Fe-bLf nanoformulation targets survivin to kill colon cancer stem cells and maintains absorption of...

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10.2217/NNM.14.132 © 2014 Future Medicine Ltd

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Fe-bLf nanoformulation targets survivin to kill colon cancer stem cells and maintains absorption of iron, calcium and zinc

Jagat R Kanwar*,1, Ganesh Mahidhara1, Kislay Roy1,Sreenivasan Sasidharan2,Subramanian Krishnakumar3,Neerati Prasad4, Rakesh Sehgal5 & Rupinder K Kanwar*,1

1Nanomedicine, Laboratory of Immunology & Molecular Biomedical Research (LIMBR), School of Medicine (SoM), Faculty of Health, Deakin University, Waurn Ponds, Victoria 3217, Australia2Institute for Research in Molecular Medicine (INFORMM), Universiti Sains Malaysia, 11800 Pulau Pinang, Malaysia3Department of Nanobiotechnology, Vision Research Foundation, Kamalnayan Bajaj Institute for Research in Vision & Ophthalmology, Chennai, India4Department of Pharmacology, Drug Metabolism & Pharmacokinetics Division (DMPK), University College of Pharmaceutical Science, Kakatiya University, Warangal, Andhra Pradesh, 506009, India5Department of Medical Parasitology, Postgraduate Institute of Medical Education & Research, Chandigarh, 160012 India*Authors for correspondence:[email protected]@deakin.edu.au

Aim: To validate the anticancer efficacy of alginate-enclosed, chitosan-conjugated, calcium phosphate, iron-saturated bovine lactoferrin nanocarriers/nanocapsules (NCs) with improved sustained release and ability to kill tumor cells, as well as cancer stem cells. Materials & methods: The stability, nanotoxicity of the modified nanoformulation was evaluated and their anticancer efficacy was re-examined. Their mechanism of internalization was studied and we identified the role of various miRNAs in absorption of these NCs/iron in various body parts of mice. We determined the effect of these NCs on survivin, stem cell markers, red blood cell count, iron, calcium and zinc concentration in mice, determined the antiangiogenic properties of these NCs and studied their effect on cancer stem-like cells. Results: Spherical NCs (396.1 ± 27.2 nm) exceedingly reduced viability of Caco-2 cells (32 ± 2.83%). The NCs also showed effective internalization and reduction of cancer stem cell markers in triple-positive CD133, survivin and CD44 cancer stem-like cells. Mice treated with the NCs showed no nanotoxicity and did not develop any tumors in xenograft colon cancer models. We found that the serum iron, zinc and calcium absorption were increased. DMT1, LRP, transferrin and lactoferrin receptors were responsible for internalization of the NCs. Different miRNAs were responsible for iron regulation in different organs. Interestingly, NCs inhibited survivin and its different isoforms. Conclusion: Our results confirmed that NCs internalized and changed the expression of selected miRNAs that further enhanced their uptake. The NCs activated both extrinsic, as well as intrinsic apoptotic pathways to induce apoptosis in cancer cells and cancer stem cells, without inducing any nonspecific nanotoxicity. Apart from inhibiting angiogenesis and stem cell markers, NCs also maintained iron and calcium levels.

Original submitted 4 May 2014; Revised submitted 25 June 2014

Keywords:

Colorectal cancer is a malignancy associated with the incidents of high morbidity and mor-tality. It is the fourth most common form of cancer and the third leading cause of cancer-related deaths with approximately 0.7 million deaths per year worldwide [1]. Some naturally occurring compounds such as plant/animal-derived proteins, flavonoids and polyphenols have been shown to prevent the development of colon cancer, potentially with minimum side effects [2]. Bovine lactoferrin (bLf) is a

pleiotropic natural protein that exhibits anti-bacterial activity [3–7], regulates cell growth, differentiation [8], myelopoiesis, embryonic growth [9], endothelial cell adhesion [10], and production of cytokines and chemokines, thereby implementing immune functions [11]. It was also reported that bLf induces cel-lular proliferation and cell survival in osteo-blasts [12]. Transitional metal elements such as copper, zinc, manganese and selenium were found to bind in the characteristic lobes

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iron-saturated bovine lactoferrin (Fe-bLf)

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induce apoptosis by downregulating survivin

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to induce apoptosis by targeting survivin in cancer cells...

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Research Article Kanwar, Mahidhara, Roy et al.

present in the tertiary structure of the protein [13]. The siderophilic (iron-loving) nature of the bLf is due to its globular structure consisting of two iron-binding sites per molecule [14]. Lf shares 60–80% sequence identity with transferrin (Tf) [15] and Tf receptors (TfRs) are overexpressed in several human carcinomas including breast, ovary and brain [16]. Suppression of LRP expres-sion by using antisense oligonucleotides diminished Lf internalization, suggesting that LRP1 plays a role in some of the cellular actions of Lf [17].

Previous studies from our laboratory have shown that natural bLf and iron-saturated forms of bLf dif-fer in their ability to augment cancer chemotherapy. By supplementing natural bLf (15%), apo-bLf (4%) and Fe-bLf (50% iron-saturated) in mice, which were sub-sequently challenged subcutaneously (s.c.) with tumor cells, we found that Fe-bLf was a potent natural adju-vant for augmenting cancer chemotherapy [18]. We also reported that Lf forms were able to impart antioxidative effects and, therefore, reduced the ability of cancer cells to protect them from oxidative stress [6,19]. Not many studies have been conducted with nanoformulated Lf for anticancer purposes. It has been reported that Lf nanoliposomes were more significant than Lf in inhib-iting human tumor cell proliferation [20]. Nanotechnol-ogy is also known to reduce the side effects of anticancer drugs/proteins [21]. Needle-free systemic delivery of the protein drugs can be achieved by the oral route, which offers a convenient alternative for the painful regime of targeting anticancer biomacromolecules against cancer.

The present study is a continuation of our previous report where we established that alginate-enclosed, chi-tosan-coated, calcium phosphate nanocarriers (NCs) encapsulating Fe-bLf (AEC-CP-Fe-bLf), when given orally, were predominantly present in tumor tissues and helped in the absorption of iron [22]. In this study, we prepared similar nanoformulations with slight modifi-cations (alginate-enclosed chitosan-conjugated calcium phosphate, Fe-bLf nanocarriers/nanocapsules [AEC-CCo-CP-Fe-bLf NCs]) for improved sustained release that were able to induce apoptosis in cancer cells, as well cancer stem cells. We also studied the mechanism involved in internalization and anticancer efficacy of these NCs.

Materials & methodsPreparation of Fe-bLfIron-saturated bLf was prepared according to the previous method developed in our laboratory [18].

Preparation of AEC-CCo-CP-Fe-bLf NCsIn our previous study, calcium phosphate nanocore was loaded with Fe-bLf using electrostatic interaction, post which a coating of 0.01% w/w of chitosan and 2% w/w

of alginate were added [23]; however, slight modifications were made this time. Briefly, calcium phosphate solu-tion was prepared and 0.01% w/w chitosan in acetate buffer was added under constant stirring and 0.01% of cross-linking agent, sodium tripolyphosphate was added dropwise. Constant stirring at 6000 rpm for 12 h was done to ensure that nanoformulation obtained the size of 200–250 nm. Fe-bLf was then chemically con-jugated to the chitosan nanocores [24]. A total of 150 lethyl(dimethyl aminopropyl) carbodiimide (EDC)/N-hydroxysuccinimide (NHS) solution was used to initi-ate the cross-linking reactions. Fe-bLf/EDC/NHS in a weight ratio of 1:1.84:0.23 were added to a NC sus-pension at pH 5.5 and conjugated by stirring at 6000 rpm for 72 h at 4°C. The unbound Fe-bLf/EDC/NHS was removed by washing and the nanocores were coated with 1% w/v alginate solution and calcium chloride, with 0.6% mass ratio of calcium/alginate. The final obtained NCs were washed and lyophilized for fur-ther characterization. All these experiments were car-ried out at 4°C as to protect the polymeric and protein components in the formulation.

