Biodistribution Analysis of Peptide-Coated Magnetic Iron Nanoparticles: A Simple and Quantitative Method

Abstract

Biodistribution tracks compounds or molecules of interest in vivo to understand a compound’s anticipated efficacy and safety. Nanoparticles deliver nucleic acid and drug payloads and enhance tumor permeability due to multiple properties such as high surface area to volume ratio, surface functionalization and modifications. Studying the in vivo biodistribution of nanoparticles documents the effectiveness and safety and facilitates a more application driven approach for nanoparticle development that allows for more successful translation into clinical use. In this study, we present a relatively simple method to determine the biodistribution of magnetic iron nanoparticles in mice. In vitro, cells take up Branched Amphiphilic Peptide coated Magnetic Nanobeads (BAPc-MNBs) like their counterpart i.e., Branched Amphiphilic Peptide capsules (BAPCs) with a hollow water-filled core. Both BAPc-MNBs and BAPCs have widespread application as a nano-delivery systems. We evaluated the BAPc-MNBs tissue distribution in wildtype mice injected intravenously (i.v.), intraperitoneally (i.p.) or orally gavaged to understand the biological interactions and to further the development of branched amphiphilic peptides-based nanoparticles. The magnetic nanoparticles allowed collection of the BAPc-MNBs from multiple organs by magnetic bead sorting followed by a high throughput screening for iron content. When injected i.v., nanoparticles distributed widely to various organs before elimination from the system via the intestines in feces. The spleen accumulated the highest amount of BAPc-MNBs in mice administered the NPs i.v. and i.p. but not via oral gavage. Together, these data demonstrate not only that the magnetic sorting allowed quantification of the BAPc-MNBs but also identified the distribution of BAPc-MNBs after distinct administration methods.

Graphical Abstract

1. Introduction

Highly desirable nanoparticles (NPs) effectively deliver drugs, nucleic acids and theragnostic agents with application in multiple fields including cancer therapy^1–3, vaccine delivery⁴, and gene therapy.⁵ The high surface area to volume ratio of NPs facilitates delivery of higher drug loads and higher tumor accumulation due to the enhanced permeability⁶ and retention effect. Importantly, NPs may be engineered for controlled release of drugs and targeted therapy. Although the field of nanotechnology has advanced significantly, only incremental advances occurred in the translation to clinical use.⁷ NPs encounter a very complex environment in vivo, which influences the tissue distribution, bioavailability, and clearance from the system.^{8, 9} Studying the bio-interactions and biodistribution allows development of an application driven nanoparticles rather than a formulation driven approach. The application driven approach increases the clinical use and success rate of nanoparticles compared to a formulation driven approach.¹⁰ Besides, the majority of NPs accumulate in the liver which may lead to hepatotoxicity.^11–13 Thus, biodistribution studies are of great importance in the growing field of nanomedicine.

The cationic Branched Amphiphilic Peptides self-assemble in an aqueous solution to form peptide bilayer delimited spherical capsules called Branched Amphiphilic Peptide Capsules (BAPCs).^14–18 BAPCs are promising biodegradable¹⁴ nano-delivery systems that deliver dsRNA, siRNA, plasmid DNA, mRNA¹⁹ and radioactive nuclides.^{15, 16, 18, 20} Conjugating branched amphiphilic peptides to the surface of metallic nanoparticles such as gold²¹ and iron oxide nanoparticles²² expanded the applications of the peptide-based delivery system and provided opportunities to study the surface properties of the peptide bilayer delimited nanoparticles. Our previous study demonstrated that the branched amphiphilic peptide forms a bilayer on 50 nm magnetic iron nanobeads that are monodispersed in water and saline. Importantly, both endothelial and macrophage cell lines endocytose the Branched Amphiphilic Peptide bilayer coated Magnetic NanoBeads (BAPc-MNBs).²² In the current article we explore the biodistribution of BAPc-MNBs in mice using a novel assay.²²

Previous studies indicated that without altering viability of mammalian cells in vitro, multiple endocytic cellular routes facilitate the uptake of BAPc-MNBs in a time dependent manner.²² Thus, BAPc-MNBs are suitable candidates for in vivo applications. The initial interactions, the overall pharmacokinetics and the biodistribution of NPs depend on the method of evaluation, and the tissues evaluated. Current biodistribution evaluation methods include qualitative whole animal visualization methods such as Infrared (IR) imaging, Computed Tomography (CT), Magnetic Resonance Imaging (MRI), Positron Emission Tomography (PET) or histopathology where the tissue is harvested and analyzed for nanoparticle traces.⁷ However, small animal IR, CT, MRI and PET are not available in many locations. In addition, conventional histopathology uses different stains or fluorescence tracking agents only available for specific types of nanoparticles and ligands Electron microscopy obtains detailed subcellular localization of nanoparticles with only limited use in studying in vivo distribution of nanoparticles. Quantitative biodistribution analyses utilize other expensive, frequently unavailable techniques such as Atomic Absorption Spectroscopy (AAS) and quantitative MRI.⁸ Biodistribution of drug containing nanoparticles are measured by the drug load in specific tissues. However, the drug distribution may not be synonymous to the nanoparticle distribution since the drug can be released prematurely and diffuse additional to tissue sites after nanoparticle deposition. Another popular quantitative biodistribution analysis method uses nanoparticles extracted from tissues followed by elemental analysis by ICP-MS that requires a mass spectrometer.²³ Thus, a simple, easy quantitative and low-tech method for quantifying NPs is needed.

