Unlocking the Potential of Camel Milk-Derived Exosomes as Novel Delivery Systems: Enhanced Bioavailability of ARV-825 PROTAC for Cancer Therapy

Abstract

This study investigates the use of camel milk-derived exosomes (CMEs) as carriers for ARV-825, an anticancer agent targeting bromodomain-containing protein 4 (BRD4), in oral chemotherapy. CMEs were isolated and characterized, and ARV-825-loaded CME formulations were prepared and evaluated through various in vitro and in vivo tests. The ARV-825-CME formulation exhibited an entrapment efficiency of 42.75 ± 5.05%, a particle size of 136.8 ± 1.94 nm, and a zeta potential of -32.75 ± 0.70 mV, ensuring stability and sustained drug release. In vitro studies showed a 5.4-fold enhancement in drug release kinetics compared to the free ARV-825 solution. Permeability studies indicated a 3.2-fold increase in apparent permeability, suggesting improved cellular uptake. Cytotoxicity assays demonstrated potent anticancer activity, with IC50 values decreasing by 1.5 to 2-fold in cancer cell lines SF8628 DIPG and H1975R (resistant to Osimertinib). In vivo pharmacokinetic studies in Sprague-Dawley rats revealed superior systemic absorption and bioavailability of ARV-825 from CMEs, with a 2.55-fold increase in plasma concentration and a 5.56-fold increase in AUC. Distribution studies confirmed absorption through the ileum. This research highlights the potential of CMEs as a promising delivery platform for ARV-825, enhancing its therapeutic efficacy and offering a novel approach to cancer treatment.

Keywords: ARV-825; anticancer; camel milk exosomes; oral bioavailability.

Figure 1

Nanoparticle tracking analysis and Western blotting of exosomes. (A) Overlap of particle size distribution graphs of exosomes in scatter and fluorescence mode. (B) Zeta potential curve of exosomes and ARV-825-CME. (C) Particle size distribution graph of control exosomes before formulation. (D) Particle size distribution graph of ARV-825-CME. (E) Western blots analysis of exosomes showing expression of proteins HSP70, Alix, CD63 and CD81. (F) TEM image of CME, (G) TEM image of ARV-825-CME.

Figure 2

Proteomics analysis of protein cargo of CMEs. (A) Principal component analysis (PCA) plot for CMEs compared to Cow and Goat milk; (B) Heatmap of differentially expressed proteins (DEPs); (C) Volcano plot of DEPs for Camel milk group compared to the Goat milk group; (D) Volcano plot of DEPs for Camel milk group compared to the Cow milk group; (E) Venn Diagram of all the identified proteins in CMEs compared to Cow and Goat milk. (F) Heatmap of overlapped proteins.

Figure 3

(A) An in vitro release study in a pH 7.4 release medium showed significantly increased drug release from the ARV-825-CME formulation compared to the control ARV-825 solution placed in a dialysis bag. (B) In vitro cell permeability assay showing enhanced apparent permeability coefficient (Papp) of ARV-825-CME as compared to ARV-825 solution (control). Results were expressed as Mean ± SD (n = 3). **** p < 0.0001. (C) In vitro release study in pH 1.2.

Figure 4

(A) In vitro cytotoxicity studies. Line graph showing concentration-dependent cell viability of H1975R lung cancer cells resistant to Osimertinib and SF8628 DIPG cells when treated with CMEs (0–2 × 10¹¹ particles/mL) in 2D and 3D cultures. Results were expressed as % in Mean ± SD (n = 4). (B) ARV-825’s IC₅₀ in H1975R and SF8628 cells in 2D and 3D cultures when treated with ARV-825 solution and ARV-825-CME formulation.

Figure 5

Microscopic (cryosection) images of H1975-R tumors showing internalization of exosomes by the tumor 4 h after oral administration in mice of (A) PBS, fluorescent, (B) PBS, brightfield, (C) SYTO RNASelect stained exosomes, fluorescent, and (D) SYTO RNASelect stained exosomes, brightfield. The scale bar represents 10 µm.

Figure 6

(A) The pharmacokinetic study graph shows significantly increased T_1/2, T_max, C_max, AUC, and MRT in the ARV-825-CME group compared to the control ARV-825 solution group. (B) Table showing the PK data for parameters T_1/2, T_max, C_max, AUC, MRT, Vd, and CL for ARV-825 solution vs. ARV-825-CME. Results were expressed as Mean ± SD (n = 5).

Figure 7

(A) Microscopic fluorescent images of intestinal tissue sections showing bright fluorescence at 0.5, 1, 4, and 4 h for duodenum, ileum, jejunum, and plasma samples, respectively, after administration of stained exosomes. The scale bar represents 20 µm. (B) Graph showing fluorescence intensities of intestinal and plasma samples from 0 to 4 h after administration of stained exosomes. (C,D) Graphs showing ARV-825 amount (C) and distribution of ARV-825 (D) in intestinal and plasma samples from 0 to 4 h after administration of stained ARV-825-CME. Results were expressed as Mean ± SD (n = 3). ** p < 0.01, * p < 0.05, ns is ‘not significant’, p < 0.001 (if not mentioned in the bar graph (D)).