Chemotherapeutic Drug Delivery Nanoplatform Development: From Physicochemical to Preclinical Evaluation

Abstract

Through this study, the synergistic behavior of small-molecular-weight, amphiphilic surfactant molecules and the triblock copolymer Pluronic 188 was extensively evaluated based on their ability to formulate nanocarriers with novel properties for the delivery of class II and IV (biopharmaceutical classification system) chemotherapeutic compounds. The combination of four different surfactants at multiple weight ratios and twelve initially formulated nanosystems resulted in four hybrid delivery platforms, which were further studied in terms of multiple physicochemical characteristics, as well as their stability in protein-rich media (fetal bovine serum/phosphate-buffer saline). Finally, we obtained a single final nanoformulation that exhibited a high loading capacity (%EE ≥ 75%) and a sustained drug release profile under physiological conditions (model drug methotrexate), without altering the original physicochemical characteristics of the carrier. With a mean hydrodynamic radius (Rh) of less than 70 nm, a polydispersity index of 0.219, and no protein complexation, the system is a suitable candidate for in vivo, intravenous, and/or intramuscular administration. The cytotoxicity and genotoxicity of both loaded and unloaded carriers were evaluated through the examination of the upregulation or downregulation of apoptosis-related pathways. Multiple conventional 2D and 3D spheroidal conformations were used for these assessments, including HEK293, HCT-116, and MCF-7 cell lines, the results of which stressed the safety and biocompatibility of the empty nanocarrier. Additionally, experiments on Caenorhabditis elegans were conducted to evaluate the system's in vivo toxicity, focusing on developmental stages, egg-laying behavior, and locomotion. Nanosystems studied in terms of chemotherapeutic encapsulation have mostly focused on the physiochemical aspect of the development of such novel delivery platforms, with only few exceptions proceeding step-by-step from cellular 2D to 3D to in vivo experimentation. The present study offers a holistic view of the behavior of such a novel system, advancing our understanding of the capabilities of polymeric/surfactant-based nanodelivery platforms.

Keywords: 3D cell culture; C. elegans; MTS assay; Pluronic 188; real-time PCR; thin-film hydration.

Figure 1

Comparative size distribution graphs of Pluronic 188–Tween 80 (90:10) nanosystems with MTX encapsulated from 0.1 mg/mL up to 0.3 mg/mL and the filtered unloaded system of Pluronic 188–Tween 80 (90:10).

Figure 2

Size distribution graphs after incubation in physiological conditions. (A) The system’s stability assessment after 1 h incubation. (B) Incubation in fetal bovine serum where after 1 h the system was continuously monitored.

Figure 3

In vitro drug release study of MTX (303 nm) in PBS media at 37 °C.

Figure 4

HEK293 adhesive cell line viability vs. different concentrations of the Pluronic 188–Tween 80 nanosystem, along with free methotrexate at equal concentrations as encapsulated in nano-dispersions. Each graph accounts for the dispersion of the viability percentage via the standard deviation. Results after a 24 h incubation.

Figure 5

Cell viability vs. different concentrations of the Pluronic 188–Tween 80 nanosystem, along with free methotrexate at equal concentrations as encapsulated in nano-dispersions: (A) HCT-116 adherent human colon cancer cell line, (B) MCF-7 adherent human breast adenocarcinoma cell line, and (C) SK-BR3 estrogen-independent adherent human breast cancer cell line.

Figure 5

Cell viability vs. different concentrations of the Pluronic 188–Tween 80 nanosystem, along with free methotrexate at equal concentrations as encapsulated in nano-dispersions: (A) HCT-116 adherent human colon cancer cell line, (B) MCF-7 adherent human breast adenocarcinoma cell line, and (C) SK-BR3 estrogen-independent adherent human breast cancer cell line.

Figure 6

Relative viability (%) vs. the concentration (μg/mL) of (A) Pluronic 188–Tween 80–MTX (9:1:0.2)-loaded micelles in multiple cell line 2D cultures and (B) equal concentration of free MTX.

Figure 7

Cell viability expressed for the same nanosystem in relation to cell type incubation. Results after 24 h exposure, with concentrations ranging from 25 μg/mL to 200 μg/mL: (A) Pluronic–Tween 80 (90:10) filtered, unloaded nanosystems; (B) Pluronic–Tween 80 (90:10) nanosystems loaded with 0.2 mg/mL MTX; and (C) incubation with free MTX.

Figure 7

Cell viability expressed for the same nanosystem in relation to cell type incubation. Results after 24 h exposure, with concentrations ranging from 25 μg/mL to 200 μg/mL: (A) Pluronic–Tween 80 (90:10) filtered, unloaded nanosystems; (B) Pluronic–Tween 80 (90:10) nanosystems loaded with 0.2 mg/mL MTX; and (C) incubation with free MTX.

Figure 8

Cell culture viability vs. different concentrations of Pluronic 188–Tween 80 filtered nanosystems, loaded with the antitumor compound, methotrexate, and unloaded. The results shown are for HCT-116 3D spheroidal culture.

Figure 9

Fold change in mRNA levels in Casp3 and IL6 in a 2D culture of MCF-7 breast cancer adherent cell line for Pluronic 188–Tween 80 hybrid nanosystems, both loaded (right) and unloaded (left).

Figure 10

Representative plate images of (A) control (1) plate and (B) egg laying when nematodes where exposed to Pluronic 188–Tween 80–MTX nanosystems at a concentration of 150 μg/mL.

Figure 11

Toxicity assessment in 1-day and 5-day adult wild-type nematodes.