Formulation, development and evaluation of hyaluronic acid-conjugated liposomal nanoparticles loaded with regorafenib and curcumin and their in vitro evaluation on colorectal cancer cell lines

Abstract

Colorectal cancer is one of the major causes of global cancer, with chemotherapy and radiation therapy being effective but limited due to low specificity. Regorafenib, a multikinase inhibitor, provides hope to patients with metastatic colorectal cancer and was approved by the FDA in 2012. However, due to resistance issues and adverse events, its efficacy is compromised, necessitating further refinement. Meanwhile, curcumin, a compound of turmeric, exhibits anticancer effects through antioxidant and anti-inflammatory actions, induction of the apoptosis, arrest of cell cycle, inhibition of angiogenesis, and modulation of signaling pathways. Unfortunately, its clinical utility is limited by its poor bioavailability, pointing towards innovative drug delivery strategies for enhanced efficacy in colorectal cancer treatment. Hyaluronic acid (HA)-decorated liposomes (LIPO) have been developed to target colorectal cells through an overexpressed CD44 receptor, increasing antitumor and antimetastasis efficacy. This study investigates the possibility of loading curcumin (CUR) or regorafenib (REGO) into a liposomal formulation for passive and HA-actively targeted treatment, evaluating its critical quality attributes (CQA) (size, zeta potential, polydispersity index) and cytotoxic activity in the HT29 colorectal cancer cell line. The average particle size of the plain liposomes and those decorated with HA was 144.00 ± 0.78 nm and 140.77 ± 1.64 nm, respectively. In contrast, curcumin-loaded plain liposomes and HA-decorated liposomes had 140 ± 2.46 nm and 164.53 ± 15.13 nm, respectively. The prepared liposomes had a spherical shape with a narrow size distribution and an acceptable zeta potential of less than -30 mV. The encapsulation efficiency was 99.2 % ± 0.3 and 99.9 ± 0.2 % for HA-decorated and bare regorafenib loaded. The % EE was 98.9 ± 0.2 % and 97.5 ± 0.2 % for bare liposomal nanoparticles loaded with curcumin and coated with curcumin. The IC₅₀ of free REGO, CUR, REGO-LIPO, CUR-LIPO, REGO-LIPO-HA and CUR-LIPO-HA were 20.17 ± 0.78, 64.4 ± 0.33, 224.8 ± 0.06, 49.66 ± 0.22, 73.66 ± 0.6, and 27.86 ± 0.49 µM, respectively. The MTT assay in HT29 cells showed significant cytotoxic activity of the HA-decorated liposomal formulation compared to the base uncoated formulation, indicating that hyaluronic acid-targeted liposomes loaded with regorafenib or curcumin could be a promising targeted formulation against colorectal cancer cells.

Keywords: CD44; Colorectal cancer; Curcumin; Hyaluronic acid; Liposome; Regorafenib; Targeted drug delivery.

Fig. 1

Structure and mechanism of action. (a) The mechanisms of regorafenib anti-tumor activity. Regorafenib blocks multiple active pathways involved in angiogenesis, tumor cell survival, cell proliferation, differentiation, apoptosis, and extracellular matrix formation. This offers potential explanations for its anti-tumor effectiveness in colorectal cancer. Created with BioRender.com. (b) Chemical structure of regorafenib. Created with chem-space.com. (c) Chemical structure of curcumin. Created with chem-space.com..

Fig. 2

The basic structure and schematic illustration of regorafenib or curcumin-loaded HA-decorated liposomes. The CD44 receptor, which is overexpressed in colorectal tumor cells, is specifically liganded by the HA molecule. Cellular uptake of liposomes was enhanced by CD44-mediated endocytosis. Created with BioRender.com.

Fig. 3

Hyaluronic acid activation and conjugation with DOPE through the formation of amide bonds. Created with chem-space.com.

Fig. 4

Average particle size distribution obtained by DLS. (a) REGO-LIPO, (b) REGO-LIPO-HA, (c) CUR-LIPO, and (d) CUR-LIPO-HA. DLS, dynamic light scattering; HA, hyaluronic acid; REGO, regorafenib; CUR, curcumin; LIPO, liposome. The results are presented as mean ± SD of triplicate measurements.

Fig. 5

Liposomal morphology and encapsulation efficiency. (a) Transmission electron microscopy image of (A) REGO-LIPO, (B) REGO-LIPO-HA, (C) CUR-LIPO, (D) CUR-LIPO-HA. Scale bar: 200 nm, 500 nm. (b) The % encapsulation efficiency values of regorafenib and curcumin’s HA-coated and uncoated liposomal formulations with the results presented as mean ± SD of triplicate measurements.

Fig. 6

Schematic diagram of in vitro release of REGO and CUR from bare-LIPO and LIPO-HA compared with the free drug form at pH 7.4 represented as % leaked at different time points up to 96 h.

Fig. 7

Relative cell viability of HT29 colorectal cancer cells treated with Free REGO, Free CUR, REGO-LIPO, CUR-LIPO, REGO-LIPO-HA and CUR-LIPO-HA for 72 h (cell viability % vs logarithmic molar concentration (μM)). The results are presented as mean ± SD of the measurements in triplicate.

Fig. 8

FT-IR spectrum; transmittance (%T) against the wavenumber (1/cm). [a]: (A) DOPE-HA, (B) DOPE, and (C) HA. [b]: (A) REGO-LIPO-HA, (B) CUR-LIPO-HA, and (C) BLANK-LIPO-HA. The peak at 1635 cm^-1 represents the amide bond formation.