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 IC 50 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.

Encapsulation of Iron-Saturated Lactoferrin for Proteolysis Protection with Preserving Iron Coordination and Sustained Release

“…As mentioned in the introduction, such a high value for EE% outperforms other recently developed systems reporting EE% of 50–75% [ 36 , 37 , 38 ]. The high LC% value obtained is superior to the majority of the previously reported systems for Lf encapsulation and at a level similar to the innovative systems developed by Kanwar et al (0.17 mg of protein per 1 mg of nanocapsules) [ 53 ] or Kiryukhin et al (0.11 mg of protein per 1 mg of microparticles) [ 54 ].…”

Nanoencapsulation of oral‐pharmaceutical lactoferrin using chitosan and the evaluation of stability against trypsin and pepsin and its antibacterial effect

“…Because proteins and peptides have low absorption due to denaturation during absorption and passage through the intestinal wall during oral consumption, nanopolymers overcome these limitations and increase their oral absorption [29]. Chitosan is a health‐safe biopolymer with beneficial properties for adhesion to biological mucosa in the body [28]. This polymer has a positive and stable charge in a neutral and acidic environments and as a carrier, has the ability of permeability and biocompatibility for hydrophilic compounds such as water‐soluble proteins [30, 31].…”

“…Alginate was used exclusively to protect Lf‐chitosan nanoparticles against acidic gastric environment. When the nanoparticles enter the intestine, the alginate layer is discarded by the intestinal cells to absorb the final nanoparticles (Lf + chitosan) [28].…”

“…The interaction of proteins with nanoparticles leads to the formation of a dynamic nanoparticle-protein complex and can induce structural changes in the adsorbed proteins that may alter their function, so choosing a protein carrier is very important [28].…”

Lactoferrin: A Glycoprotein Involved in Immunomodulation, Anticancer, and Antimicrobial Processes

“…In the same way, both forms of Lf can modulate some apoptotic molecules, including p53, and completely inhibit the expression of survivin, a multifunctional protein involved both in the apoptotic inhibition and in the regulation of the cell cycle, which promotes resistance to cancer cells in chemotherapy and radiotherapy [ 77 , 78 ]. On the other hand, it has been reported that treatment with nanoparticles of calcium phosphate loaded with bovine saturated Lf is able to decrease the size of the tumors in murine models [ 79 ]. Alternatively, specific bioactive peptides from Lf have also been used to test their antitumor effect, such as lobe C from hLf, which was used against breast cancer, promoting cellular apoptosis and generating significant growth arrest in MDA-MB-231 cells [ 80 ].…”