Previously, we demonstrated that human metastatic cervical cancer cells¹⁸ readily take up BAPCs and delivered an HPV-16 oncoprotein encoding DNA to successfully reduce tumor cell proliferation in mice.²⁰ Therefore, based on the evidence, BAPCs have tumor penetrating abilities and the enhanced permeability effect of NPs. We hypothesized that melanoma tumor cells take up BAPc-MNBs take up in tumor bearing mice.

In the current study, we evaluated the BAPc-MNBs tissue distribution in wildtype mice injected intravenously (i.v.), intraperitoneally (i.p.) or orally gavaged to understand the biological interactions of the peptide NP for further development of branched amphiphilic peptides-based nanoparticles. We also established a new relatively facile method that does not require the use of high-end instruments for determining biodistribution of iron oxide nanoparticles. NP tissue distribution varied with route of application and were eliminate via the intestines and feces.

2. Materials and methods

2.1. Chemical reagents and materials

Ethanol (99% pure, Sigma, ChromSolv, Denatured ethanol), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (Acros Organics, ThermoFisher), Magnetic nanobeads (Ocean Nanotech, San Diego, CA), Trifluoroethanol (TFE) (Tokyo Chemical Industry Ltd.), 3-(2-Pyridyl)-5,6-di(2-furyl)-1,2,4-triazine-5’, 5”-disulfuric acid disodium salt (Ferene-s, Sigma Ultra), Proteinase K (Sigma-Aldrich), Matrigel Matrix^® (Corning), Isoflurane (Akorn Animal Health), Dulbecco’s modified Eagle’s medium (Gibco, Sigma-Aldrich), Opti-MEM-I (Gibco, Sigma-Aldrich), L-glutamine (GlutaMAX, Sigma-Aldrich) B16F10 melanoma cell line (ATCC), pH 7.4 phosphate buffered saline (PBS) with Ca²⁺ and Mg²⁺, L-ascorbic acid (Sigma-Aldrich), acetate buffer (Glacial Acetic acid, Fisher Scientific).

2.2. Synthesis of Branched Amphiphilic Peptide- Magnetic Nanobeads (BAPc-MNBs)

Branched amphiphilic peptide coated – magnetic nanobeads (BAPc-MNBs) were synthesized as described in Natarajan et al.²² Briefly, the magnetic nanobeads were coated with a peptide bilayer by forming one peptide layer at a time on the surface of the 50nm nanoparticles. The peptide monolayer was formed on the surface by covalently binding the cysteine residue on the C-terminus of the peptide bis-(Ac-FLIVIGSII)-KKKKK-C-CONH₂, to the maleimide groups on the magnetic nanobead surface in 75% Ethanol: HEPES solvent to prevent assembly of the peptides into spherical capsules. After washing off the excess peptides, the second peptide layer formation on the peptide monolayer coated MNBs was promoted by adding two-fold excess of bis-(Ac-FLIVIGSII)-KKKKK-CONH₂ and adding water. The peptides self-assemble on the surface of the nanoparticles in the presence of water due to hydrophobic interactions. After sitting for 20–30 minutes on the magnetic rack, the BAPc-MNBs were carefully collected and was concentrated on a rotavapor with a 40 °C water bath. BAPc-MNBs were re-dispersed in water alone. After overnight incubation at 4 °C the BAPc-MNBs were extruded through a sterile 0.22 μM syringe filter (Millex-GS, Millipore-Sigma). This sterilizes the BAPc-MNBs and excludes any large aggregated BAPc-MNBs. The concentration of BAPc-MNBs was adjusted appropriately after quantification by ferene-s assay as stated below, such that the same lot of BAPc-MNBs were administered as high and low dose to the mice.

2.3. In vivo studies.

C57Bl/6 mice (Jackson Laboratory) were bred and maintained in the Division of Biology at Kansas State University. Male and female C57Bl/6 mice were kept in a 12 h light/dark cycle with constant access to rodent food and water. Four-month-old male BALB/c mice were purchased from Charles River, individually housed at NWRC and maintained on a 12 h light/dark cycle with access to standard mouse chow and other forms of enrichment such as apple slices. All procedures were approved by the Institutional Animal Care and Use Committee (IACUC) and were in compliance with the Animal Welfare Act.