Characterization of AEC-CCo-CP-Fe-bLf NCsThe average size of the NCs, in various steps of their preparation viz. calcium phosphate Fe-bLf, chito-san formulation and alginate-enclosed particles were determined by dynamic light scattering (DLS), using Zetasizer (Malvern Instruments, Worcestershire, UK). DLS was also used to study the stability of NCs. Pro-tein release studies were conducted to determine the release profile of the NCs. The number of NCs/mg/ml was obtained using Zetasizer and was found to be 2.8 × 105/mg. Surface morphology of these particles was determined by transmission electron microscopy (Jeol, Tokyo, Japan). X-ray diffraction was also performed (X’pert3 MRD XRD, Panalytical, NSW, Australia).

Cell viability assayTACS MTT assay kit (Sigma Aldrich, NSW, Austra-lia) was used for cell viability analysis as per the manu-facturer’s instructions and measured using measured ASYS expert plus UV plate reader at 550 nm.

NC uptake mechanismThe uptake of NCs in Caco-2 cells was studied using the flow cytometer BD FACS canto II (BD Biosciences, NSW, Australia) as described in the Supplementary data (see online at www.futuremedicine.com/doi/suppl/10.2217/nmm.14.132).

NP uptake in sorted colon cancer cellsThe triple-positive cells (Supplementary data) were seeded in 8-well slides and once confluent, were treated

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Fe-bLf nanoformulation targets survivin to kill colon cancer stem cells Research Article

with (0.01%) rhodamine-labeled Fe-bLf NCs for 3 h. The slides were prepared and viewed using a confocal microscope.

Tumor spheroid assay70% confluent triple-positive Caco-2 cells were har-vested from the culture dishes by trypsin/EDTA and suspended in DMEM (10% fetal bovine serum). Single cell suspension of cells in the concentration of 1 × 103 cells/well was added to 96-well plates, previ-ously coated with 1% (w/v) agarose, which prevents cell adhesion to the wells, and further incubated for 7 days at 37°C with 5% CO

2; they formed uniform

spheroids in all the wells [25,26]. The spheroids were fur-ther treated with NCs (9.01 × 105) corresponding to 3200 g of Fe-bLf up to 96 h.

Preparation of diet containing AEC-CCo-CP-Fe-bLf NCs for oral therapy in vivoThe dose was standardized earlier [18], where we found that 1.2% (w/w) or 12 g Fe-bLf/kg of diet showed no tumor development. The same dose was then used in our recently published study with a nanoformu-lated Fe-bLf diet [22], with similar findings. The diet was mixed with the appropriate amount of autoclaved milliQ water and made into pellets of suitable sizes, to be able to give as a treatment diet. Equal quantities of this diet was given to mice each time to normalize the dosage in metabolic cages.

Induction of tumor & in vivo studiesFor in vivo studies, athymic nude mice (6–8 weeks) were s.c. injected with 1 × 106 Caco-2 cells and CD133-, CD44- and EpCAM-positive Caco-2 cells sorted using magentic microbeads (MACS, Milt-enyi Biotec, Australia; stem cell model, in left flank). Mice were divided into control and treatment groups. Bodyweight, behavioral changes and tumor sizes were observed every 5 days. Tumor volume was cal-culated using the formula ½ L (mm) × W2, where L is the length of the tumor and W is the width of the tumor, as measured by a vernier callipers. Animals were humanely killed once the tumors reached 1.0 cm in diameter, in accordance with animal ethics rules. Mice were sacrificed post-treatment period, and tissues were harvested. Tissues were snap frozen and stored in -80°C until further use.

Tissue processing, hematoxylin & eosin staining pathology & immunohistochemistryTissues were thawed and washed with phosphate-buff-ered saline from the interior (lumen), fixed using 4% paraformaldehyde, frozen in optimum cutting temper-ature (OCT) compound, stored at -20°C and sectioned

in cryotome for 5- m-thin sections. The cryotomic sections were fixed using ice-cold acetone solution (1:1 mixture of acetone and distilled water). The sec-tions were rehydrated in distilled water and subjected to hematoxylin and eosin or immunohistochemical staining (Supplementary data).

Biodistribution of AEC-CCo-CP-Fe-bLf NCs in miceIn order to determine the biodistribution of AEC-CCo-CP-Fe-bLf NCs to mice tumor, blood, brain, kidney, heart, spleen, lungs, eye, bones, intestine, mus-cle and liver were stained with Cibacron brilliant red (CBR)-3BA, a dye specific to chitosan. Staining proce-dures were carried out as previously reported [27], with slight modifications [28]. Tissue sections of different organs from AEC-CCo-CP-Fe-bLf NC-treated mice were stained with CBR-3BA and the percentage level of red stain was quantified from five different sections of the same organ.

CD31 & Prussian blue double stainingTo determine angiogenic vasculature in the tissue of interest, 10- m frozen sections were immunostained with goat anti-CD-31 antibody and probed as per previously published protocol [22].

Real-time PCRReal time analysis of apoptotic genes, Lf receptor genes, survivin splice variants and stem cell markers were car-ried out as described in the Supplementary data and according to the protocol given in the SybrGreen RT-PCR kit (BioRad, NSW, Australia) [29].

Regulation of miRNA & genes involved in iron metabolism by AEC-CCo-CP-Fe-bLf NCsGene expression of receptors regulated in the translo-cation of NCs was analyzed using RNA isolated from mice tissues using the Trizol method [30,31]. To quan-tify miRNAs in mouse small intestine, total RNA from homogenized mouse small intestine was isolated using isolation kit (Sigma Aldrich). cDNA was gen-erated from 2 g RNA using TaqMan® MicroRNA Reverse Transcription Kit (Applied Biosystems, Vic-toria, Australia), according to the manufacturer’s protocol.

Estimation of iron, calcium & zincThe serum iron concentrations were determined using a previously published procedure [18], whereas the serum calcium concentrations were determined using a Fura-2/acetoxymethyl ester as previously published [23,32]. The serum zinc concentration was determined using atomic absorption spectrophotometry [33].

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Hi Janet, We have included some information in the supplementary data (as suggested by one of the reviewers).

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findings. The amount of nano-diet consumed daily by each animal was also determined (supplementary data). The diet...



ELISA of cytokine & nitrite production in the small intestineCytokines expressed in mouse serum were determined using the Single Analyte ELISA Kits (Qiagen, Victoria, Australia) for IL-1 , IL-6, IL-18, IL-12, IL-10, IL-15, IFN-1 , IFN-1 , IL-2, IL-4, IL-5, IL-17, IL-13, IFN- ,IL-19, GM-CSF, IL-7, IL-11, IL-3, TGF- and NO, and followed as per the manufacturer’s instructions and as described earlier [18,28,34,35]. In brief, after euthanizing mice, blood was collected via cardiac puncture and trans-ferred into unheparanized tubes. The blood was allowed to clot by incubating the tubes at room temperature for 20 min. Blood clot was removed by centrifugation at 2000 × g for 10 min at 4°C, and the supernatant was carefully transferred into a fresh tube and stored imme-diately at -80°C. The regulation of serum cytokines from wild-type or nude mice and AEC-CCo-CP-Fe-bLf NCs and control diets were determined (pg/ml). Quan-tification of nitrite, indicative of NO production, was carried out by the Griess reaction (Sigma Aldrich). The results are expressed as the mean nitrite concentration ( M/ml) ± standard error of the mean.

TUNEL assayTUNEL assay was performed with in situ cell death detection kit (Roche, NSW, Australia), as per the manufacturer’s protocol.

