Oral administration of iron-saturated bovine lactoferrin–loaded ceramic nanocapsules for breast cancer therapy and influence on iron and calcium metabolism

Abstract

We determined the anticancer efficacy and internalization mechanism of our polymeric–ceramic nanoparticle system (calcium phosphate nanocores, enclosed in biodegradable polymers chitosan and alginate nanocapsules/nanocarriers [ACSC NCs]) loaded with iron-saturated bovine lactoferrin (Fe-bLf) in a breast cancer xenograft model. ACSC-Fe-bLf NCs with an overall size of 322±27.2 nm were synthesized. In vitro internalization and anticancer efficacy were evaluated in the MDA-MB-231 cells using multicellular tumor spheroids, CyQUANT and MTT assays. These NCs were orally delivered in a breast cancer xenograft mice model, and their internalization, cytotoxicity, biodistribution, and anticancer efficacy were evaluated. Chitosan-coated calcium phosphate Fe-bLf NCs effectively (59%, P≤0.005) internalized in a 1-hour period using clathrin-mediated endocytosis (P≤0.05) and energy-mediated pathways (P≤0.05) for internalization; 3.3 mg/mL of ACSC-Fe-bLf NCs completely disintegrated (~130-fold reduction, P≤0.0005) the tumor spheroids in 72 hours and 96 hours. The IC₅₀ values determined for ACSC-Fe-bLf NCs were 1.69 mg/mL at 10 hours and 1.62 mg/mL after 20 hours. We found that Fe-bLf-NCs effectively (P≤0.05) decreased the tumor size (4.8-fold) compared to the void NCs diet and prevented tumor recurrence when compared to intraperitoneal injection of Taxol and Doxorubicin. Receptor gene expression and micro-RNA analysis confirmed upregulation of low-density lipoprotein receptor and transferrin receptor (liver, intestine, and brain). Several micro-RNAs responsible for iron metabolism upregulated with NCs were identified. Taken together, orally delivered Fe-bLf NCs offer enhanced antitumor activity in breast cancer by internalizing via low-density lipoprotein receptor and transferrin receptor and regulating the micro-RNA expression. These NCs also restored the body iron and calcium levels and increased the hematologic counts.

Keywords:

• oral delivery
• Fe-bLf
• miRNA
• xenograft
• breast cancer

Supplementary materials

Figure S1 Time dependent internalization of NCs.

Abbreviations: ACSC, alginate-enclosed chitosan-coated calcium phosphate; CSC, chitosan-coated calcium phosphate; Fe-bLf, iron-saturated bovine lactoferrin; NCs, nanocapsules/nanocarriers.

Figure S2 Determining the percentage viability in MDA-MB2-31 cells with NC treatments at 10 h.

Abbreviations: ACSC, alginate-enclosed chitosan-coated calcium phosphate; CSC, chitosan-coated calcium phosphate; Fe-bLf, iron-saturated bovine lactoferrin; h, hours; NC, nanocapsule/nanocarrier.

Figure S3 Determining the percentage viability in MDA-MB2-31 cells with NC treatments at 20 h.

Abbreviations: ACSC, alginate-enclosed chitosan-coated calcium phosphate; CSC, chitosan-coated calcium phosphate; h, hours; Lf, lactoferrin; NC, nanocapsule/nanocarrier.

Figure S4 Determining the clonogenic potential of MDA-MB-231 with NC treatments.

Notes: (A) Clonogenic potential of MDA-MB-231 was performed. (B) It was revealed that the CSC-Fe-bLf NCs led to a significant reduction (*P≤0.05) in 12 and 24 h and a highly significant reduction (**P≤0.005) in 48, 72, and 96 h.

Abbreviations: CSC, chitosan-coated calcium phosphate; Fe-bLf, iron-saturated bovine lactoferrin; h, hours; NCs, nanocapsules/nanocarriers.

Figure S5 Agarose gel images of the Q-RT-PCR (quantitative real time polymerase chain reaction) products.

Abbreviation: LRP, low-density lipoprotein receptor.

Figure S6 Agarose gel images of the Q-RT-PCR (quantitative real time polymerase chain reaction) of TfR and its isoforms.

Abbreviation: TfR, transferrin receptor.

Table S1 Comparison of physicochemical and biological characters in CSC-Fe-bLf NCs and ACSC-Fe-bLf NCs

Acknowledgments

The authors thank the Australia–India Strategic Research Fund and National Health and Medical Research Council for financial support. The authors thank Dr Nick Branson for his assistance with animal work and for his technical support and guidance with the animal experimental procedures. The authors also thank the upper animal house staff, in particular T Thorpe, A Cooper, and B Newell for providing help in handling the animals.

Disclosure

The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of the manuscript. The authors report no other conflicts of interest in this work.