2.4. Intravenous injection of nanoparticles in C57BL/6 mice with or without tumors

C57Bl/6 mice (n=39) were anaesthetized using isoflurane (2–3% in oxygen) prior to the injection of 100 μL of nanoparticles dispersed in 0.15 M saline i.v. Mice were randomly assigned to one of 4 groups. The mice received either (i) low dose of BAPc-MNBs (2×10¹⁰), (ii) high dose BAPc-MNBs (1×10¹¹), (iii) Control MNB beads (2×10¹⁰) without peptide coating or saline (Table 1). Each group receiving MNB consisted of 8 mice while the saline control group contained 6 mice. The deeply anesthetized animals were exsanguinated and euthanized by cervical dislocation prior to collecting the tissues (spleen, lungs, kidneys, heart, brain, intestines, thymus, tumor, blood, and urine,) either 24 h or 48 h after injection. The organs were weighed, washed in saline, snap frozen in liquid nitrogen, and stored at −80°C for further analysis. The treatments that the mice were subjected to is indicated in Table 1. Feces were collected every 12 h over 48 h from additional mice injected with saline (N=3), low dose (n=3) or high dose (n=3) BAPc-MNBs.

Table 1.

Treatment of C57BL/6 mice with magnetic nanoparticles

Treatment	Without tumor		With tumor		Total
	24 h	48 h	24 h	48 h
Saline Control	6		6		12
2×1010 BAPc-MNBs (Low dose)	4	4	4	4	16
1×1011 BAPc-MNBs (High dose)	4	4	4	4	16
2×1010 control beads (Low dose)	4	4	4	4	16
					60

Addition C57Bl/6 mice (n=30) of the same groups were injected into melanoma tumor containing mice. B16F10 (ATCC) mouse melanoma tumor cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% OptiMEM-I^®, 5% FBS, 5% NuSerum, and 2mM L-Glutamine. Two million (2 × 10⁶) B16F10 cells suspended 1:1 in Matrigel were injected sub-cutaneous on the ventral side in the thoracic region of the mice. The mice were weighed, and tumor growth monitored daily. Tumor size was measured and recorded using vernier calipers as (length × width²)/2. The NPs were injected between 7 to 8 days after tumor cell injection and the tumor was excised 24 h or 48 h after injection of NPs and the ex-vivo tumor volume (Length × Width × Height) was calculated.

2.5. Oral gavage mice

Eighteen (n=18) in BALB/c mice animals were fasted overnight with access to water ad lib. Mice were lightly anesthetized with isoflurane and gavaged with high dose (2×10¹¹) of BAPc-MNBs in a max volume of 200 μL. Mice were euthanized at 24 h post-gavage (n= 6), at 48 h post-gavage (n= 6) or at 48 h post-sham gavage (n=6). Tissues were removed snap frozen and shipped on dry ice to Kansas State University for analysis.

2.6. Intraperitoneal injected mice

For i.p. injections, wildtype Balb/c mice (n=18) were briefly restrained, appropriate landmarks identified, and the lower right quadrant sterilized with isopropyl alcohol. Using a 25–30-gauge needle, high dose (2×10¹¹) BAPc-MNBs were administered to mice in a volume of ≤ 1% kg body weight (n = 12) or saline was administered to the control group (n = 6). Mice were returned to their individual cages and monitored until normal behavior was observed. Organs were harvested from each treatment group at 24 h or 48 h after administration.

2.7. Quantification of BAPc-MNBs by Ferene-s assay in mouse tissue

The tissues were homogenized in 0.1M tris buffered saline (TBS) containing 1% Tween-20 (Sigma-Aldrich) using a pre-programmed gentleMACS^™ dissociator (Miltenyi Boitec). The tissue homogenate was collected in 1.5mL Eppendorf vials and placed on a magnetic separator (Permagen) overnight for separation and collection of magnetic nanoparticles. The magnetic beads were resuspended in DI water containing five μL of 5 mg/mL Proteinase K (Sigma-Aldrich) to digest any protein bound to the NPs. The magnetic nanobeads were aliquoted into a 96 well plate and placed on handheld magnets overnight for separation at 37 ℃. The magnetic beads were washed in DI water and quantified using the ferene-s assay as described previously in Natarajan et al.²² adapted to the Ferene-s chromophoric assay described in Hedayati et al.²⁴ The feces collected were weighed in 1.5mL pre-weighed tubes and treated with 300 μL of 5N HCl followed by heating for 30 min at 95°C and 700 μL of DI H₂O was added to each tube. The debris was spun down on a benchtop centrifuge and the supernatant was collected for Fe-content analysis. Briefly, the 3-(2-Pyridyl)-5,6-di(2-furyl)-1,2,4-triazine-5’, 5”-disulfuric acid disodium salt working solution was prepared by mixing 10 mL of 5 times working buffer, 2 g L-ascorbic acid in 11 mL 2 M Acetate Buffer, and 500 μL of 0.5 M Ferene-s in DI H₂O (0.5 g in 2 mL water). The volume was subsequently brought up to 50 mL with DIH₂O. Ferene-s working solution (200 μL/well) was added and allowed to incubate overnight. The iron was quantified using the standard curve generated for each 96 well plate using Microsoft Excel. The statistical analysis was performed using GraphPad Prism. The iron content was normalized using the weight of the tissue.

