Unlocking Nature's Potential: Ferritin as a Universal Nanocarrier for Amplified Cancer Therapy Testing via 3D Microtissues

Abstract

In the existing development of extensive drug screening models, 3D cell cultures outshine conventional 2D monolayer cells by closely imitating the in vivo tumor microenvironment. This makes 3D culture a more physiologically relevant and convenient system in the regime of preclinical drug testing. In the nanomedicinal world, nanoconjugates as nanocarriers are largely hunted due to their capability of precisely binding to target cells and distributing essential dosages of therapeutic drugs with enhanced safety profiles. Thus, for boosted drug availability, the evolution from conventional drug treatment to combination therapies and last switching to drug carriers has gained significant progression in cancer cure. In contrast to conventional engineered nanoparticles, herein, we successfully designed biomolecule (ferritin)-based drug nanoconjugates effective both as a single drug (valproic acid-VPA) and twin-drug (valproic acid/doxorubicin-Dox) carriers, which dramatically enhance the proficiency of the tumor therapeutic modality. To question the reported adjuvant drug property of VPA, we progressed utilizing at first VPA alone as an effective yet exclusive tumor therapy when delivered via some carrier molecule, in particular protein. Subsequently, we paralleled this comprehensive investigation output to compare and test the coloading strategy of drugs and observe the synergistic and/or additive behavior of VPA in conjugation with other anticancer agents (Dox) while given via a carrier molecule. To approach this, VPA and/or Dox molecules were encapsulated into the ferritin (F) cavity using a thermosensitive synthesis method by maintaining the temperature at 60 °C. The successful encapsulation of drugs in the protein nanocage was confirmed through various characterization techniques. The F-VPA/F-VPA-Dox nanoconjugates exhibited similar morphology and structural characteristics to the hollow ferritin cage and showed significant cytotoxicity than the naked drugs when tested on physiologically relevant 3D spheroid models. Precisely, our first designed carrier nanoconjugate, i.e., F-VPA, offered more than a 3-fold increased intratumoral drug concentration than free VPA and significantly suppressed tumor growth after a single-dose treatment. However, our second modeled carrier nanoconjugate, viz. F-VPA-Dox, revealed an extended median survival period and lesser toxicity when administered at a much more effective dose (∼3-5 μM), in 3D tumor spheroid models of various cancer cell lines. All in all, importantly, ferritin nanoconjugates exhibited an enhanced tumor inhibition rate with a single-dose treatment, which further confirms the benefits of the active targeting property of these nanocarriers. Moreover, these nanocarriers also offer to deliver a significant dose of the therapeutic drug into tumor cells, alongside tremendous biocompatibility and safety profiles in numerous tumor 3D spheroid models.

Keywords: 3D microtissues; drug nanoconjugate; ferritin; nanocarrier materials; protein; spheroids.

Scheme 1. Route of Protein–Drug Nanoconjugate Synthesis Using Thermal-Induced Switch of the Drug Entry Channel in Ferritin Nanocages and Tumor Killing Mechanism

Figure 1

Characterization of F-VPA and F-VPA-Dox nanoconjugates. (a) Size distribution and (b) zeta potential of ferritin alone, F-VPA, and F-VPA-Dox determined by DLS, (c) TEM image of the synthesized nanoconjugate F-VPA, (d) the mean graphical size obtained from TEM images using the ImageJ software for F-VPA and (e,f) likewise for F-VPA-Dox, respectively, (g) UV–visible spectrophotometric analysis of F-VPA and (h) F-VPA-Dox, (i) size exclusion chromatography (SEC) to confirm the drug conjugation with protein (ferritin), and (j,k) circular dichroism (CD) spectroscopic analysis of ferritin alone, F-VPA, and F-VPA-Dox nanoconjugates.

Figure 2

(a) Drug loading of VPA in F-VPA at increasing concentrations, (b) drug loading of VPA at an increasing concentration (0.2–1 mM) and Dox at a constant concentration of 0.7 mM in F-VPA-Dox, (c) in vitro release analysis of the F-VPA nanoconjugate in PBS (pH 7) and glycine buffer (pH 4) over the period of 24 h, (d) cumulative drug release of VPA and Dox from the F-VPA-Dox nanoconjugate at different pH values, (e) circular dichroism indicating structure alteration of ferritin to release VPA in neutral to acidic conditions, and (f) structure alteration in ferritin to release VPA and Dox at different pH values indicated from CD.

Figure 3

Hemolysis test of ferritin–drug nanoconjugates. Images of centrifuged RBC solutions and the percentage hemolysis of F-VPA/F-VPA-Dox after incubation with different concentrations of nanoconjugates. PBS was used as negative control, whereas Triton X-100 and deionized water were used as positive controls.

Figure 4

Optimization of nanoconjugate drug doses (F-VPA and F-VPA-Dox) on 2D monolayer cancer models (a) MCF-7, (b) C4-2, and (c) HT-29, (d) with increasing concentrations. (e) Live–dead cell-stained images with Calcein-AM (green) and PI (red) showing live cells in green and dead cells in red after drug conjugate treatment. (f) Testing the interference of ferritin as a carrier protein with cytotoxic behavior of drug in increasing concentrations.

Figure 5

(a) Schematic of 3D spheroid formation depicting the layout from cell seeding until spheroid formation using customized microwell platforms. (b–d) Optimization of spheroid growth and size with respect to varying cell number seeded for each cell line.

Figure 6

Flow cytometric analysis of viable cells in 3D spheroid models after treatment with drug nanoconjugates for F-VPA and F-VPA-Dox in comparison to alone drug controls (Dox and VPA) for (a) MCF-7, (b) C4-2, and (c) HT-29.

Figure 7

Drug treatment on 3D spheroid models. (a) Schematics of a comparison between 2D monolayer and 3D spheroid model with altered drug penetration to the deep tumor regions. (b) Bright-field imaging of MCF-7 spheroids for 4 consecutive days after drug treatment. (c) Graphical representation of drug effects on spheroid size over the period of 4 days. (d) Live–dead cell-stained images of spheroids at the end of drug treatment showing live cells in the periphery and dead cells in the core.

Figure 8

Bright-field imaging of (a) C4-2 and (d) HT-29 spheroids for 4 consecutive days after drug treatment. (c,f) Live–dead cell- stained images of spheroids at the end of drug treatment showing live cells in the periphery and dead cells in the core; and (b,e) graphical representation of drug effects on spheroid size over the period of 4 days.