Fabrication principles and their contribution to the superior in vivo therapeutic efficacy of nano-liposomes remote loaded with glucocorticoids

Abstract

We report here the design, development and performance of a novel formulation of liposome- encapsulated glucocorticoids (GCs). A highly efficient (>90%) and stable GC encapsulation was obtained based on a transmembrane calcium acetate gradient driving the active accumulation of an amphipathic weak acid GC pro-drug into the intraliposome aqueous compartment, where it forms a GC-calcium precipitate. We demonstrate fabrication principles that derive from the physicochemical properties of the GC and the liposomal lipids, which play a crucial role in GC release rate and kinetics. These principles allow fabrication of formulations that exhibit either a fast, second-order (t(1/2) ~1 h), or a slow, zero-order release rate (t(1/2) ~ 50 h) kinetics. A high therapeutic efficacy was found in murine models of experimental autoimmune encephalomyelitis (EAE) and hematological malignancies.

Figure 1. MPS physicochemical parameters relevant to GC remote loading.

(a)Comparing surface tension of MPS (♦) and DEXP (□) as a function of GC concentration. (b) Octanol/buffer log distribution of MPS (using 20 mM HEPES at the specified pH as buffer).

Figure 2. Calcium-MPS interaction studies.

(a) Change of turbidity (OD at 600 nm) of 20 mM MPS solution at pH 7.4 generated by mixing MPS with different salt solutions: calcium acetate (♦), calcium chloride (▪), sodium acetate (▴), and sodium chloride (•). (b) X-ray diffraction pattern of calcium acetate (blue), MPS (red) and calcium-MPS (black) powders. (c) Cryo-TEM images of nSSL (white a) before and (white b) after MPS loading (reprinted with permission from Langmuir and Arthritis and Rheumatism [27]).

Figure 3. Studying the kinetics of nSSL acetate release and MPS encapsulation during nSSL remote loading, and the effect of intraliposome salt composition on MPS loading.

(a)Reduction in level of extraliposome MPS during remote loading of 20 mM (▴) and 6.66 mM MPS (×). Extraliposome acetate increase induced by remote loading of 6.66 mM MPS (•) and 20 mM MPS (♦). As a control, acetate release from nSSL with no MPS present in the bulk medium (▪). (b) Effect of intraliposome salt composition on MPS remote loading.

Figure 4. Effect of nSSL lipid compositions and external medium composition on kinetic order and rate of MPS release from nSSL-MPS at 37°C.

(a) Release of MPS from nSSL composed of: HSPC/CHOL/2000-PEG-DSPE (55/40/5 mole ratio, DSPC being a high T_m PC) (▴) (reprinted with permission from Arthritis and Rheumatism [27]);DSPC/CHOL/2000-PEG-DSPE (55/40/5 mole ratio), when incubated at 37°C in plasma (♦); nSSL composed of HSPC/CHOL/2000-PEG-DSPE (55/40/5 mole ratio, HSPC being a high Tm PC), at 37°C in PBS buffer (pH 7.4) (▪); nSSL composed of POPC/CHOL/2000-PEG-DSPE (55/40/5 mole ratio, POPC being a low T_m PC) at 37°C in plasma (□). Inset Table — Calculated release kinetic parameters. (b) Scheme describing the mechanism of kinetic order and release rate: 1. In nano-liposomes of all lipid compositions (applies to all liposomes!) used in this study, calcium-MPS salt inside the nano-liposomes is in an intraliposome pH- and temperature-dependent equilibrium (K₁) between its precipitated salt form, and its soluble dissociated and un-dissociated forms. Since salt solubility increases with rise in temperature from 4°C to 37°C thereby shifting the equilibrium to increased level of the dissociated products. 2. MPS dissociates from its calcium salt is in equilibrium (K₂) between its protonated (uncharged) and unprotonated (charged) form. This equilibrium is dependent on K₁, as the intraliposome pH, is slightly alkaline, only a small fraction MPS is in the protonated neutral form. 3. Protonated (uncharged) MPS can transverses the lipid bilayer in both directions effluxing (k ₃) and influxing (k _-3). The net direction of movement is dependent on various conditions including lipid membrane composition, drug physicochemical properties (such as partition coefficient and polar to non-polar surface area, pH, gradient, dilution, motion, mixing and others Zucker et al . For the efflux/influx reactions equilibrium is not reached. The effect of interplay between liposome bilayer lipid composition and the temperature is mediated by bilayer level of “free volume”, as discussed by Stein , and reviewed by Barenholz and Cevc . The results described by Fig 4a clearly demonstrate that for nSSL based on high-T_m PCs (HSPC or DSPC) there is a slow, zero-order kinetics MPS release, while for the nano-liposomes based on the low-T_m DOPC, release rate is fast, second-order. This difference agrees well with the fact that the low T_m-based nSSL has much higher level of liposome membrane free volume (for more in depth discussion on this issue see Results section entitled: “Release kinetics as a function of liposome lipid composition and bulk medium composition”).

Figure 5. In vivo pharmacokinetic study in mice of ³H-nSSL-MPS and of free MPS.

Pharmacokinetics of free MPS (♦), MPS of nSSL-MPS (▴), ³H-nSSL of nSSL-MPS (▪), and the ratio between encapsulated MPS and its carrier ³H-nSSL (□).The change in this ratio with time is used as a method to calculate in vivo MPS release rate from ³H-nSSL-MPS.

Figure 6. Therapeutic activity of nSSL-MPS in mice induced with EAE (multiple sclerosis model) and in a BCL-1 B-cell leukemia mice animal model (cancer model).

(a) Comparison of the therapeutic efficacy of nSSL-MPS and non-liposomal (free) MPS in acute EAE mice model. SJL/J mice (n = 10) were treated by IV injections on days 10, 12, 14 with: nSSL-MPS 50 mg/kg BW (▴), free MPS 50 mg/kg BW (♦), and dextrose 5% (control) (▪). (b) Comparison of therapeutic efficacy of nSSL-MPS with clinically available drugs, Copaxone and Betaferon on acute EAE mouse model. SJL/J mice (n = 10) were treated by IV injections on days 8, 11 and 14 with: nSSL-MPS 50 mg/kg BW (▴); Copaxone 250 µg (▾); Betaferon 2000 units (•); dextrose 5% (control) (▪). (c) Comparison of therapeutic efficacy of nSSL-MPS and two types of interferon- beta on MOG peptide-induced chronic EAE mouse model. C57Bl/6 mice (n = 10) were treated by IV injections on days 12, 14, and 16 with: nSSL-MPS 50 mg/kg BW (▴); Avonex 2000 units (▾); Betaferon 2000 units (•); dextrose 5% (control) (▪). Inset Tables — For all of the above experiments detailed disease-characteristics are given. (d) Survival curve of mice induced with BCL-1 B-cell leukemia. BALB/c mice (n = 10) were IV injected on days 5, 9, 12 and 16 with either free MPS 5 mg/kg BW (▴), nSSL-MPS 5 mg/kg BW (•) free MPS 25 mg/kg BW (Δ dashed line) or nSSL-MPS 50 mg/kg BW (○). The control group (n = 6) did not receive any treatment (▪). (e) Survival curve of mice induced with J6456 mouse T-cell lymphoma cells. BALB/c mice (n = 10) were IV injected on days 5, 9, 12 and 16 with either free MPS 50 mg/kg BW (▴ dotted line) or nSSL-MPS 50 mg/kg BW (○). The control group (n = 6) did not receive any treatment (▪).