2.8. Dynamic light scattering (DLS) and Zeta potential analysis

BAPc-MNBs (50 nm) and control beads were resuspended in sterile DDI H₂O to a final concentration of 6 × 10¹¹ particles/mL. The BAPc-MNBs were diluted to 1.2 × 10⁹ in Dulbecco’s Modified Essential Media (DMEM) with or without 10% FBS. Dynamic light scattering (DLS) and zeta potential (ZP) analyses were performed for nanoparticles in 10 mm path length cuvettes (Sarstedt^® Standard Cuvettes)) for DLS and Malvern Panalytical Folded Capillary Zeta Cell for ZP measurement, respectively, on a Zetasizer Nano ZSP (Malvern Instruments Ltd., Westborough, MA).

2.9. Transmission Electron Microscopy (TEM)

Five μL of undiluted NPs samples (50 nm BAPc-MNBs) were spotted on Parafilm paper. Individual grids (Lacey F/C 200 mesh Au) were carefully placed on the surface of each NP sample for ~ 5 min. Grids were then sequentially washed with 20 μL deionized water on the parafilm. Excess sample or water were removed by gently putting the side edge of grids in contact with Kim wipes. Grids were allowed to dry overnight at ~ 50°C in petri dishes. For imaging, grids were mounted in specimen holders specific for TEM. Conditions for imaging were set to 25 KV on a SEM Model S-4800 (Hitachi) or adjusted occasionally according to quality of images.

3. Result and Discussion

3.1. BAPc-MNBs characterization and high throughput screening of tissue for Fe content

Previously, BAPc-MNBs exhibited a hydrodynamic size of ~200 nm by DLS analysis and approximately 50 nm by TEM analysis.²² In this study, we synthesized, purified, and characterized BAPc-MNBs by TEM and Dynamic Light Scattering and Zeta Potential Analysis similar to our previous studies and in physiological media, DMEM and DMEM + FBS. The zeta potential and polydispersity index for BAPc-MNBs in are indicated in Table 2. Similar to previous results, BAPc-MNBs appeared to be in homogenous solution in any solution tested with the polydispersity index of <0.2 in water, <0.3 in DMEM and 0.33 in DMEM with FBS. The zeta potential indicated positively charged nanoparticles in water (+23mV) that became slightly negatively charged in DMEM (−7.7) without or with FBS (−10.0). The zeta potential results suggest that in physiological media, the BAPc-MNBs are coated with a possible corona effect in sera. These data suggest that in physiological media, BAPc-MNBs endocytosis may be receptor-mediated rather than charge-mediated.

Table 2.

BAPc-MNB analysis of hydrodynamic diameter, polydispersity index and Zeta potential

	Hydrodynamic diameter (nm)	Polydispersity Index	Zeta potential (mV)
DMEM	74.34 ± 17.07	0.31 ± 0.02	−3.4 ± 0.28
DMEM+10% FBS	16.83 ± 0.06	0.27 ± 0.003	−0.73 ± 0.67
BAPc-MNB in DMEM	594.98 ± 656.45	0.28 ± 0.08	−7.67 ± 0.31
BAPc-MNB in DMEM + FBS	1452 ± 1177.39	0.33 ± 0.05	−10.03 ± 0.05

3.2. Tissue distribution of in wild type C57BL/6 mice

The screening of mouse tissue for Fe was performed as described in methods and as depicted in Scheme 1. After administering low (~2 ×10¹⁰) and high dose (1×10¹¹) nanoparticles i.v. to mice, we quantitatively assessed the biodistribution of BAPc-MNBs based on whole organ iron content at 24 h and 48 h post injection and expressed it as microgram of iron detected per gram of tissue/organ. As each organ contains distinct iron quantities normally, saline injections (black bars) represent the background iron in the tissue in all figures. Most organs from saline treated animals contained an average of 1.73–3.86 ± 0.829–1.768 (SEM) μg Fe/g of tissue. The intestine contained 6.48±3.319 μg and as expected, the spleen contained significantly more with an average of 11.57 ± 3.83 μg Fe/g of tissue. At 24 h after injection, both low and high dose BAPc-MNBs localized primarily in the spleen with higher levels at 24 h and significantly reduced levels at 48 h (Figure 1B, D). The average iron content detected was proportional to the dose of BAPc-MNBs injected i.e., the organs of high dosed mice contained more iron per gram than the low dosed mice. The iron content ranged from ~75 μg/g −350 μg/g in the spleen and from ~10 μg/g −20 μg/g in the lungs and heart. Compared to saline, at 24 h the lungs, heart, and intestines contained significant NPs (Figure 1A, C, black bars vs blue and red bars). In addition, the intestines of mice injected with high dose BAPc-MNBs contained significantly higher iron content than saline 48 h after injection. Thus, BAPc-MNBs localized to the spleen, lungs, heart and intestines in significantly high amounts when injected i.v. in C57BL/6 mice.

