Novel Nanotherapeutic Systems Based on PEGylated Squalene Micelles for Enhanced In Vitro Activity of Methotrexate and Cytarabine

Abstract

Nanomedicine has garnered significant attention due to the advantages it offers in the treatment of cancer-related disorders, some of the deadliest diseases affecting human lives. Conventional medication formulations often encounter issues of instability or insolubility in biological environments, resulting in low bioavailability. Nanocarriers play a crucial role in transporting and safeguarding drugs at specific sites of action, enabling gradual release under particular conditions. This study focuses on methotrexate (MTx) and cytarabine (Cyt), essential antitumoral drugs, loaded into PEGylated squalene micellar structures to enhance therapeutic effectiveness and minimize drawbacks. The micelles were prepared using ultrasound-assisted methods in both water and phosphate buffer saline solutions. Evaluation of drug-loaded micelles encompassed parameters such as particle size, colloidal stability, surface charge, morphology, encapsulation efficiency, drug loading capacity, and in vitro release profiles under simulated physiological and tumoral conditions. In vitro cell inhibition studies conducted on MCF-7 and HeLa cell lines demonstrated higher antitumoral activity for the drug-encapsulated micelles compared to free drugs. The encapsulation effectively addressed the burst effect, providing sustained release for at least 48 h while enhancing the drug's protection under physiological conditions.

Keywords: HeLa; MCF-7; PEGylated squalene; antitumoral; controlled release; cytarabine; drug delivery; methotrexate; micelles.

Scheme 1

Synthesis pathway of the SQ-PEG₁₅₀₀-NH-Boc copolymer.

Figure 1

Overlapping ¹H-NMR (CDCl₃, 400 MHz) spectra of (a) SQ-CHO; (b) SQ-COOH; (c) H₂N-PEG₁₅₀₀-NH₂; (d) H₂N-PEG₁₅₀₀-NH-Boc; (e) SQ-PEG₁₅₀₀-NH-Boc.

Figure 2

Overlapping ATR-FTIR spectra of (a) SQ-COOH; (b) H₂N-PEG₁₅₀₀-NH-Boc; (c) SQ-PEG₁₅₀₀-NH-Boc. Where 1: νN-H; 2: νC-H (alkyl); 3: νC=O; 4: νC-O (carboxyl); 5: νC-N; 6: νC-O (ether); 7: overlapping of νO-H and νN-H.

Figure 3

CMC study of SQ-PEG₁₅₀₀-NH-Boc in PBS solutions with a pH value of 7.4 in the presence of pyrene using the spectrofluorimetric method. (a) Normalized emission spectrum of pyrene in an aqueous solution; (b) normalized emission spectra of pyrene in the presence of different concentrations of SQ-PEG₁₅₀₀-NH-Boc (ranged between 3 × 10⁻⁸ M and 5 × 10⁻³ M), λ_ex = 334 nm; (c) Boltzmann sigmoidal fitting of I_E/I₃ as a function of logC (mg/mL, concentration of SQ-PEG₁₅₀₀-NH-Boc).

Scheme 2

Schematic representation of SQ-PEG₁₅₀₀-NH-Boc micelles during the deconstruction-reformation process (green is squalene moiety and red is PEG).

Figure 4

Colloidal features of SQ-PEG₁₅₀₀-NH-Boc micelles in PBS with a pH of 6.5 and 7.4: (a) hydrodynamic diameter distributions; (b) average zeta potential plots. The mass concentration of the SQ-PEG₁₅₀₀-NH-Boc copolymer was 1.25 mg/mL at 23 °C.

Figure 5

STEM morphological analysis of SQ-PEG₁₅₀₀-NH-Boc: (a) STEM image of SQ-PEG₁₅₀₀-NH-Boc micelles at 1 µm scale; (b) size distributions plot of SQ-PEG₁₅₀₀-NH-Boc micelles (n = 120 micelles).

Scheme 3

Schematic representation of the experimental procedure for obtaining the MTx-loaded micelles in an aqueous solution by the nanoprecipitation method. Where SQ-PEG = SQ-PEG₁₅₀₀-NH-Boc.

Scheme 4

Schematic representation of the experimental procedure for obtaining the Cyt-loaded micelles in aqueous solution by the nanoprecipitation method. Where SQ-PEG = SQ-PEG₁₅₀₀-NH-Boc.

Figure 6

Overlapping the absorbance spectra recorded in the PBS solutions with a pH value of 7.4: (a) absorbance spectra of unloaded SQ-PEG₁₅₀₀-NH-Boc micelles, free MTx and MTx-loaded SQ-PEG₁₅₀₀-NH-Boc micelles; (b) absorbance spectra of unloaded SQ-PEG₁₅₀₀-NH-Boc micelles, free Cyt and Cyt-loaded SQ-PEG₁₅₀₀-NH-Boc micelles.