CD31, CD105 double staining for angiogenesisFor CD31 and CD105 double staining, 10- m fro-zen sections were preincubated in 1% rabbit and horse serum and then incubated for 60 min with goat anti-CD-31 and rabbit anti-CD105 (1:100) antibodies at 37°C. After gentle washes, the sections were incubated for 60 min with donkey anti-goat IgG–Alexa488 and goat anti-mouse IgG–Alexa568-conjugated secondary antibodies. Permanent mounting media was used to fix the cover slips and analyzed in confocal microscopy with photomultiplier voltage corresponding to 488 and 568 nm.

Statistical analysisResults are expressed as mean values ± standard devia-tion of at least three experiments unless otherwise stated. For the statistical evaluation of numerical data, student’s t-test was used for evaluating the significance. The value of p < 0.05 denotes statistical significance, whereas p < 0.005 denotes results that are highly significant.

ResultsPhysicochemical characterization of AEC-CCo-CP-Fe-bLf NCsThe NCs were found to have a hydrodynamic diam-eter of 205 ± 15 nm for CCo-CP-Fe-bLf inner core

(Figure 1A). As observed by DLS and transmission electron microscopy, the size of the NCs increased to 396.1 ± 27.2 nm after alginate coating (AEC-CCo-CP-Fe-bLf NCs). The stability studies using DLS con-firmed that particle size (350 ± 37.22nm), with a poly-dispersity index of 0.106 and zeta potential of -0.217 was increased after lyophilization (396.1 ± 27.2 nm), with a polydispersity index of 0.068 and zeta poten-tial of -1.29. In total, 15% of the NCs were found to degrade at pH 2 (corresponding to stomach) leaving 85% of NCs intact. The shedding of the alginate layer at pH 8 (corresponding to intestine) was observed as the particle size was reduced to 220 ± 10 nm (Figure 1B).A total of 1 mg of Fe-bLf was present in 2.8 × 105 NCs, therefore 3.57 ng of Fe-bLf was calculated to be pres-ent on each CCo-CP-Fe-bLf NC, with a loading effi-ciency of 25.8% (Table 1). The protein release studies conducted revealed that AEC-CCo-CP-Fe-bLf NCs had an improved sustained release profile when com-pared with AEC-CP-Fe-bLf NCs (Figure 1C). X-ray diffraction patterns analyzed to predict the crystallin-ity in the nanoformulation and to examine the inter-actions between the ceramic and polymeric ingredients (Figure 1D) revealed that the crystallinity of the cal-cium phosphate after the nanopowder formation was lowered. Diffraction patterns for chitosan and sodium alginate were not detected, which meant that chitosan and sodium alginate obtained amorphous structure in the NCs.

Internalization & cytotoxicity of AEC-CCo-CP-Fe-bLf NCsA logarithmic curve was plotted using the cell viability values from 10 and 20 h and the concentration of NCs; using the equation from each curve, the correspond-ing IC

50 values were also calculated. The IC

50 at 10 h

for void NCs was 1.05 × 109 NCs, for AEC-CCo-CP-Fe-bLf was 3.36 × 106 NCs and for CCo-CP-Fe-bLf was 2.16 × 105 NCs. Higher cytotoxic activities were observed for all doses of CCo-CP-Fe-bLf NCs than AEC-CCo-CP-Fe-bLf NCs (Figure 1E). Meager cell viability (32 ± 2.83%) was observed with 106 NCs compared with 72.5 ± 4.2% with the same concentra-tion of void NCs at 10 h. The IC

50 values at 20 h of

void NCs were 1.09 × 107 NCs, for AEC-CCo-CP-Fe-bLf was 2.74 × 105 NCs and for CCo-CP-Fe-bLf was 1.53 × 105 NCs (Figure 1F). Inhibition of clathrin endocytosis using chlorpromazine showed a consider-able reduction in the uptake compared with the void-loaded NCs, while indomethacin showed no difference (Figure 1G). There was a complete reversal of absorp-tion when treated with antibodies to LRPs and TfR indicating the absorption may be TfR and LfR medi-ated. The confocal images obtained post 3 h incubation

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with rhodamine-labeled AEC-CCo-CP-Fe-bLf NCs showed that the NCs were able to effectively internal-ize in the triple-positive sorted Caco-2 cells (shown by arrows) and showed remarkable downregulation of all the stem cell marker expressions (Figure 1H).

In vitro & in vivo antitumor efficacy of AEC-CCo-CP-Fe-bLf NCsThe in vitro anticolon cancer stem cell efficacy of Fe-bLf NCs was determined using 3D colonosphere using the triple-positive sorted Caco-2 cells. A total disinte-gration of the tumor spheroid was observed at 72 and 96 h post the NC treatment, showing that AEC-CCo-CP-Fe-bLf NCs effectively inhibited tumor spheroid growth (Figure 2A) The images were analyzed using Image J software (NIH, MD, USA) and the mean tumor spheroid size was calculated and plotted in a graph (Supplementary data). Mice were fed with either the AIN93G diet containing 3.38 × 109 NCs having 12 g Fe-bLf per kg of diet (1.2% w/w); or with a same amount of void NCs, or control diet. In the treatment model, developed tumors were treated after 35 days with Fe-bLf-loaded AEC-CCo-CP-FebLf NCs diet and void NCs diet. All tumor-bearing mice in both the xenograft colon cancer and stem cell models (Figure 2B) rejected tumors in 3–4 weeks, while the tumors kept growing in mice fed on the control NCs diet.

After 2 weeks of pretreatment, tumors were induced by s.c. injection of 1 × 106 Caco-2 cells. The NC diet was continued throughout this study. Within a period of 20 days, the control group of mice developed tumors with sizes of approximately 432 mm3, void NCs showed slight delay in tumorigenesis with tumor sizes of approx-imately 210 mm3, whereas no tumor development was observed in the case of AEC-CCo-CP-Fe-bLf NC-treated mice (Figure 2B). The study was further extended to observe in vivo antitumor efficacies of pretreatment containing AEC-CCo-CP-Fe-bLf NCs in comparison to intraperitoneal taxol injections. The treatment groups were further divided into two groups: one where the NC diet was continued throughout the study and another where the NC diet was switched off to the normal diet after the second challenge with cancer cell injec-tion (Caco-2 2nd control). In the case of control mice, tumor volume was increased periodically with respect to time. As shown in Figure 2D, tumors were regressed by injecting taxol, once they reached a diameter of approxi-mately 300 mm3. The taxol-treated group and Fe-bLf NC treatment groups were re-challenged with 1 × 106

Caco-2 cells. Notably, solid tumors appeared in a period of 5 days in taxol-treated groups, whereas the Fe-bLf NC treatment groups did not show any tumors. Within a period of 5 weeks, tumors in the control group reached approximately 700 mm3 (Figure 2D).

Evaluation of nanotoxicity for AEC-CCo-CP-Fe-bLf NCsThere was no effect in bodyweight in treatment groups when compared with controls. In fact, average body-weight of treatment groups (17.86 g) was more than controls (16.22 g) and the mice remained healthy (Figure 2E). Average bodyweight of all mice before the treatment was found to be 14.22 g. Fourier transform infrared spectroscopy was performed on mice tissues (liver, brain, intestine, heart, kidney and lungs), as previously reported, for arsenic-induced toxicity in the liver of fish [36]. Major biomacromolecules including protein, lipid, nucleic acids, which have a characteris-tic amide (3293 and 1543 cm-1), methyl (CH

3 stretch-

ing: 2958 cm-1; CH2 bending: 1451 cm-1) and phos-

phate symmetric stretch (1230 cm-1) vibrations have shown the same frequencies in the control, as well as the treated groups (Figure 2F). It is known that chemo-therapy causes hematologic suppression and anemia [18]. Thus, we also analyzed the hematologic cell count, red blood cell (RBC) count, serum iron, zinc and cal-cium content in control versus Fe-bLf NCs treatment mice groups. The analysis revealed (Table 2) substan-tial increase in total hematologic cell count (1.23-fold) and RBC count (1.17-fold). The serum iron, zinc and serum calcium deposits were also found to be increased (1.27-, 1.21- and 1.15-fold, respectively) compared with control mice, therefore suggesting an enhance-ment in the immune system and body metabolism of the Fe-bLf NC-treated mice.