Scheme 1.

Schematic depiction of the extraction of Magnetic Nanobeads from whole organ and High throughput screening for Iron content in whole organ. (Schematic created using ©Biorender)

Figure 1. Tissue distribution of BAPc-MNBs injected i.v. in C57BL/6 mice.

Fe content per gram ± SEM of whole organ harvested 24 h (blue and red bars) and 48 h (yellow and green bars) after i.v. injection with 2×10¹⁰ (low dose) BAPc-MNBs (A, B) and with 1×10¹¹ (high dose) BAPc-MNBs (C, D). Black bars represent Fe content per gram of organs from saline control treated mice. n ≥ 3, statistical hypothesis was tested with 2-way ANOVA (A, C) and 1-way ANOVA (B, D) statistical analysis, Dunnett’s multiple comparison test. p-value: * <0.05, ** <0.01, *** <0.001, **** < 0.0001 compared to saline control.

The magnetic nanoparticles could not be detected in significant amounts in the blood (Supplementary Figure 1). Since the intestines showed significantly higher iron content after i.v. injection at either 24 h (low dose BAPc-MNBs) or 48 h (high dose BAPc-MNBs), it was likely that the NPs were being excreted in the feces. To test this hypothesis, feces were collected every 12 h from 3 mice per treatment group- saline control, low dose BAPc-MNBs and high dose BAPc-MNBs i.v. injected mice. The Fe content in feces of mice injected with low dose BAPc-MNBs was less than or equal to saline control suggesting that either all NPs were sequestered in other organs, or the iron was excreted prior to 24 h as suggested by the slightly (but not significant) elevated levels at the 12 h time point and decreased at 24 h prior to returning to saline levels at 36 and 48 h. Based on the data analyses presented in Figure 1A and B, the most plausible explanation is that BAPc-MNBs are sequestered in the organs at least up to 24h after i.v. injection and thus, not detected in feces. By 48h BAPc-MNBs (Fig. 1C and D) were not detected in significant levels in organs or feces of the low dosed mice which indicates that the Fe levels were below the detectable limits of the assay.

The Fe content in the feces was significantly higher than saline control after 24 h of injection in mice dosed with 10¹¹ (high dose) of BAPc-MNBs (Figure 2) and was maintained for at least 48 h after injection. This is consistent with the observed increase in iron content in the mouse intestines (Figure 1C) and suggests that BAPc-MNBs are eliminated via feces. By extension, it is likely that the BAPCs with the hollow water filled core may also be excreted in the feces.

Figure 2. Iron content in mouse Feces -.

Fe content per gram ± SEM of feces was determined in mouse feces collected at 12 h intervals after administering BAPc-MNBs at low (2E10^10; red bar) or high (10¹¹; green bar) dose. Black bars represent Fe content per gram of feces from saline control treated mice. n = 3, 2-way ANOVA statistical analysis, Tukey’s multiple comparison test for statistical hypothesis testing. p-value: * <0.05, ** <0.01, *** <0.001, **** < 0.0001

3.3. Tissue distribution of BAPc-MNBs in melanoma tumor bearing C57BL/6 mice

Previous studies suggested that BAPCs target tumor cells.^{18, 20} BAPc-MNBs have the same surface chemistry as BAPCs with a hydrodynamic size of ~200 nm. These characteristics enhance the permeability effect⁶ increasing tumor uptake and therefore, BAPc-MNBs may target tumor cells as well. To test this hypothesis, mice were injected with B16F10 melanoma cells 7–8 days prior to injecting BAPc-MNBs for determining NP tissue distribution at 24–48 h after injection. At both time points, tumor containing mice displayed a similar tissue distribution as the non-tumor bearing mice despite some changes in quantity. A significant proportion of BAPc-MNBs were localized in the spleen followed by the lungs and heart (Figure 3 A, B, D, E). The major difference between the tumor and non-tumor mice was the quantity of localized magnetic nanoparticles. The distribution of beads was not consistent with the dosage of NPs administered since the spleen, lungs, liver and excretory organs showed comparatively similar levels of iron content, at 24 h and 48 h after injection of low or high doses of BAPc-MNBs.

