Potent Suppression of Prostate Cancer Cell Growth and Eradication of Cancer Stem Cells by CD44-targeted Nanoliposome-quercetin Nanoparticles

Abstract

The bioavailability of quercetin, a natural compound, is hindered by low solubility, limited absorption, and restricted systemic availability. Therefore, encapsulating it in biocompatible nanoparticles presents a promising solution. This study aimed to target prostate cancer stem cells (CSCs) overexpressing CD44+ receptors as well as cancer cells, employing quercetin-loaded hyaluronic acid-modified nanoliposomes (LP-Quer-HA). Synthesized via a green ethanol injection method, these nanoliposomes had an average diameter of 134 nm and an impressive loading efficiency of 96.9%. Human prostate cancer cells were treated with either 10 μM of free quercetin or the same concentration delivered by LP-Quer-HA for 72 hours. Free quercetin reduced androgen-resistant PC3 cell viability by 16%, while LP-Quer-HA significantly increased cell death to 60%. It induced apoptosis, upregulating cytochrome c, Bax, caspases 3 and 8, and downregulating survivin and Bcl-2 expression. Compared to free quercetin, LP-Quer-HA upregulated E-cadherin expression while inhibiting cell migration and reducing the expression of fibronectin, N-cadherin, and MMP9. Treatment of PC3 cell tumor spheroids with LP-Quer-HA decreased the number of CD44 cells and expression of CD44, Oct3/4 and Wnt. Moreover, LP-Quer-HA inhibited p-ERK expression while increasing p38/MAPK and NF-κB protein expression. In androgen-sensitive LNCaP cells, LP-Quer-HA efficacy was notable, reducing cell viability from 10% to 52% compared to free quercetin. Utilizing HA-modified nanoliposomes as a quercetin delivery system enhanced its potency at lower concentrations, reducing the CD44+ cell population and effectively impeding prostate cancer cell proliferation and migration. These findings underscore the potential of quercetin-loaded cationic nanoliposomes as a robust therapeutic approach.

Keywords: Liposomes; Nanoparticles; Neoplasms; Prostate cancer; Quercetin; Therapeutic use.

Figure 1. The physicochemical characteristics of nanoliposomes.

(A) Scanning electron microscope image of liposomal formulations. (B) Dose-dependent cell viability of PC3 cells 72 hours, incubation performed in the equivalent concentration of Quer loaded HA-modified nanoliposomes. (C) 10 µM Quer and liposomal Quer formulations treated PC3, (D) LNCaP cells, and (E) ARPE-19 cell viability. (F) Microscopic visualization of PC3 cells, 10 µm Quer, liposomal Quer, and HA-modified Quer loaded nanoliposomes after 72 hours incubation period. (G) Relative survivin mRNA expression levels were determined by RT-qPCR in PC3 cells. Target gene expressions were normalized to that of GAPDH. UT, untreated; LP, nanoliposomes; LP-HA, hyaluronic acid-modified nanoliposomes; Quer, quercetin; LP-Quer, quercetin-loaded nanoliposomes; LP-Quer-HA, hyaluronic acid-modified, quercetin-loaded nanoliposomes. *P < 0.05 vs. UT, **P < 0.001 vs. all groups, ^‡P < 0.05 vs. LP-HA.

Figure 2. The characterization of liposomal formulations.

Size distribution and zeta potential (A) plots of liposomal formulations. (B) NMR spectra of HA and formulation of LP-Quer-HA. NMR, nuclear magnetic resonance spectroscopy; LP, nanoliposome; LP-Quer-HA, hyaluronic acid-modified, quercetin-loaded nanoliposomes.

Figure 3. Apoptosis induction of liposomal treatment.

(A) Live, dead and apoptotic cell distribution of PC3 cells were quantitatively assessed by annexin V/propidium iodide staining using image-based cytometer. (B) Each column represents the mean ± regulation of apoptotic genes. All data were performed in triplicate and values are means of the two different experiments ± standard deviation. Apoptotic cells detected by Hoechst staining. (C) RT-qPCR mRNA expressions of BAX, APAF, Caspase 3, Caspase 8 were analyzed 72 hours after treatment. (D) Treatment cells with liposomal Quer significantly inhibited the expression levels of antiapoptotic Bcl-2 protein, and (E) upregulated apoptotic cytochrome c protein. UT, untreated; LP, nanoliposomes; LP-HA, hyaluronic acid-modified nanoliposomes; Quer, quercetin; LP-Quer, quercetin-loaded nanoliposomes; LP-Quer-HA, hyaluronic acid-modified, quercetin-loaded nanoliposomes. *P < 0.001 vs. UT, ^#P < 0.001 vs. LP-Quer-HA, ^‡P < 0.001 vs. Quer, **P < 0.001 vs. all groups.