Histopathology studies after NC treatmentThe hematoxylin and eosin-stained sections involved in Fe-bLf NCs internalization including the intes-tine, brain, organs involved in the reticuloendothelial system, including the liver and kidney, showed no abnormality. The anatomy of controlled versus treated sections retained their integrity, showing negligible damage to these organs with the treatment (Figure 3A).A substantial amount of Fe-bLf-loaded NCs were vis-ible around the vascular region of the tumor and brain tissue slices (Figure 3B) using Prussian blue and CD31 double staining. Consistently, reduced immunohisto-chemical reactivity in the case of control tissues was verified using the same staining procedure. Biodistri-bution studies of AEC-CCo-CP-Fe-bLf NCs after oral administration in the diet showed high uptake of the NCs in tumor. Sections of various mice organs were stained with CBR-3BA dye and the percentage of red stain observed quantified from five different sections of the same organ. The NCs showed low uptake in muscle, spleen, eye and stomach. High uptake of NCs was observed in blood, liver, brain and intestine apart from the tumor (Figure 3C).

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Figure 1. Characterization and in vitro efficacy of alginate-enclosed chitosan-conjugated calcium phosphate, iron-saturated bovine lactoferrin nanocarriers/nanocapsules (facing page). (A) Dynamic light scattering spectrometric observation and transmission electron microscopy analysis of NCs confirmed the size of CP-Fe-bLf ceramic nanocores (160 ± 11 nm), the size of CCo-CP-Fe-bLf NCs (205 ± 15 nm) and of AEC-CCo-CP-Fe-bLf NCs (396.1 ± 27.2 nm). (B)Using dynamic light scattering, it was observed that there was an increase in particle size post freeze drying. It was also observed that 15% of NCs were degraded at pH 2 and that the alginate layer was shed off from the NCs at pH 8. (C) The protein release studies revealed that AEC-CCo-CP-Fe-bLf NCs had improved sustained release profile when compared with AEC-CP-Fe-bLf NCs. (D) X-ray patterns indicated the characteristic diffraction pattern for CP-Lf at 11.4, 13.6, 22.4, 25.1, 29.7 and 47.2. (E) The growth inhibition was measured by MTT assay after 10 h and (F) 20 h of the treatment. (G) Mechanism of internalization for Fe-bLf NCs. (H) AEC-CCo-CP-Fe-bLf NCs (shown by arrows) internalized in triple-positive sorted Caco-2 stem-like cells (CD133-, EpCAM- and CD44-positive), having high cellular expression of survivin and lowered the expression of survivin and stem cell markers. Data represent mean ± standard error of the mean. Experiments were repeated three-times independently with similar results.Ab: Antibody; ACSC: Alginate-enclosed chitosan-coated; AEC: Alginate enclosed; bLf: Bovine lactoferrin; CCo: Chitosan conjugated; CP: Calcium phosphate; NC: Nanocarrier/nanocapsule; NP: Nanoparticle; TfR: Transferrin receptor.

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Immunohistochemical observation of NCs after oral deliveryIn the case of treatment groups, characteristic immuno-reactivity of bLf was seen. As represented in the Figure 4A, the tumor showed maximum immunore-activity corresponding to Fe-bLf, intestine (treatment group) showed intense bLf immunoreactivity corre-sponding to the proposed transcytosis of the NCs, fol-lowed by mild immunoreactivity in the brain, kidney and liver. Concentrations of Fe-bLf were determined from tissue lysates (Figure 4B) and the analysis showed maximum Fe-bLf content in tumor (two- to four-fold higher), bone (twofold higher), blood (twofold higher) and intestines (twofold higher). These results showed superior allocation of Fe-bLf and NCs in muscle, blood, liver, bone, spleen, kidney, lung and intestine when compared with our previous study [22]. Western blotting of the tissue homogenates (Figure 4C) from intestine, spleen, liver, kidney, brain, bone, muscle and eye from control, as well as treatment groups confirmed the Fe-bLf-specific immunoreactivity. Densitometric analysis of the Western blots showed heightened local-ization of Fe-bLf in the tumor tissue (2.3 ± 0.02-fold increase) followed by intestine (1.5 ± 0.02-fold).

Real-time analysis showing mechanism of internalization & apoptosisIn order to understand the mechanism of NC uptake, Lf receptor gene expression in the tissue homogenates were

measured by quantitative real-time PCR (RT-PCR) and normalized with respect to the GA3PDH house-keeping gene. The treatment group demonstrated an upregulation in DMT1 receptor (stomach: 1.33-fold; intestine: 2.57-fold; liver: threefold; spleen: 3.125-fold; and brain: 2.25-fold), TFR1 (stomach: 1.11-fold; intes-tine: threefold; liver: 2.08-fold; spleen: 1.83-fold; and brain: 1.25-fold), TFR2 (intestine: threefold; liver: 2.27-fold; spleen: 2.5-fold; and brain: 1.11-fold) and LFR (intestine: 2.75-fold; liver: 1.91-fold; and spleen: 2.25-fold) in treated tissues (Figure 5A). Previous stud-ies have reported that Lf is a LRP and Tf ligand [37].This receptor was therefore analyzed by RT-PCR anal-ysis in tumors and other organ tissues. Although LRP1mRNA was overexpressed in all treated tissues, the level of LRP2 in tumor tissues, as well as in the brain and intestine was elevated much higher (Figure 5B). We found that LRP2 mRNA was elevated 3.4 ± 0.06-fold (p < 0.005) in the liver, and 1.8 ± 0.03-fold (p < 0.005) in the brain, whereas it was 4.9 ± 0.05-fold (p < 0.005) in the intestine compared with Caco-2 cells treated with NCs. Furthermore, RT-PCR analysis of various genes in control versus treated tissues indicated downregu-lation of antiapoptotic factors including survivin and Bcl-2 family, and upregulation of proapoptotic Bax, Fas and caspases-9 and -3 as represented in Figure 5C.We found that antiapoptotic factor survivin was down-regulated in treatments compared with control cells and tumors from treatment groups by 0.46 ± 0.15-fold.

Table 1. Characteristics of alginate-enclosed, chitosan-conjugated, calcium phosphate, iron-saturated bovine lactoferrin nanocarriers/nanocapsules.

Samples Efficiency (%) Loading content (%)

Number of NCs per ml (0.1% w/v suspension)

Amount of protein per NCs (ng)

Size (nm)

Void ACSC – – 4.92 × 104 – 322.0 ± 15.1

AEC-CCo-CP-Fe-bLf NCs – – 4.00 × 104 3.57 396.1 ± 27.2

CCo-CP-Fe-bLf NC 80 25.6 2.80 × 105 3.57 205.0 ± 15.0

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10.2217/NNM.14.132 Nanomedicine (Epub ahead of print)

Figure 2. In vivo anticancer efficacy and determination of nanotoxicity. (A) Representative images from tumor spheroid assay showed that AEC-CCo-CP-Fe-bLf NCs disintegrated the multicellular tumor spheroid in 72 and 96 h. (B) Antitumor efficacy of the AEC-CCo-CP-Fe-bLf NCs diet and void NC diet were tested on mice models injected either with cancer cells or cancer stem cells. (C) Antitumor efficacy of the treatments of AEC-CCo-CP-Fe-bLf NCs in comparison with the void NCs or control mice. (D) Ability of Fe-bLf NCs to prevent tumor recurrence in mice. (E) Average bodyweight of the all mice groups to determine any abnormality in weight. (F) Fourier transform infrared spectroscopic observation of ctrl versus tre tissues showed no toxicity with respect to characteristic peaks corresponding to protein, lipids and nucleic acids. Data represent mean ± standard error of the mean (n = 5).ACSC: Alginate-enclosed chitosan-coated; AEC: Alginate enclosed; bLf: Bovine lactoferrin; CCo: Chitosan conjugated; CP: Calcium phosphate; ctrl: Control; Lf: Lactoferrin; NC: Nanocarrier/nanocapsule; tre: Treated.

