Multidomain drug delivery systems of β-casein micelles for the local oral co-administration of antiretroviral combinations

Abstract

The antiretroviral (ARV) cocktailrevolved the treatment of the human immunodeficiency virus (HIV) infection. Drug combinations have been also tested to treat other infectious diseases, including the recentcoronavirus disease 2019 (COVID-19) outbreak. To simplify administration fixed-dose combinationshave been introduced, however, oral anti-HIV therapy still struggles with low oral bioavailability of many ARVs.This work investigated the co-encapsulation of two clinically relevant ARV combinations,tipranavir (TPV):efavirenz (EFV) anddarunavir (DRV):efavirenz (EFV):ritonavir (RTV),within the core of β-casein (bCN) micelles. Encapsulation efficiency in both systems was ~100%. Cryo-transmission electron microscopy and dynamic light scattering of the ARV-loaded colloidaldispersions indicatefull preservation of the spherical morphology, and x-ray diffraction confirm that the encapsulated drugs are amorphous. To prolong the physicochemical stabilitythe formulations were freeze-driedwithout cryo/lyoprotectant, and successfully redispersed, with minor changes in morphology.Then, theARV-loaded micelles were encapsulated within microparticles of Eudragit® L100, which prevented enzymatic degradation and minimized drug release under gastric-like pH conditionsin vitro. At intestinal pH, the coating polymer dissolved and released the nanocarriers and content. Overall, our results confirm the promise of this flexible and modular technology platform for oral delivery of fixed dose combinations.

Keywords: Antiretrovirals; Colloidal dispersion; Combination therapy; Darunavir, efavirenz and ritonavir; Nanoparticle-in-microparticle delivery system (NiMDS); cryo-TEM; β-casein micelles (bCN).

Graphical abstract

Fig. 1

Chemical structure of (A) EFV, (B) DRV, (C) TPV and (D) RTV.

Fig. 2

LM and DIC images showing floating crystals of free drug combinations: (A) TPV:EFV, at 1:1 mol ratio, and (B) DRV:EFV:RTV, at 8:6:1 mol ratio, and complete solubilization after encapsulation in bCN micelle: (C) bCTE, at 1:8:8 protein:drugs mole ratio, and (D) bCDER, at 1:8:6:1 protein:drugs mole ratio. bCN concentration is 10 mg mL⁻¹, and compositions according to Table 1.

Fig. 3

Right: Size distribution expressed as hydrodynamic diameters, of drug-free and drug-loaded bCN micelles (solid lines), with a mean diameter 25 ± 2 nm growing to 35 ± 1 nm and 37 ± 2 nm after encapsulation. No influence of drying on the mean diameter was observed after resuspension the dry dispersions in PBS to the original compositions (doted lines). The corresponding solutions were transparent both before (a1, b1, c1) and after (a2, b2, c2) lyophilization. Left: Turbidity (upper panel) and Z-potential (lower panel) of drug-free and drug-loaded bCN micelles before and after lyophilization and resuspension shows no change in the overall electro-kinetic potential suggesting good stability of the colloidal dispersion. The results from three independent experiments are presented as mean values ± S.D.

Fig. 4

(A) Cryo-TEM images of bCTE (1:8:8 protein:drugs mole ratio): (A1) fresh and (A2) after freeze-drying and resuspension to the original concentration. (B) Cryo-TEM images of bCDER (1:8:8 protein:drugs mole ratio): (B1) fresh and (B2) after freeze-drying and resuspension to the original concentration.

Fig. 5

(A) Cryo-TEM shows uniform bCDER micelles (1:8:6:1, protein:drugs mole ratio) after lyophilization and resuspension to a 5-fold higher concentration (50 mg mL⁻¹) than the original one. (B) The colloidal dispersion remains transparent.

Fig. 6

Representative XRD spectra of free drugs (A to D) compared to bCN (E) and bCN with encapsulated drugs (F to G). (A) EFV, (B) TRP, (C) DRV, (D) RTV (E) bCN micelles, (F) bCTE, and (G) bCDER. Measurements clearly show no peaks of drugs in the presence of bCN micelles indicating encapsulated drugs in an amorphous form.

Fig. 7

LM micrographs of free drug combinations (A1) TPV:EFV and (B1) DRV:EFV:RTV showing dissolution occur at acidic pH 2.0 and (A2) TPV:EFV and (B2) DRV:EFV:RTV showing reprecipitation at neutral pH 6.5. Cuvettes showing appearance of dispersion in both the conditions. (C) HR-SEM micrographs of (C1) EbCTE and (C2) EbCDER showing spherical round shape microparticles.

Fig. 8

DRV, EFV and RTV release profile from bCDER (solid line) and EbCDER (dotted line) under (A) gastric pH condition; pH 2.0 and (B) intestinal pH condition; pH 6.8. The results from two independent experiments are presented as mean values ± S.D.