Peg-Grafted Liposomes for L-Asparaginase Encapsulation

Abstract

L-asparaginase (ASNase) is an important biological drug used to treat Acute Lymphoblastic Leukemia (ALL). It catalyzes the hydrolysis of L-asparagine (Asn) in the bloodstream and, since ALL cells cannot synthesize Asn, protein synthesis is impaired leading to apoptosis. Despite its therapeutic importance, ASNase treatment is associated to side effects, mainly hypersensitivity and immunogenicity. Furthermore, degradation by plasma proteases and immunogenicity shortens the enzyme half-life. Encapsulation of ASNase in liposomes, nanostructures formed by the self-aggregation of phospholipids, is an attractive alternative to protect the enzyme from plasma proteases and enhance pharmacokinetics profile. In addition, PEGylation might prolong the in vivo circulation of liposomes owing to the spherical shielding conferred by the polyethylene (PEG) corona around the nanostructures. In this paper, ASNase was encapsulated in liposomal formulations composed by 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) or 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) containing or not different concentrations of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N [methoxy (polyethylene glycol)-2000] (DSPE-PEG). Nanostructures of approximately 142-202 nm of diameter and polydispersity index (PDI) of 0.069 to 0.190 were obtained and the vesicular shape confirmed by Transmission Electron Microscopy (TEM and cryo-TEM). The encapsulation efficiency (%EE) varied from 10% to 16%. All formulations presented activity in contact with ASNase substrate, indicating the liposomes permeability to Asn and/or enzyme adsorption at the nanostructures' surface; the highest activity was observed for DMPC/DSPE-PEG 10%. Finally, we investigated the activity against the Molt 4 leukemic cell line and found a lower IC₅₀ for the DMPC/DSPE-PEG 10% formulation in comparison to the free enzyme, indicating our system could provide in vivo activity while protecting the enzyme from immune system recognition and proteases degradation.

Keywords: L-asparaginase; acute lymphoblastic leukemia; liposome; nanocarrier; nanoreactor; pegylated liposome.

Figure 1

Dynamic light scattering profile of liposome formulations. Z-average values for increasing concentrations of DSPE-PEG. # formulations showed significant difference compared to PEGylated liposomes. #, ## formulations showed significant difference compared to DOPC/DSPE-PEG 5% and DMPC/DSPE-PEG 5%, respectively. Data are presented as mean ± SD (n = 3 independent experiments), (Two-way ANOVA, α = 0.05, **** p < 0.0001, Turkey test).

Figure 2

Physical stability of liposomal formulations stored at 4 °C. (A,B): Z-average and PDI, respectively, of DOPC, DOPC/DSPE-PEG 5% and DOPC/DSPE-PEG 10% formulations; (C,D): Z-average and PDI, respectively, of DMPC, DMPC/DSPE-PEG 5% and DMPC/DSPE-PEG 10% formulations. n = 3 independent experiments. Mean ± SD.

Figure 3

Physical stability analysis of liposomal formulations stored at 37 °C. (A,B): Z-average and PDI, respectively, of DOPC, DOPC/DSPE-PEG 5% and DOPC/DSPE-PEG 10% formulations; (C,D): Z-average and PDI, respectively, of DMPC, DMPC/DSPE-PEG 5% and DMPC/DSPE-PEG 10% formulations. n = 3 independent experiments. Mean ± SD, Tukey’s test, ** p < 0.01, **** p < 0.0001 (Between 3° to 40° day; 15° to 40° day).

Figure 4

Chromatogram of Size Exclusion Chromatography. (A) L-asparaginase solution (5 mg/mL), peak 1 residues flow-through; peak 2 probably corresponds to aggregated protein and peak 3 refers to pure and soluble ASNase solution (area corresponding to 30.65 mAU·mL). (B) ASNase-DOPC/DSPE-PEG 5% liposome system, peak 1 flow-through; peak 2 correspond to ASNase-DOPC/DSPE-PEG 5% liposomes and peak 3 ASNase non-encapsulated (area corresponding to 27.37 mAU·mL). n = 4, isocratic elution with PBS (pH 7.4).

Figure 5

Dynamic light scattering profile of blank liposomes and ASNase-loaded liposome formulations after purification (n = 4).

Figure 6

Encapsulation efficiency EE (%) of ASNase in liposomes. EE% were calculated based on the area of the chromatographic peak corresponding to the free ASNase before encapsulation and after purification by size exclusion chromatography, n = 4. Mean ± SD.

Figure 7

Transmission electronic microscopy (TEM). Formulations: (A). DMPC; (B). DOPC; (C). DMPC/DSPE-PEG 5%; (D). DOPC/DSPE-PEG 5%; (E). DMPC/DSPE-PEG 10% e (F). DOPC/DSPE-PEG 10%. Magnification of 25,000× (E) and 62,000× (A–F). The bars indicate size of 200 nm (E) and 100 nm others. Black arrows indicate fragments of lamellae or fragmented liposomes white arrows indicate clusters of liposomes.

Figure 8

Cryogenic electron microscopy of DMPC liposomes. (A): DMPC formulation. (B): ASNase-DMPC/DSPE-PEG 10% (C): DMPC/DSPE-PEG 10%. Magnification of 62,000×. The bars indicate size of 100 nm.

Figure 9

Cryogenic electron microscopy of DOPC liposomes. (A): DOPC formulation. (B): ASNase-DOPC/DSPE-PEG 5% (C): DOPC/DSPE-PEG 5%. Magnification of 25,000× (A) and 62,000× (B,C). The bars indicate size of 100 nm (B,C) and 200 nm (A).

Figure 10

Ammonia generation in the presence of ASNase-loaded liposomes (Nessler’s assay results). # Formulation presented significant difference related to others’ formulations. n = 6 independent experiments. The error bars correspond to standard deviation. Two-way ANOVA, Turkey´s test, α = 0.05.

Figure 11

In vitro analyses of formulation. In vitro cytotoxicity of free ASNase (A), ASNase-DMPC (B) and ASNase-DMPC/DSPE-PEG 10% (C) formulations against MOLT-4 cells. The error bars correspond to standard deviation.