Figure 3. Tissue distribution of BAPc-MNBs injected i.v. in B16F10 melanoma tumor bearing mice –

Iron content ± SEM in major organs (A, D), spleen (B, E) and tumor (C, F) was determined 24 h (blue and red bars) and 48 h (yellow and green bars) after injecting low dose (2×10¹⁰) (A, B, and C) of BAPc-MNBs and high dose (1×10¹¹) (D, E, and F) of BAPc-MNBs. Black bars represent Fe content per gram of organs from saline control treated mice. n = 4, 2-way ANOVA (A, D) and 1-way ANOVA (B, C, E, and F) statistical analysis, Dunnett’s multiple comparison test for statistical hypothesis testing. p-value: * <0.05, ** <0.01, *** <0.001, **** < 0.0001

Although significantly elevated compared to saline, the spleen of mice treated with a high dose of BAPc-MNBs for 24 h showed significantly (p < 0.001) fewer NPs in the melanoma tumor bearing mice in comparison to the mice without tumors (Figure 1, 3). Iron content was increased in tumors harvested 24 h after treatment of mice with low dose of BAPc-MNBs only (Figure 3C, F).

3.4. Tissue distribution of BAPc-MNBs injected i.p. and administered orally in BALB-c mice

To examine the effect of route of administration, BAPc-MNBs were injected i.p. or orally gavaged into additional mice. Compared to i.v. injection, i.p. or oral administration of BAPc-MNBs resulted significantly fewer NPs collected from all tissues. After 24 h post i.p. injection, no organs tested showed significantly more Fe compared to saline control, although a few BAPc-MNBs were detected in the spleen. At 48 h post i.p. injection, the majority of BAPc-MNBs were detected in the spleen and to some extent in the stomach and intestines (p=0.0725) (Figure 4A, B, C). At 48 h after administration BAPc-MNBs accumulated significantly only in the spleen and not detected in significant amounts in other organs tested.

Figure 4. Tissue distribution of BAPc-MNBs injected IP in BALB/c mice –

Iron content ± SEM in major organs (A), the spleen (B) and the gastrointestinal system (C) was determined 24 h (blue bars) and 48 h (yellow bars) after injecting high dose (2×10¹¹) of BAPc-MNBs. Black bars represent Fe content per gram of organs from saline control treated mice. n = 5, 2-way ANOVA (A, C) and 1-way ANOVA (B) statistical analysis, Dunnett’s multiple comparison test for statistical hypothesis testing. p-value: * <0.05, ** <0.01, *** <0.001, **** < 0.0001

BAPc-MNBs administered gavaged orally were only significantly increased in lungs after 24 h (Figure 5A). No significant difference was observed between saline treated and BAPc-MNBs gavaged mice in any other major organs including the stomach and intestines. (Figure 5A, B, C).

Figure 5. Tissue distribution of BAPc-MNBs administered orally to BALB/c mice –

Iron content ± SEM in major organs (A), spleen (B) and gastrointestinal system (C) was determined 24 h (blue bars) and 48 h (yellow bars) after administering high dose (2×10¹¹) of BAPc-MNBs. n = 5, 2-way ANOVA (A, C) and 1-way ANOVA (B) statistical analysis, Dunnett’s multiple comparison test for statistical hypothesis testing. p-value: * <0.05, ** <0.01, *** <0.001, **** < 0.0001.

3.5. Effect of Branched Amphiphilic Peptide coating on tissue distribution of magnetic nanobeads

As BAPCs appear to target tumors^{18, 20} and changed the quantity of NPs detected in the spleen (Figure 3B, E) in the presence of melanoma, we hypothesized that the peptide bilayer alters the biodistribution of the MNBs. To test this hypothesis, we injected 2×10¹⁰ MNBs or BAPc-MNBs prior to analyzing the biodistribution. In the absence of tumors at 24 h, mice injected with control beads i.e., no peptide bilayer coating contained significantly fewer beads in the spleen (Figure 6A). No other organs were significantly different from control beads in the absence of tumors at 24 h or 48 h (Figure 6A, B). However, control beads significantly increased in the intestines compared to saline at 24 h and 48 h after injection (Supplementary Figure 1)

Figure 6. Tissue distribution of BAPc-MNBs vs control beads injected i.v. in C57BL/6 mice with or without tumors –

Fe content per gram ± SEM of whole organ harvested 24 h and 48 h after i.v. injection with 2×10¹⁰ (low dose) BAPc-MNBs (A, C Black; B, D purple bars) and control beads (A, C white; B, D magenta bars) in mice without (A, B) and with tumors (C, D). n ≥3, statistical hypothesis was tested with 2-way ANOVA statistical analysis, Dunnett’s multiple comparison test. p-value: * <0.05, ** <0.01, *** <0.001, **** < 0.0001 compared to saline control.