Figure 7

DLS analysis of drug-loaded SQ-PEG₁₅₀₀-NH-Boc micelles at 1.25 mg/mL in PBS with pH values of 6.5 and 7.4 at an ambient temperature of 25 °C. (a) Particle size distributions of MTx-loaded SQ-PEG₁₅₀₀-NH-Boc micelles; (b) particle size distributions of Cyt-loaded SQ-PEG₁₅₀₀-NH-Boc micelles; (c) zeta potentials of MTx-loaded SQ-PEG₁₅₀₀-NH-Boc micelles; (d) zeta potentials of Cyt-loaded SQ-PEG₁₅₀₀-NH-Boc micelles. Data are presented as mean ± SD (n = 3).

Figure 7

DLS analysis of drug-loaded SQ-PEG₁₅₀₀-NH-Boc micelles at 1.25 mg/mL in PBS with pH values of 6.5 and 7.4 at an ambient temperature of 25 °C. (a) Particle size distributions of MTx-loaded SQ-PEG₁₅₀₀-NH-Boc micelles; (b) particle size distributions of Cyt-loaded SQ-PEG₁₅₀₀-NH-Boc micelles; (c) zeta potentials of MTx-loaded SQ-PEG₁₅₀₀-NH-Boc micelles; (d) zeta potentials of Cyt-loaded SQ-PEG₁₅₀₀-NH-Boc micelles. Data are presented as mean ± SD (n = 3).

Figure 8

STEM morphological analysis of drug-loaded SQ-PEG₁₅₀₀-NH-Boc micelles. (a) STEM image of MTx-loaded micelles; (b) STEM image of Cyt-loaded micelles and their size distribution plots; (c) size distribution of MTx-loaded micelles (n = 120 micelles); (d) size distribution of Cyt-loaded micelles (n = 120 micelles).

Figure 8

STEM morphological analysis of drug-loaded SQ-PEG₁₅₀₀-NH-Boc micelles. (a) STEM image of MTx-loaded micelles; (b) STEM image of Cyt-loaded micelles and their size distribution plots; (c) size distribution of MTx-loaded micelles (n = 120 micelles); (d) size distribution of Cyt-loaded micelles (n = 120 micelles).

Figure 9

In vitro cumulative release of MTx from the free MTx solution and MTx-encapsulated SQ-PEG₁₅₀₀-NH-Boc micelles. (a) In simulated physiological conditions (PBS with pH of 7.4 and 37 °C); (b) in simulated tumoral conditions (PBS with pH 6.5 and 39 °C). The experiment was carried out for 9 days, and the results are expressed as means ± SEM (n = 3). * p < 0.05, ** p < 0.01, and # p > 0.05 (ns) by Student’s t-test.

Figure 10

In vitro cumulative release of Cyt from the free Cyt solution and Cyt-encapsulated SQ-PEG₁₅₀₀-NH-Boc micelles. (a) In simulated physiological conditions (PBS with pH of 7.4 and 37 °C); (b) in simulated tumoral conditions (PBS with pH 6.5 and 39 °C). The experiment was carried out for 15 days, and the results are expressed as means ± SEM (n = 3). ** p < 0.01, *** p < 0.001, by Student’s t-test.

Figure 11

In vitro antitumor activity of free MTx and MTx-loaded SQ-PEG₁₅₀₀-NH-Boc micelles and unloaded SQ-PEG₁₅₀₀-NH-Boc micelles at various concentrations of MTx against (a) MCF-7 cells and (b) HeLa cells after incubation for 72 h. Results are reported as % of cell inhibition based on the untreated control cells normalized to 0% and are expressed as means ± SD (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001, and # p > 0.05 (ns) (for MTx-loaded micelles vs. free MTx) and * p < 0.05, ** p < 0.01, *** p < 0.001 (for MTx-loaded micelles vs. unloaded micelles) by Student’s t-test.

Figure 12

In vitro antitumor activity of free Cyt, Cyt-loaded SQ-PEG₁₅₀₀-NH-Boc micelles, and unloaded SQ-PEG₁₅₀₀-NH-Boc micelles at various concentrations of Cyt against (a) MCF-7 cells and (b) HeLa cells after incubation for 72 h. Results are reported as % of cell inhibition based on the untreated control cells normalized to 0% and are expressed as means ± SD (n = 3). ** p < 0.01, *** p < 0.001, and # p > 0.05 (ns) (for Cyt-loaded micelles vs. free Cyt) and * p < 0.05, ** p < 0.01, *** p < 0.001, and # p > 0.05 (ns) (for Cyt-loaded micelles vs. unloaded micelles) by Student’s t-test.