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The antiapoptotic gene Bcl-xl was downregulated by 0.2 ± 0.03-fold. NC treatment had also increased the expression of proapoptotic genes including caspase-3 (19.01 ± 0.01-fold), caspase-8 (5.7 ± 0.13-fold) and caspase-9 (8.2 ± 0.6-fold) in the case of treated tumor tissues compared with control tumor.

Expression of various stem cell markers, miRNA & angiogenesis markersEndogenous levels of survivin expression and expres-sion of survivin splice variants in in vivo xenograft tumors was found to decrease. The AEC-CCo-CP-Fe-bLf NCs led to a nearly 9.5–10-fold decrease in expression of survivin and survivin splice vari-ants (survivin- Ex3, survivin-2B, survivin-3B and survivin-2 ; Figure 6A). Decreased expression of stem cell marker genes (CD133: 1.4-fold; CD44: 3.6-fold; CD166: 3.15-fold) and survivin (4.37-fold) was observed in the mice treated with the Fe-bLf NC diet (Figure 6B), compared with untreated mice. It was observed that miR-Let7d was highly overexpressed in the brain, whereas clearly downregulated in other body parts (intestine, liver and spleen). miR122 expression showed nearly no change in the spleen. miR196, on the other hand, was highly upregulated in the spleen and showed no change in expression in other body parts. miR200b was clearly downregulated in the liver and brain, but miR210 showed overexpression in the liver. miR214 was downregulated in the liver, but did not show much variation in any other body parts. miR320 was clearly downregulated in the intes-tine, liver and brain. miR485 showed overexpression in all body parts, whereas miR584 was only found to be overexpressed in the liver (Figure 6C). The CD31 average vessel counts were 38.8 ± 4.71 in the case of the control tumor, whereas it was 23 ± 1.3 in void NC-treated tumor. Neoangiogenic vessels were 13 ± 1.3 in the control tumor and 4.8 ± 0.48 in the case of void NC-treated tumors (Figure 6D). CD31-positive and CD105-positive blood vessel density was reduced by 14.92 ± 0.81-fold and 16.25 ± 0.37-fold, respectively, with Fe-bLf NC treatment. A substantial number of

TUNEL-positive cells were observed in the case of tumors developed in the treatment group (Figure 6E),indicating degeneration of tumor progression in these animals.

DiscussionWe report the synergistic antitumor activity of 100% Fe-bLf in monotherapy or in combination with chemo-therapeutics, to prevent tumorigenesis in allograft mice model for EL4 lymphomas, Lewis lung carcinoma or B16 melanoma tumors [18]. The study revealed that, Fe-bLf could be more effective compared with natural bLf in combating tumor. It has been also shown by previous studies that Fe-bLf exerts anticancer activi-ties by enhancing the production of Th1 and Th2 cytokines within the intestine and tumor, including TNF and IFN- , as well as nitric oxide, which have been reported to sensitize tumors to chemotherapy [18].However, we have shown recently that Fe-bLf down-regulated survivin (inhibitor of apoptosis family of protein) gene expression [38]. Therefore, this study was conducted in immunocompromised nude mice to study this alternative pathway in detail without any interfer-ence of immune cells. However, in the present study, we have also compared the cytokine levels in wild-type (C57) and nude mice for immune response/inflamma-tion with AEC-CCo-CP-Fe-bLF NC diet as these have been reported to be expressed in cells derived from nude mouse xenografts [34,35]. The analysis of various cytokines in wild-type and nude mice has been pre-sented in Table 3. As observed in this study, IL-1 levels were found to be low in wild-type mice when com-pared with nude mice. This could be because IL-1is involved in apoptosis (and is activated by caspase) [39]. Therefore, this analysis also shows that apart from boosting the immune response to fight cancer, Fe-bLf induced apoptosis in cancer since IL-1 is specifically released during apoptosis. IL-6, on the other hand, is both proinflammatory, as well as an anti-inflammatory cytokine secreted by T cells [40]. Therefore, the level of IL-6 was found to be low in nude mice (absence of T cells) when compared with wild-type mice. IL-18

Table 2. Hematologic and trace element profiles of tumor-bearing mice after nanocarrier/nanocapsule treatment.

Treatment Total hematologic cell count

RBC† Serum iron ( g/g)

Zinc ( g/dl) Serum calcium (mg/dl)

Control 7.06 ± 0.62 7.3 ± 1.13 145 ± 20 9.5 ± 3.5 7.42 ± 1.14

Void NCs 7.42 ± 1.14 7.3 ± 0.62 135 ± 25 9.0 ± 3.5 7.95 ± 1.23

ACSC-Fe-bLf NCs 8.71 ± 1.93 8.6 ± 1.91 185 ± 30 11.5 ± 5.5 8.60 ± 1.93†Mean values of whole blood cells including RBC (n × 109

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Figure 3. Biodistribution of alginate-enclosed chitosan-conjugated calcium phosphate, iron-saturated bovine lactoferrin nanocarriers/nanocapsules in mice body parts. (A) Hematoxylin and eosin staining for observation of tissue integrity in (i) control ileum (transverse section), (ii) treated ileum (transverse section), (iii) control liver (longitudinal section [LS]), (iv) treated liver (LS), (v) control kidney (LS), (vi) treated kidney (LS), (vii) control brain (LS) and (viii) treated brain (LS) were stained with hematoxylin, whereas eosin was used as a counter stain. (B) CD31 and Prussian blue double staining further confirmed internalization of NCs (ii) in brain – inset shows internalization of Fe-bLf-loaded AEC-CCo-CP NCs near CD31-stained area, (iv) treatment tumor showing double staining of CD31 and (vi) transcytosis of NCs via intestinal villi. (C) Biodistribution study of AEC-CCo-CP-Fe-bLf NCs after oral administration in the diet. Data are expressed as the percentage biodistribution of AEC-CCo-CP-Fe-bLf NCs and represented as mean ± standard deviation.AEC: Alginate enclosed; bLf: Bovine lactoferrin; CCo: Chitosan conjugated; CP: Calcium phosphate; NC: Nanocarrier/nanocapsule.

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is a proinflammatory cytokine, which is also respon-sible for cell-mediated immunity as it activates NK cells to release IFN- [41], which may account for the high expression of IL-18 in both wild-type and nude

mice. IL-12 is a proinflammatory cytokine responsible for both cell-mediated and humoral immune response, also known to stimulate TNF- , IFN- and NK cells, which may account for its increased expression

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in both wild-type mice and nude mice [41]. IL-5 is a proinflammatory cytokine produced by helper T cells and mast cells in response to allergy for activation of eosinophils and allergic immunity [42]. Therefore, as observed in our study, the levels of IL-5 with the AEC-CCo-CP-Fe-bLf NCs group were lower than control diet in wild-type as well as nude mice, indicating that these NCs were safe for oral delivery and did not induce any allergy in either nude or wild-type mice. IL-13 is another proinflammatory cytokine produced by T helper cells to mediate the allergic immunity and inflammation [43]. We found that the levels of IL-13

were lower in the AEC-CCo-CP-Fe-bLf NCs group than control diet in wild-type as well as nude mice, indicating that these NCs did not induce any allergy or related inflammation in mice. GM-CSF is released by macrophages, T cells, mast cells and NK cells. It plays a major role in the immune/inflammatory cascade for fighting infection [44]. We found that the GM-CSF levels were lower in AEC-CCo-CP-Fe-bLf NCs group when compared with control diet group in both wild-type mice, as well as nude mice. Therefore, the cyto-kine analysis conducted by us proved that the AEC-CCo-CP-Fe-bLf NCs were suitable for oral delivery

Figure 4. Allocation of iron-saturated bovine lactoferrin released from nanocarriers/nanocapsules in various body parts of mice. (A) Immunohistochemical localization of AEC-CCo-CP-Fe-bLf NCs with lactoferrin antibody. Tumor, ileum (transverse section), kidney (longitudinal section), liver (longitudinal section) and brain (longitudinal section) sections from (i–v) control and (vi–x) treatment groups were shown. (B) Fluorescent signal of tissue extracts after 24 h of oral administration were analyzed. NCs were labeled with coumarin-6 (60 mg/kg). (C) Fe-bLf was observed in intestine and tumor tissues and faint bands in brain, kidney, bone, eye and liver tissues (lanes 1–5: kidney, liver, brain, tumor, intestine-treated; lanes 6–10: control tissues; lanes 11–13: treated bone, muscle and eye; and lanes 14–16 represent the corresponding controls).AEC: Alginate enclosed; bLf: Bovine lactoferrin; CCo: Chitosan conjugated; CP: Calcium phosphate; NC: Nanocarrier/nanocapsule.

