Biodegradable manganese-doped hydroxyapatite antitumor adjuvant as a promising photo-therapeutic for cancer treatment

Abstract

The efficiency of a cancer therapy agent depends on its ability to eliminate tumors without endangering neighboring healthy tissues. In this present study, a novel multifunctional property enriched nanostructured system was synthesized on manganese-doped hydroxyapatite (Mn-HAp) conjugated with counter folic acid (FA) IR-783 fluorescence dye. The tailored synthesis of nano rod-shaped Mn-HAp nanoparticles with high surface area allows to conjugate FA/IR-783 dye which enhanced retention time during in vivo circulation. The drug-free Photothermal Photodynamic therapy mediated cancer treatment permits the prevention of collateral damages to non-cancerous cells. The safe HAp biomaterial matrix allows a large number of molecules on its surface due to its active different charge moieties (Ca²⁺/PO₄ ^3-) without any recurrence toxicity. The doped Mn allows releasing of Mn²⁺ ions which triggered the production of toxic hydroxyl radicals (•OH) via Fenton or Fenton-like reactions to decompose H₂O₂ in the tumor sites. Herein, IR-783 and FA were selected for targeted fluorescence imaging-guided photothermal therapy. 6The PTT performance of synthesized nanostructured system shows enhanced potential with ∼60°C temperature elevation with 0.75 W∙cm^-2 power irradiated within 7 min of treatment. PDT activity was also observed initially with Methylene Blue (MB) as a targeted material which shows a drastic degradation of MB and further in vitro studies with MDA-MB-231 breast cancer cell line show cytotoxicity due to the generated reactive oxygen species (ROS) effect. FA/IR-783 conjugated Mn-HAp nanoparticles (2.0 mol% Mn-HAp/FA-IR-783) show significant tumor-specific targeting and treatment efficiency while intravenously injected in (tail vain) BALB/c nude mice model without any recurrence. The synthesized nanostructured system had ample scope to be a promising Photo-Therapeutic agent for cancer treatment.

Keywords: antitumor adjuvant; cancer treatment; manganese doped hydroxyapatite; photodynamic therapy; photothermal therapy.

FIGURE 1

Schematic representation of fluorescence imaging guided Photothermal Photodynamic therapeutic efficacy of Mn-HAp/FA-IR-783 nanoparticles.

FIGURE 2

(A) XRD analysis of HAp and Mnx-HAp (x = 0.5, 1.0, and 2.0 mol%) nanoparticles (B) UV-Vis Diffusive Reflectance Spectroscopy analysis of HAp and Mnx-HAp (x = 0.5, 1.0, and 2.0 mol%) nanoparticles (C) Raman shift and (D) FTIR analysis of HAp and Mnx-HAp (x = 0.5, 1.0, and 2.0 mol%) nanoparticles (E) Thermogravimetric and differential thermal analysis of HAp and Mnx-HAp (x = 0.5, 1.0, and 2.0 mol%) nanoparticles (F–I) FE-TEM study of HAp and Mnx-HAp (x = 0.5, 1.0, and 2.0 mol%) nanoparticles and (K–N) their corresponding HR-TEM analysis with inter atomic distance measurement (J) EDS mapping for “Mn” (O) EDS mapping for “Ca”.

FIGURE 3

Photodynamic effect of Mn-HAp nanocomposites. (A) UV–vis absorption spectra of Methylene Blue (MB) mixed with HAp and Mnx-HAp/FA-IR-783 (x = 0.5, 1.0, and 2.0 mol%) nanoparticles treatments (Inset: synthesized HAp and Mnx-HAp (x = 0.5, 1.0, and 2.0 mol%) nanoparticles and) (B) PDT effect on MB solution (C) The mechanism of cellular ROS generation due to manganese ions (D) Detection of O₂ production due to the PDT effect (reaction with DCFH-DA) of synthesized nanoparticles (each scale bar is 20 micro meter).

FIGURE 4

(A) MTT assay of HAp and Mnx-HAp/FA-IR-783 (x = 0.5, 1.0, and 2.0 mol%) nanoparticles at different concentration (50–250 μg/ml) with MDA-MB-231 breast cancer cell line. (B) FACS study of HAp, 2.0 mol %Mn-HAp/FA-IR-783 (0.5 W cm⁻², 0.75 W cm⁻², and 1.0 W cm⁻²) post PTT/PDT treatment effect on MDA-MB-231 cell line. Data were presented as mean ± S.D. (n = 5, *significant p < 0.05 compared to control, as statistically significant) (C) MTT assay of HAp and Mnx-HAp/FA-IR-783 (x = 0.5, 1.0, and 2.0 mol%) nanoparticles with different time interval (24 h, 48 h, and 72 h) with MDA-MB-231 breast cancer cell line. (D) Confocal Laser scanning microscopy of AO/PI stained control (green fluorescent without any nanoparticle) and (E) post PTT/PDT treated MDA-MB-231 (dead cells red fluorescence) cell line (each scale bar represents 20 μm). (F) Hemolysis study of 2.0 mol% Mn-HAp/FA-IR-783 nanoparticles with different concentration (50–250 μg mL⁻¹).

FIGURE 5

(A) In vivo tumor targeted fluorescence imaging study of 2.0 mol% Mn-HAp/FA-IR-783 nanoparticles (B) In vitro study of 2.0 mol% Mn-HAp/FA-IR-783 nanoparticles.

FIGURE 6

(A) Photothermal efficiency measurement of 2.0 mol% Mn-HAp/FA-IR-783 nanoparticles irradiated with a 0.75 W cm⁻² laser (808 nm) for 7 min (B) Laser ON/OFF study for 5 cycles to determine the nanoparticles performance stability (C) IR images of initial and final PTT treatment (D) PTT/PDT treatment of MDA-MB-231 tumor bearing BALB/c nude mice and their corresponding IR images.

FIGURE 7

(A) Harvested organs (Kidney, Liver, Spleen, Heart, Lungs, Tumor) from the experimental animals (B) H and E stained organs imaged under microscope (each scale bar is 50 micro meter) (C) Biodistribution of “Mn” in the harvested organs (Kidney, Liver, Spleen, Heart, Lungs) and tumor of the mice treated with 2.0 mol% Mn-HAp/FA-IR-783 nanoparticles for 24 h and 4 weeks post treatment. (D) Tumor volume in different groups following PTT/PDT therapies. Data were represented as mean ± S.D. (n = 3, *significant p < 0.05) (E) after PTT treatments, the mice’s body weight.