Figure 4. Hyaluronic acid-modified nanoliposomes effects on stemness properties.

(A) Number of CD44+ cancer stem cells in Quer alone and HA-modified liposomal Quer treated cells were counted after magnetic separation. (B) Spheroid formation assay performed in serum-free media and captured (×10) at the end of the incubation period. (C) LP-Quer-HA suppresses CD44 and (D) Oct3/4 mRNA expression. RT-qPCR mRNA expressions were performed triplicate and GAPDH was used as housekeeping gene. Western blot results indicated that liposomal Quer treated PC3 cells shown less Wnt (E) protein expression than Quer alone. Densitometry analysis performed to quantify the results and, beta actin used as loading control. UT, untreated; LP, nanoliposomes; LP-HA, hyaluronic acid-modified nanoliposomes; Quer, quercetin; LP-Quer, quercetin-loaded nanoliposomes; LP-Quer-HA, hyaluronic acid-modified, quercetin-loaded nanoliposomes. *P < 0.001 vs. UT, **P < 0.001 vs. all groups, ^‡P < 0.05 vs. LP-HA.

Figure 5. Effect of liposomal Quer on cell migration and protein expression.

PC3 cell migration was determined by wound healing assay during 30 hours treatment in serum-free medium. (A) Hyaluronic acid-modified and non-modified liposomal Quer inhibited cell migration (×10). (B) The migration ratio (%) was calculated by measuring the wound gaps. Protein lysates were analyzed by Western blotting. Beta-actin was used as loading control. (C) Protein expression E-Cadherin, (D) N-Cadherin, (E) fibronectin, and (F) MMP-9 given as fold changes of each protein compared to loading control. The intensities of immunoreactive bands were quantified by densitometric analysis. UT, untreated; LP, nanoliposomes; LP-HA, hyaluronic acid-modified nanoliposomes; Quer, quercetin; LP-Quer, quercetin-loaded nanoliposomes; LP-Quer-HA, hyaluronic acid-modified, quercetin-loaded nanoliposomes. *P < 0.01 vs. UT, **P < 0.001 vs. all groups, ^‡P < 0.05 vs. Quer.

Figure 5. Effect of liposomal Quer on cell migration and protein expression.

PC3 cell migration was determined by wound healing assay during 30 hours treatment in serum-free medium. (A) Hyaluronic acid-modified and non-modified liposomal Quer inhibited cell migration (×10). (B) The migration ratio (%) was calculated by measuring the wound gaps. Protein lysates were analyzed by Western blotting. Beta-actin was used as loading control. (C) Protein expression E-Cadherin, (D) N-Cadherin, (E) fibronectin, and (F) MMP-9 given as fold changes of each protein compared to loading control. The intensities of immunoreactive bands were quantified by densitometric analysis. UT, untreated; LP, nanoliposomes; LP-HA, hyaluronic acid-modified nanoliposomes; Quer, quercetin; LP-Quer, quercetin-loaded nanoliposomes; LP-Quer-HA, hyaluronic acid-modified, quercetin-loaded nanoliposomes. *P < 0.01 vs. UT, **P < 0.001 vs. all groups, ^‡P < 0.05 vs. Quer.

Figure 6. The effects of liposomal Quer on different signaling pathways.

PC3 cells were treated with liposomal Quer, HA-modified liposomal Quer and, free Quer (10 µM) for 72 hours, isolated protein samples were then subjected to Western blotting. (A) Liposomal Quer regulates p38 MAPK, (B) p-ERK, (C) NF-ĸB p105, and (D) NF-ĸB p50. UT, untreated; LP, nanoliposomes; LP-HA, hyaluronic acid-modified nanoliposomes; Quer, quercetin; LP-Quer, quercetin-loaded nanoliposomes; LP-Quer-HA, hyaluronic acid-modified, quercetin-loaded nanoliposomes. *P < 0.001 vs. UT, ^#P < 0.001 vs. LP-Quer-HA, ^‡P < 0.001 vs. Quer, **P < 0.001 vs. all groups.