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Figure 5. Gene expression analysis to determine the mechanism of internalization and cytotoxicity. (A) Real-time (RT) analysis of Lf receptor gene expression measured by quantitative RT-PCR showed an upregulation in DMT1, TFR1, TFR2 and LFR in treated tissues. (B) RT-PCR analysis for LRP1 and LRP2 receptors showed LRP1 expression in the liver, brain, intestine and tumor cells. (C) RT-PCR analysis for expression of various proapoptotic and antiapoptotic genes both in vivo and in vitro.*p 0.005.AEC: Alginate enclosed; bLf: Bovine lactoferrin; CCo: Chitosan conjugated; CP: Calcium phosphate; NC:Nanocarrier/nanocapsule; tre: Treated.

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and did not induce any allergy or inflammation in neither wild-type (C57) nor nude mice.

Taking into account that the rapid renal clearance [45–47] of peptides imposes development of new strate-gies for enhanced bioavailability, we employed nano-technology to orally deliver Fe-bLf. Previously, we reported that AEC-CP-Fe-bLf NCs have additional advantages in encapsulation and delivery of anticancer peptides [22]. In this study, we were able to replicate the previously synthesized nanoformulation with only better drug allocation ability. The zeta potential of the NCs was -1.29; however, once the NCs were dissolved at pH >7 (in the intestine), the zeta potential becomes neutral or close to neutral [22]. We claim that the NCs synthesized in the present study have better sustained release as the maximum protein release observed in our previous study was observed within 12-h period; how-ever, in the present scenario we have observed a much

slower release of Fe-bLf ( 85% in 40 h). We have also shown higher concentrations of Fe-bLf (ng/ml) in the tissue lysates from various body parts of mice when compared with the previous study (Figure 4A), Hence, we claim superior allocation ability of these NCs. The stability studies conducted in this study revealed that the NC size increased after freeze drying and that 15% of the NCs were degraded at pH 2, whereas the algi-nate layer was shed from the NCs at pH 8, therefore exposing the chitosan layer and thereby increasing their mucoadhesiveness. The stability study was performed with NCs that were stored at 4°C for over 7 weeks.

Any anticancer therapeutic must be able to signifi-cantly eradicate cancer cells, as well as cancer stem cells in order to prove effective against the cancer. Previ-ously published studies have shown that sorting cells using magnetic microbead columns works efficiently [48]. The cancer stem cells isolated using microbeads

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were not cultured for more than 12 h. They were fro-zen in liquid nitrogen and revived only when needed to be used for an assay. We found encouraging results from expression of CD133, EpCAM, survivin and CD44 in Fe-bLf NC-treated cells. There is always a question on stem cell markers as their expression dif-fers in different cancer cell types; however, CD133, CD44 and EpCAM have been widely studied and well proclaimed as stem cell markers in colorectal cancer [49–51]. Therefore, this study clearly implemented that the prepared AEC-CCo-CP-Fe-bLf NCs could be used for therapy against cancer cells, as well as cancer stem cells in colon cancer. The in vitro antitumor efficacy of Fe-bLf NCs was also determined using 3D tumor spheroid model. Tumor spheroids are used mainly to determine the effectiveness of any therapeutic as they mimic the in vitro tumor growth [52].

In order to re-examine the anticancer efficacy of the newly synthesized nano-encapsulated Fe-bLf, NCs containing 12 g Fe-bLf (1.2% w/w) per kg of diet were administered to experimental mice. The amount of protein supplementation was in accordance with the recommendations by the commercial health and food companies and previously published study [22]. There is evidence regarding mild toxicity of chitosan deriva-tives and solution form of chitosan at low pH and its salts [53], careful handling of chitosan promises health benefits [54]. Sodium alginate was declared as generally regarded as safe for oral administration/consumption by the US FDA in 1973 [55]. The dosage of Lf recom-mended by health and food companies is 3 g of Lf per day [18]. Thus, any questions of nanotoxicity could be nullified and the synthesized NC system could be treated as biocompatible, biodegradable and neutra-ceutic. The nanotoxicity of NCs was evaluated in the present study using tissue histology studies from the liver, spleen, kidney, brain and ileum, which proved that the NCs do not impose toxicity to these organs. Neurotoxicity, which could be the side effect of many present day medications, was infinitesimal in the case of these NCs, as confirmed by the mild immune reactivity in brain tissues. Fourier transform infrared spectroscopy specified that the stretching and bending vibrations corresponding to proteinaceous, lipid and nucleotide components remained the same between control versus treatment groups, relieving further support for the absence of nanotoxicity. The in vitro cytotoxicity and apoptosis studies performed in the previous study showed no significant cytotoxic-ity or apoptosis in healthy (FHs 74 Int cells) [22]. We also compared the number of RBCs in the treatment groups and in control mice and found that AEC-CCo-CP-Fe-bLf NC-treated mice were healthy with improved blood cell counts.

A number of in vivo studies were conducted to vali-date the effect of AEC-CCo-CP-Fe-bLf NCs and void NCs on tumor size. The results mimicked the in vitroresults and were in accordance with our previously pub-lished results [22]. In addition, the phenomenal increase in anticancer efficacy of Fe-bLf incorporated in AEC-CCo-CP NCs could be attributed to improved delivery and controlled release of the protein. We also exam-ined the effect of Fe-bLF NCs on tumor recurrence and found that the subsequent challenge of all mice groups with cancer cells showed no signs of tumorigenesis in the Fe-bLf NC treatment groups. Co-localization of Prussian blue staining along with CD31-positive stain-ing was observed to be accumulated into tumor tissues, which indicated enhanced permeability and retention effect. Furthermore, histochemical staining in organs involved in the reticuloendothelial system such as the liver, kidney and spleen showed positivity, which supported the biodegradability sites for these NCs.

Previous studies have shown that Lf led to an increase in the expression of LRP1 receptor to medi-ate endocytosis [56]. We also showed that Fe-bLf, when taken orally, led to higher LRP1 level in the intestine and tumor tissues compared with untreated cells. Real-time PCR analysis confirmed downregulation of survivin and Bcl2, whereas proapoptotic Bax, Fas and caspases were upregulated and promoted apopto-sis in the tumors. This was further confirmed by the TUNEL assay. Taken together, these results give infor-mation regarding the mechanism of internalization and induction of apoptosis for AEC-CCo-CP-Fe-bLf NCs (Figure 6). Fe-bLf worked better when compared with AEC-CCo-CP-Fe-bLf NCs in downregulating the stem cell markers CD133, CD44 and CD166 (in tumor tissues); however, the data was obtained at 24 h period. Thus, Fe-bLf NCs may have failed to impart considerably higher effect due to their sustained release profile. The confocal images showed clearly that the Fe-bLf NCs co-localized with the stem cell markers and lowered their expression. The level of EpCAM was observed to be higher in both Fe-bLf and Fe-bLf NCs than control. The effect of Fe-bLf on EpCAM needs to be verified further and therefore, this phenomenon may be of interest for several ongoing studies.

