration

Figures & data

Figure 1 Chemical structure of chlorambucil.

Table 1 Formulation components of the lipophilic mixture of the final lipid nanospheres

Lipophilic mixture	Monostearin (mg·mL^−1)	LCT (mg·mL^−1)	MCT (mg·mL^−1)
Lipophilic mixture 1	40	20	–
Lipophilic mixture 2	40	10	10
Lipophilic mixture 3	40	–	20

Abbreviations: LCT, long-chain triglycerides; MCT, medium-chain triglycerides.

Table 2 Formulation components of the surfactant mixture of the final lipid nanospheres

Surfactant mixture	E80 (mg·mL^−1)	Lutrol F 68 (mg·mL^−1)	Tween 80 (mg·mL^−1)
Surfactant 1	20	10	30
Surfactant 2	20	20	20
Surfactant 3	20	30	10

Table 5 Drug loading amount and entrapment efficiency (EE%) of the optimized lipid nanospheres (formulation composed of lipophilic mixture 2: MCT: 10 mg·mL⁻¹, LCT: 10 mg·mL⁻¹, monostearin 40: mg·mL⁻¹, and surfactant mixture 1: E80: 20 mg·mL⁻¹, Tween 80: 30 mg·mL⁻¹, Lutrol F 68:10 mg·mL⁻¹)

Sample number	DA (mg·mL^−1)	EE%
1	0.72	99.28
2	1.07	99.83
3	1.43	98.57

Abbreviations: DA, drug load; LCT, long-chain triglycerides; MCT, medium-chain triglycerides.

Table 3 Effect of different lipophilic mixtures on lipid nanospheres droplet size and entrapment efficiency (EE%); aqueous phase composed of surfactants 1 (E80: 20 mg·mL⁻¹, Tween 80: 30 mg·mL⁻¹, Lutrol F 68: 10 mg·mL⁻¹), and 0.7 mg drugs added per mL of lipid nanospheres

Sample number	DA (mg·mL^−1)	MDS	EE%
Lipophilic mixture 1	0.48	130 ± 0.018	80.32 ± 0.01
Lipophilic mixture 2	0.43	127 ± 0.017	75.28 ± 0.01
Lipophilic mixture 3	0.52	129 ± 0.018	68.11 ± 0.01

Abbrevations: DA, drug load; MDS, mean droplet size.

Table 4 Effect of composition of surfactants on lipid nanospheres droplet size and entrapment efficiency (EE%); lipid mixtures composed of lipophilic mixture 2 (MCT: 10 mg·mL⁻¹, LCT: 10 mg·mL⁻¹, monostearin 40: mg·mL⁻¹), 0.7 mg drugs added for per mL of lipid nanospheres

Sample number	DA (mg·mL^−1)	MDS	Entrapment efficiency(%)
Surfactant 1	0.48	127 ± 0.017	61.22 ± 0.004
Surfactant 2	0.47	133 ± 0.018	55.33 ± 0.015
Surfactant 3	0.48	141 ± 0.019	52.66 ± 0.100

Abbreviations: MCT, medium-chain triglycerides; MDS, mean droplet size.

Figure 2 Mean particle size diameters of formulations after reconstitution with an optimized combination of cryoprotectants.

Table 6 Effects of cryoprotectants on morphological characteristics of reconstituted lipid nanospheres from freeze-dried suspension (cryoprotectant concentration %)

No.	Sucrose	Trehalose	Mannitol	Glucose	Physical propertiesa	Speed of redispersionb
1					+++	−
2	15				++	−
3				15	−	−
5		7.5	7.5		+	−
6		10	5		+	++
7	7.5		7.5		+++	+
8	5	5	5		+++	+
9	5	5		5	+	++
10	5		10		+++	++
11		5	10		−	+++

a Notes: The physical properties were graded from excellent to bad (+++ > ++ > + > −);

b The speed of redispersion was graded from fast to slow (+++ > ++ > + > −).

Figure 3 A transmission electron microscopy image of 2% phosphotungstic acid-stained lipid nanospheres (pH adjusted to 7.4 with 0.1 mol/L⁻¹ NaOH after preparation) (×40,000, bar = 200 nm).

Figure 4 Differential scanning calorimetry scan of lyophilized lipid nanospheres (LNS) powder heating from 30°C to 120°C at a rate of 10°C·min⁻¹. 1) The physical mixture material; 2) Placebo LNS; 3) Drug-loaded LNS (containing 0.7 mg·mL⁻¹ of the model drug).

Figure 5 Mean plasma log concentration–time profiles after iv administration of lipid nanospheres of chlorambucil (CHL-LNS) and free chlorambucil (CHL) at a dose of 10 mg·kg⁻¹ in mice.

Note: Results are shown as mean ± SD.

Table 7 Pharmacokinetic parameters after iv administration of chlorambucil (CHL) injection and lipid nanospheres (CHL-LNS) to mice

		CHL (± SD; %)	CHL-LNS (± SD, %)
t1/2α	min	0.985 ± 0.477	16.143 ± 13.935
t1/2β	min	17.365 ± 1.983	23.369 ± 4.311
V1	L·kg^−1	0.018 ± 0.017	0.217 ± 0.175
CL	L·min^−1·kg^−1	0.011 ± 0.003	0.008 ± 0
AUC (0–t)	mg·L^−1·min	1426.87 ± 262.332	2087.778 ± 129.56
AUC (0–∞)	mg·L^−1·min	1790.917 ± 368.64	2473.315 ± 107.02
MRT (0–t)	min	16.019 ± 1.109	23.733 ± 3.196

Abbreviations: AUC, area under the concentration time curve; CL, clearance; V1, steady-state apparent volume of distribution; MRT, mean retention time.

Figure 6 Distribution profiles in various organs after iv administration of lipid nanospheres of chlorambucil (CHL-LNS) and free chlorambucil (CHL) at a dose of 10 mg·kg⁻¹ in mice.

Note: Bars represent standard error of 3 determinations.