In most tissues, tumor bearing mice injected with control beads showed significantly less iron than those without tumor (Figure 6C, D). Like the findings above, the spleen contained the most control beads at both time points and tumor bearing mice contained significantly more BAPc-MNBs than control beads (Figure 6C, D). Compared to control beads, iron content increased significantly in tumors harvested 24 h after treatment of mice with low dose of BAPc-MNBs only (Figure 6C, D). The presence of melanoma tumors significantly increased the peptide bilayer containing BAPc-MNBs compared to control beads in the lungs, heart and intestines at 24 h but only the lungs remained significantly different at 48 h (Figure 6C, D). Thus, BAPc-MNBs localized to tumors, spleen, lungs and heart in significantly higher amounts when injected i.v. in C57BL/6 mice when compared to the control beads without the peptide coating.

4. Discussion

4.1. Development of BAPc-MNBs nanoparticles

Branched Amphiphilic Peptides Capsules (BAPCs) are promising nano-delivery systems which have successfully delivered nucleic acids such as eGFP encoding plasmid DNA¹⁵ in a variety of cell lines, anti-HPV-16 DNA vaccines²⁰ in tumor bearing mice, dsRNA¹⁶ and CRISPR-Cas9 in insects. The BAPc-MNBs expand the applications of the branched amphiphilic peptides as they possess the magnetic properties of the iron oxide nanoparticles and the surface properties of the branched amphiphilic peptides. In this study we determined the biodistribution of BAPc-MNBs in vivo.

This study developed a facile method to assess the distribution of the BAPc-MNBs in mouse organs. Briefly, the magnetic iron oxide nanoparticles were recovered from homogenized mouse organs using a rare earth magnet. The recovered nanoparticles were quantified using the spectrophotometric ferene-s assay developed by Hedayati et al.²⁴ and previously used in our study to determine cellular uptake of magnetic nanoparticles in vitro.²² We determined that intravenously administered BAPc-MNBs cleared rapidly from the bloodstream, accumulated mainly in spleen, lungs, heart and excreted through the intestine. In comparison to oral delivery, i.p. injections distributed BAPc-MNBs more successfully with variations in tissue distribution in mice with and without subcutaneously injected melanoma tumors.

4.2. A simple and accurate method of determining biodistribution- ferene-s quantification

The ferene-s quantification method showed similarity to the biodistribution profiles determined by other methods such as imaging/staining^{9, 25} and ICP-MS^{26, 27} after i.v. injection of iron oxide NPs. Sharma et al.²⁸ also showed that iron oxide NPs with different surface modifications (CM, Dextran, PEG-PEI coated) cleared the bloodstream within 24 h. In addition, the highest concentration of NPs was observed in the spleen and liver while the lungs showed only positively charged nanoparticles (PEG-PEI coated) similar to BAPc-MNBs. The positively charged BAPc-MNBs were also observed in the lungs and spleen but the liver did not show significant number of NPs during the quantitative analysis using the ferene-s assay. It is unclear currently if the difference is due to the surface coating or the assay.

Kunte et al.¹⁹ found a similar biodistribution in mice injected i.v. with hollow water filled core BAPCs tagged with IRDye-800CW using fluorescence reflectance imaging of individual organs after euthanization or multispectral optoacoustic tomography of whole animals. Significant numbers of BAPCs accumulated in liver, lungs and spleen up to 24 h after injection. Therefore, the in vivo biodistribution profile of BAPCs and BAPc-MNBs is similar supporting the fact that surface properties of NPs significantly affect their behavior in vivo. The imaging techniques used to determine BAPCs distribution support the quantitative method used in this study for BAPc-MNBs. However, the ferene-s quantitative method used in this study detected BAPc-MNBs in mice up to 48 h after injection and detected low quantities of BAPc-MNBs in lungs, heart, intestines, and feces while MSOT and IR-dye based imaging techniques detect Therefore, the method presented here facilitates detection of the nanoparticles in vivo quantitatively without the limitations of imaging techniques that rely on a dye or high-end imaging instrumentation.

Visual observation during the sorting of magnetic beads from the tissues clearly indicated presence of BAPc-MNBs either in the liver or the liver circulatory system as indicated in Figure S3 where the BAPc-MNBs magnetically sorted as visible along the sides of the collection tube. Thus, limitation of this quantitative spectrophotometric assay included false negative results. While no one technique is perfect, using complementary methods will help build confidence in the results obtained.