It is known that the maintenance and control of cellular iron homeostasis is critical to avoid iron defi-ciency and iron toxicity. This task is mainly coordinated by a family of cytosolic RNA proteins known as iron regulatory proteins. As described in our previous study [18], binding and release of iron by bLf is accompanied mainly by the domain movements that close or open the iron-binding sites in the N -and C-lobes. The dis-sociation of iron from bLf occurs only at very low pH (2.2), whereas the protein remains intact at higher pH

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Fe-bLfAEC-CCo-CP-Fe-bLf NCs diet

AEC-CCo-CP-Fe-bLf NCs diet

Control diet untreated

Control diet untreated

i iii

iv v vi

vii ixviii

ii

Figure 6. Alginate-enclosed chitosan-conjugated calcium phosphate, iron-saturated bovine lactoferrin nanocarriers/nanocapsules inhibits survivin, stem cell markers and angiogenesis. See facing page for legend.



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Figure 6. Alginate-enclosed chitosan-conjugated calcium phosphate, iron-saturated bovine lactoferrin nanocarriers/nanocapsules inhibits survivin, stem cell markers and angiogenesis (facing page). (A) Endogenous expression of survivin and survivin splice variants were analyzed and found to be decreased using quantitative PCR from tumor tissues. (B) Decreased expression of stem cell marker genes (CD133, CD44 and CD166) was observed in the mice treated with the AEC-CCo-CP-Fe-bLf NCs diet. (C) Using real-time PCR, the fold change in expression of various miRNA responsible for iron metabolism was studied in the intestine, liver, spleen and brain with treatments of Fe-bLf NCs. (D) Visualization of blood vessel density in representative tumor sections was observed under confocal microscope. (E) TUNEL assay performed on the tumor sections. Experiments were repeated three-times and the images represent five different fields.AEC: Alginate enclosed; bLf: Bovine lactoferrin; CCo: Chitosan conjugated; CP: Calcium phosphate; FITC: Fluorescein isothiocyanate; NC: Nanocarrier/nanocapsule; trt: Treatment.



ranges. Only the stomach has a pH level of 2.2, but as mentioned in the above study, 20% iron saturated Lf is easily digested in the stomach, whereas 100% Fe-bLf is not easily digested. Moreover the alginate and chitosan outer coating in our NC system protects pH-based deg-radation of Fe-bLf.

In our previously published report [18], we have also found that 1.2% (w/w) diet of Fe-bLf raised the iron lev-els from 0.0053% to just 0.0069%, which was statisti-cally insignificant (p 0.05.) It has also been shown in this study and previously [22] that Fe-bLf enters the cells

mainly by LRP and TfR receptors, which are mainly overexpressed in tumor. Moreover, Fe-bLf seems to fol-low an additional internalization mechanism in tumors by invading the tumor vasculature (enhanced perme-ability and retention effect). In addition, as shown in the present study, apart from the tumor, the majority of Fe-bLf is localized in the spleen and liver; both of these organs are capable of metabolizing the iron. Therefore, there is no question of increase in the metal ion depo-sition. In pharmacokinetic studies conducted in our recently published study [22], we have shown that oral

Table 3. Comparative cytokines levels in wild-type C57 and nude mice for immune cell differentiation or activation of immune cells for immune responses/inflammation.

Cytokines (pg/ml)

Wild-type mice Nude mice

AEC-CCo-CP-Fe-bLf NCs (pg/ml)

Control diet (pg/ml) AEC-CCo-CP-Fe-bLf NCs (pg/ml)

Control diet (pg/ml)

IL-1 5 ± 2 10 ± 2 32 ± 6 10 ± 2

IL-6 285 ± 48 200 ± 25 85 ± 8 110 ± 5

IL-18 58 ± 31 5 ± 2 55 ± 13 5 ± 2

IL-12 650 ± 120 250 ± 20 70 ± 20 50 ± 12

IL-10 345 ± 35 234 ± 21 35 ± 5 34 ± 6

IL-15 50 ± 5 32 ± 5 20 ± 5 22 ± 5

IFN-1 45 ± 5 25 ± 5 25 ± 5 20 ± 5

IFN-1 16 ± 5 10 ± 4 12 ± 5 6 ± 2

IL-2 125 ± 15 20 ± 5 25 ± 5 13 ± 4

IL-4 24 ± 6 14 ± 6 12 ± 3 4 ± 2

IL-5 2 ± 0 8 ± 3 2 ± 0 7 ± 2

IL-17 10 ± 3 3 ± 1 5 ± 3 4 ± 1

IL-13 7 ± 2 15 ± 5 5 ± 2 10 ± 4

IFN- 15 ± 2 5 ± 2 5 ± 3 2 ± 0

IL-9 10 ± 2 12 ± 5 6 ± 2 10 ± 3

GM-CSF 22 ± 5 26 ± 5 8 ± 3 16 ± 4

IL-7 27 ± 5 16 ± 5 7 ± 2 6 ± 2

IL-11 11 ± 5 13 ± 5 6 ± 2 7 ± 3

IL-3 5 ± 5 12 ± 5 4 ± 2 6 ± 3

TGF 20 ± 5 10 ± 5 12 ± 4 6 ± 2

NO ( M/ml) 25 ± 5 20 ± 5 12 ± 4 10 ± 4

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Figure 7. Mechanism involved in internalization of alginate-enclosed chitosan-conjugated calcium phosphate, iron-saturated bovine lactoferrin nanocarriers/nanocapsules and their anticancer efficacy (facing page). See facing page for legend.

GI tract

Liver

Shielding

from

acidic pH

Duodenum

Shedding of alginate layer

in intestinal lumen

Intestinal

cells

DMT1

LRPs

Microvilli

Bloodstream

Capillaries

TissueEnhanced permeability

and retention effect

TFR

LFR

Single cell

Endocytosis

NC uptake

ACSC-NC-Fe-bLf

FAS

TfREpCAM CD44

CD133

LRP

Controlled

release

Pro-caspase-8

Pro-caspase-9

Caspase-8

Caspase-3

Caspase-9

Cytochrome C

DNA fragmentation

Mitochondrial

depolarization

Survivin and

survivin

isoforms

Lysosome

Bcl-2 Bcl-xl Bax

ApoptosisDNA

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Figure 7. Mechanism involved in internalization of alginate-enclosed chitosan-conjugated calcium phosphate, iron-saturated bovine lactoferrin nanocarriers/nanocapsules and their anticancer efficacy (facing page). (A) Representation of Fe-bLf NCs, where it is shown that since iron absorption receptors are present in the small intestine, the maximum uptake of NCs occurs there via the DMT1and LRP receptors. The NCs enter the bloodstream and reach the tissues via enhanced permeability and retention effect where they are finally taken up by the individual cells, mainly via the transferrin and the lactoferrin receptors or by pinocytosis. (B) bLf is a ligand for TfR and LRP receptors; the CCo-CP-Fe-bLf NCs that are formed from AEC-CCo-CP-Fe-bLf NCs in the intestine are endocytosed into the cancer cells using these receptors. Once inside, Fe-bLf downregulates antiapoptotic genes, stem cell markers and upregulate the proapoptotic genes. Since Fe-bLf is an immunomodulatory molecule, integration of extrinsic and intrinsic pathways of apoptosis led to enhanced cytotoxicity in the cancer cells.ACSC: Alginate-enclosed chitosan-coated; AEC: Alginate enclosed; bLf: Bovine lactoferrin; CCo: Chitosan conjugated; CP: Calcium phosphate; NC:Nanocarrier/nanocapsule.



delivery of Fe-bLf ensured presence of Fe-bLf 3 h post feeding, and the levels of Fe-bLf was not detectable beyond 72 h, thus ensuring clearance of protein from the body.

