(M1130-05-30) Colon Targeted Delivery of Nanosuspension Coated Granules of Albendazole tor the Localized Treatment of Colorectal Cancer

Purpose: Albendazole (ABZ) has been reported to have the activity against different types of cancers. However, poor water solubility and bioavailability limit its application as a candidate. Therefore, colon targeted delivery ABZ was developed using nanocrystal technology to improve drug effectiveness at the desired site of action. The purpose of current study was to improve dissolution rate and anticancer effect by formulating nano coated granules (NCG) of Albendazole nanosuspension (NS) and developing colon targeted delivery system via enteric-protected capsules EUDRACAP^®. Therefore, the objectives of the study are: (a) to formulate and characterize ABZ NS; (b) to prepare ABZ NCG and use EUDRACAP^® for colon targeted delivery; and (c) to evaluate ABZ NS anticancer effect against colorectal cancer cell lines.

Methods: ABZ NS was prepared using dual centrifugation nano-milling approach. In brief, 7.5% (w/v) ABZ was dispersed in sterile water containing different combinations of 1% (w/v) polymer and 0.1% (w/v) surfactant. The cooling temperature and milling speed were set at -15 °C and 1500 rpm, respectively, with 10 g of milling beads added in each milling trial. The milling time (1 or 2 h) and bead size (0.3 or 0.8 mm) were also optimized (Fig. 1). ABZ nanosuspension coated granules (ABZ NCG) were prepared in a Pan coater coating machine using a specialized misting spray nozzle. The coating process consisted of 35 g of MCC powder as a carrier, which was pre-heated for 30 minutes prior to coating process. Multiple layers of coating containing 80 mL of ABZ NS were sprayed on MCC at a rate of 5 mL/min using the misting nozzle to obtain a continuous coating membrane. An air blower with a temperature of 45 ± 2°C and exhaust temperature of 25 ± 1°C facilitated continuous drying of the nanosuspension-layered granules during the coating process. The nanosuspension coated granules were allowed to cool down for 30 minutes. Subsequently, dried ABZ NCGs passed through a #40 mesh size sieve but retained on a #20 mesh size sieve were collected for further studies. The prepared NS was characterized for the particle size, PDI and zeta potential. DSC and FT-IR were applied to characterize the solid-state properties of ABZ and freeze-dried ABZ NS batches. Scanning electron microscopy (SEM) images of ABZ, MCC and ABZ NCG were captured to observe the morphology. Dissolution studies of pure ABZ, ABZ NS and ABZ NCG were performed in 500 mL of phosphate buffer medium at pH 7.2. ABZ NCG was loaded in EUDRACAP^® to mimic colon targeted delivery. Colorectal cancer cell lines HT-29 and HCT-116 were selected to evaluate the cytotoxicity of the formulation, and 3D multicellular tumor spheroids was developed for the simulation of the in vivo solid tumor to address the complex nature of tumor physiology.

Results: After screening, Kollidon^® VA64 and SLS were found to be the best stabilizers for ABZ with particle size of 264.0 ± 15.0 nm, PDI of 0.194 ± 0.02 and zeta potential of -23.8 ± 0.57 mV (Fig. 2B). Besdies, longer milling time and smaller bead size did not decrease the particle size significantly (Fig. 2A). The drug content assay for NS was approximately 91.2% and post-granulation was approximately 96.6%. In vitro drug release data shows ~60% drug release of ABZ NS and NCG in colonic pH compared to no drug release for pure ABZ (Fig. 2E). Material Charecterization data indicated the crystallinity and integrity of ABZ NS (Fig. 2C). SEM images revealed the spherical morphology of granules (Fig. 2D). In cell cytotoxicity studies, IC₅₀ of ABZ NS was found to be significantly lower than ABZ PM in HT-29 and HCT-116 (Fig. 3A). Both VA64 and SLS displayed similar cellular morphology as control, implying their safety (Fig. 3B). Furthermore, 1.68 folds reduction in the size of 3D tumor spheroids after treatment with ABZ NS was observed compared to control. In Vitro Live/Dead Cell Assay confirmed a large number of dead cells in treatment groups compared to the control through red fluorescence (Fig. 3C).

Conclusion: In summary, ABZ NS was successfully developed with Kollidon^® VA64 and SLS as stabilizers using dual centrifugation method. The dissolution of ABZ NS and NCG in colonic pH was significantly enhanced compared to the pure ABZ. EUDRACAP^® can be utilized to prevent drug release in acidic upper GI tract and to ensure colon targeted delivery as a pH-dependent delivery system. 2D assay and 3D tumor spheroids assay confirmed its anticancer effect against colorectal cancer cell lines HT-29 and HCT-116. This study suggests that repurposing ABZ by developing nano-formulation offers an appealing approach that combines improved dissolution rate with targeted anti-colorectal cancer effects using EUDRACAP^®.


Fig. 1 Graphical representation demonstrating the preparation of Albendazole nanosuspension using dual centrifugation nano-milling for the treatment of colorectal cancer.


Fig. 2 (A) Effect of stabilizers (1% w/v), surfactants (0.1% w/v), bead size (0.3 mm and 0.8 mm) and milling time (1 h and 2 h) on the particle size and PDI. (B) DLS graphs demonstrating (a) particle size and (b) Zeta potential of the optimized ABZ NS; (c) 6 months stability study of ABZ NS stored at room temperature. (C) Solid state characterization of pure ABZ, Kollidon® VA64 and ABZ NS by (a) differential scanning calorimetry and (b) FT-IR spectra. (D) SEM images of (a) pure ABZ; (b) pure MCC; (c) ABZ NCG at 200x magnification; (d) ABZ NCG at 1000x magnification. (E) In-vitro drug release of pure ABZ, ABZ NS (7.5% w/v) and ABZ NCG with or without bile salt (0.2% w/v) in pH 7.2 medium under sink condition.


Fig. 3 (A) IC50 of ABZ, ABZ PM and ABZ NS in HT-29 and HCT-116 colorectal cancer cell lines. (B) Crystal violet images of VA64 and SLS treated (a) HT-29 cells lines and (b) HCT-116 cell lines. 1% (w/v) VA64 and 0.1% (w/v) SLS treated groups exhibited comparable cellular morphology to the control group, indicating their non-toxic property at this concentration. Scale bar: 400 μm. (C) 3D multicellular tumor spheroids of colorectal cancer cell line HCT-116. (a) Images of spheroids at day 0, 2, 4, 6, 8 and 10 days treated with ABZ and ABZ NS; (b) Area of spheroids with ABZ and ABZ NS treatments over 10 days (n=3). ****p < 0.0001; (c) Fluorescence images of HCT-116 spheroids treated with ABZ and ABZ NS. Scale bar: 400 μm.