Recently, a class of small noncoding RNA, known as miRNA, has also been implicated in the control of iron metabolism [57]. Several studies have investigated the effect of miRNA subtypes on TFR-1 expression show-ing that miRNA-320 expression can suppress TFR-1 expression and cell proliferation [58]. Some metal iron transporter genes such as DMT1 have been identified, which is under the regulation of miR-Let-7d. It has also been found that miR-Let-7d participates in regulation of iron metabolism by targeting DMT1 [59]. A study has shown that Lf gene expression and function are targeted by miR-214 in HC11 and MCF7 cells. Overexpression of miR-214 markedly decreased Lf expression and inhi-bition of endogenous miR-214 expression increased Lf expression and cellular apoptotic activities [31]. On the contrary, there are some studies that also confirm that iron chelators promote the processing of miRNA pre-cursors and that cytosolic iron can regulate the activity of miRNA pathway through PCBP2 [60]. However, it is well established that the majority of iron functions as an oxygen carrier in heme groups of hemoglobin and myoglobin in the body and that large amounts of unbound iron can be highly toxic to cells. The average iron concentration in men is 50 mg/kg and in women is 35 mg/kg. Iron is stored in cells in the form of fer-ritin, which protects cells from its toxic effects and that iron is transported in plasma mainly bound to Tf. Non-heme iron is mostly absorbed in the duodenum where ferric iron (Fe3+) in food is reduced to Fe2+ by the cyto-chrome reductase DcytB and then transported across the apical membrane into the enterocyte by DMT1 [61].The miRNA responsible for iron homeostasis in mice has been identified as the liver-specific miR-122 [62].miR-122 expression is lower in the liver; however, the decrease is statistically insignificant and it is consider-ably more lower in the intestine. According to a study, miRNA analysis has revealed that the most abundant miRNA in liver metastasis, when compared with pri-mary tumors, was miR-122. Using immunohistochemi-cal analysis, it was also shown that the expression levels

of CAT1, a negative target gene of miR-122, was lower in liver metastasis than primary tumors (p < 0.001) [63]. Therefore, according to the abovementioned study, Fe-bLf NCs lower miR-122 in the liver and intestine and therefore lower the chance of metastasis. It has also been shown that miR-584 regulates expression of the Lf receptor in Caco-2 cells and in mouse small intes-tine [30]. Considering the abovementioned information, we selected all the major miRNAs responsible or related with iron metabolism and we checked their expression in the intestine, liver, spleen and brain in order to under-stand the effect of Fe-bLf on miRNA expression. One of the key findings of this study was inferring expression of miRNA types in the abovementioned mouse body parts.

Previous reports have shown that the orally adminis-tered bLf has significantly suppressed 3LL cell-induced angiogenesis [64], which is in accordance with our previ-ous report, where Fe-bLf was shown to reduce pathologi-cal angiogenesis by downregulation of resting (CD31) and activated (CD105) blood vessels along with blood flow (DiOC7). Thus, measurement of blood vessel den-sity was carried out by counting CD31 (neoangiogen-esis: resting) and CD105 (early angiogenesis: activated) blood vessels in control and tumors that developed in NC-treated mice (treated tumors) [65]. Both untreated and taxol-treated controls (where tumors developed after the second challenge) showed high blood vessel density compared with the AEC-CCo-CP-Fe-bLf NC-treated tumors. Antiangiogenic properties of the NCs were con-firmed with a significant decrease in blood vessel density as per the treatment.

Taken together, these results indicate enhancement of antitumor efficacy of 100% iron-saturated Lf upon incorporating them in NCs in order to develop an oral therapeutic system for use in a human colon can-cer xenograft model (Figure 7). In addition, our study revealed that AEC-CCo-CP-Fe-bLf NCs not only kills the colon cancer cells and colon cancer stem cells, but also replenishes the body iron and calcium content without inducing any nanotoxicity.

ConclusionWe were able to further enhance the antitumor efficacy and bioavailability of Fe-bLf by slightly modifying the

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previously published nanoformulation. In vivo studies with respect to nanotoxicity, using a xenograft model of nude mice, Fourier transform infrared spectroscopy and tissue pathology designated the safe and biocompatible nature of these NCs. The specific receptors responsible for the uptake of Fe-bLf NCs and the specific miRNAs responsible for the iron regulation in various body parts of mice were reported here for the first time. The Fe-bLf NCs also showed substantial reduction in angiogen-esis markers and showed efficient interaction with and downregulation of cancer stem cell markers. Thus, the present nanomedicine-based protein therapy is a prom-ising therapeutic for targeting colon cancer, as well as cancer stem cells.

Future perspectiveWe have shown by means of the previously published report (refer to the text) and the present study that AEC-CCo-CP-Fe-bLf NCs form a precise system for oral therapy of colon cancer. However, the capability of these NCs should be put to further test in other cancer models and in clinical trials to validate its therapeu-tic potential. Nanomedicine is the future of medicine and therefore apt and validated NC systems must be put to trial as they bring forth modern technology and limitless therapeutic capability. In order to further increase the therapeutic potential of these NCs, they

can be further modified using targeting ligands in order to achieve cancer specific or target specific cytotoxicity.

AcknowledgementsThe authors would like to thank N Branson for his assistance

with animal work and for his technical support and guidance

thank the upper animal house staff, in particular T Thorpe,

A Cooper and B Newell for providing help in handling the

animals.

Financial & competing interests’ disclosureThe authors would like to thank the Australia–India Strate

gic Research Fund (AISRF) and National Health and Medical

Research Council (NHMRC) for financial support. The authors

have no other relevant affiliations or financial involvement

with any organization or entity with a financial interest in or

in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this

manuscript.

Ethical conduct of researchThe authors state that they have obtained appropriate in

stitutional review board approval or have followed the prin

ciples outlined in the Declaration of Helsinki for all animal

Executive summary

BackgroundIn our previous study, we have shown that polymeric ceramic nanocarriers (alginate-enclosed, calcium phosphate, iron-saturated bovine lactoferrin nanocarriers/nanocapsules [AEC-CP-Fe-bLf NCs]), when given orally, were predominantly present in tumor tissues and that they helped in the absorption of iron and may have a role in enhancing iron uptake during iron deficiency. We have also shown that these NCs have the ability to elevate the red blood cell content in mice.

AimWe intended the better biodistribution and superior anticancer efficacy of the prepared nanoformulation compared to previously synthesized AEC-CP-Fe-bLf NCs. We thoroughly evaluated the nanotoxicity and confirmed the mechanism of internalization of the Fe-bLf NCs. We also studied the effect of our NCs on iron, calcium and zinc levels and further investigated the role of miRNAs in iron regulation.

Outcomes of the studyThe synthesized nanoformulation showed effective anticancer efficacy without inducing any nanotoxicity.The Fe-bLf NCs showed substantial reduction in expression of cancer stem cell markers and tumor spheroid size in triple-positive (CD133-, EpCAM- and CD44-positive) cancer stem-like cells.The serum iron, zinc and serum calcium deposits were increased (1.27-, 1.21- and 1.15-fold, respectively) compared with control mice.LRP, transferrin and lactoferrin receptors were found to be responsible for internalization of the Fe-bLf NCs.Different types of miRNA were found to be responsible for iron regulation in different organs (miRNA-lET7D: brain; miR485: intestine; miR210: liver; and miR196: intestine).

ConclusionOur results confirmed that Fe-bLf NCs exploit various receptors (DMT1, LRP, lactoferrin receptors and transferrin receptors) and miRNAs in the body to internalize and does not induce any nonspecific nanotoxicity; it reinstates the depleted body iron and calcium levels. It has the ability to activate both extrinsic and intrinsic apoptotic pathway to induce apoptosis in cancer cells, and apart from inhibiting angiogenesis, Fe-bLf NCs also hold substantial capability to inhibit stem cell marker expression.

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