4.3. BAPc-MNBs biodistribution

Upon i.v. and i.p administration, BAPc-MNBs primarily localized to the spleen. Additionally significant amounts were found in the lungs, heart and intestines of i.v. injected mice. Positively charged nanoparticles upon parenteral administration are often sequestered by macrophages in the lungs, liver, and spleen.²⁶ This may explain the increased number of BAPc-MNBs in the spleen. Spleen is a highly vascular organ that acts as a blood filter and receives about 4.8% of the total cardiac output.^{29, 30} The blood carrying foreign molecules enters through the splenic artery and distributes further through a highly organized vascular system to the white pulp and the marginal zone. Once through the marginal zone about 90% of the total splenic blood flow passes through the adjacent venous sinuses continuous with the marginal zone while some enter the meshwork of the red pulp.²⁹ The marginal zone macrophages capture BAPc-MNBs and macrophages in the red pulp retain some for slow destruction. BAPc-MNBs persist in the spleen after 24 h of injection but decrease by ~50% by 48 h after i.v. injection indicating clearance and/or escape of BAPc-MNBs (Figure 1). The average mouse spleen weighs about 0.1 grams. The iron content in the spleen of a wild-type mouse i.v. treated with low and high dose of BAPc-MNBs for 24 h was 150 μg/g (15 μg/0.1 g) (Figure 1B) and 350 μg/g (35 μg/0.1g) (Figure 1D) which amounts to 15 μg and 35 μg net iron in the spleen, respectively. However, after i.p. administration an increase in iron content was observed in the spleen after 24 h from an average of 50 μg/g at 24 h to 90 μg/g at 48 h. In comparison to all other organs BAPc-MNBs consistently accumulated in high numbers in the spleen with both treatment doses and times. However, the total amount of splenic NPs accumulated over time differed in i.v. and i.p. injected mice. BAPc-MNBs were observed in the spleen upon i.p. injection and could be distributed via the lymphatic. Not all NPs administered were detected and only a small percentage were found in the spleen and to some extent in the intestines.

The magnetic nanoparticles could not be detected in significant amounts in the blood (Supplementary Figure 1). However, the intestines and feces of BAPc-MNBs treated mice (i.v.) contained significantly more iron levels than saline treated, indicating clearance of BAPc-MNBs from circulation within 24 h of injection and excretion via the intestines. The ferene-s assay is a water-soluble assay and thus, fat and other water insoluble compounds may interfere with the assay. Organs such as intestines,liver and feces showed a very high background in general due to their inherent nature i.e., the debris/fecal matter as well as fat content in intestines and the fat content in liver. This was also variable between animals i.e., some mice had higher backgrounds in comparison to others. Thus, the threshold for detecting magnetic nanoparticles i.e., concentration of iron being excreted in comparison to saline control is much higher. Therefore, no significant increase was observed in feces of mice dosed with 2×10¹⁰ BAPc-MNBs. Similarly, the intestine also clear control beads without the peptide bilayer (Supplementary Figure 2, Figure 6). Similarly, the enterohepatic circulation transports secreted bile from the liver to the intestines for lipid digestion and absorption of nutrients. The bile transporters are conserved between humans and mice, but the bile composition varies between the two species.³¹ The results obtained suggests that BAPc-MNBs are transported to the intestines via the enterohepatic circulation, facilitating their clearance from the system.

Treatment of localized infections and abdominal malignancies commonly use intraperitoneal injections. NPs administered i.p. diffuse across the mesothelium but may not be able to diffuse across the endothelium under the mesothelial layer due to the tight junctions. However, the stomata, larger openings in the peritoneum, allow NPs to enter the lymphatic system.³²

Animal intestines do not readily absorb NPs which is a major barrier to oral delivery.^{33, 34} The peptides and proteins are acted upon by peptidases and acidic digestive juices (pH 2) act on biocompatible molecules proving to be a major hurdle in oral delivery.³⁵ However, the end user prefers oral administration and thus, researchers continually work to design nanoparticle-drug conjugates that are effective when administered orally.³⁶ In addition, mammalian proteinases do not alter or denature BAPCs within mammalian cells.^{17, 18} BAPCs successfully delivered dsRNA to arthropods, Tribolium castaneum (red flour beetle) and Acyrthosiphon pisum (pea aphid) in liquid and solid insect diets.¹⁶ Tribolium shows a gradual increase from pH 5.6 to pH 7.5 in the anterior to posterior midgut, like mammalian intestines in pH, despite a far less complex GI system.^{37, 38} We therefore hypothesized that BAPc-MNBs cross through the acidic stomach to be absorbed into the mammalian system through the intestinal barrier similar to their counterpart BAPCs which are highly stable under varying conditions. However, the iron content detected in all other organs was not significantly different from the control except the lung at 24 h probably due to aspiration of the NPs during gavage. This indicates that the presences of undetectable levels or absence of BAPc-MNBs in the tissues analyzed. Thus, oral delivery using the gavage method was unsuccessful in delivering BAPc-MNBs to all the organs tested.

4.4. Biodistribution in presence of tumors

The presence of a subcutaneously injected melanoma tumor affected tissue distribution of i.v. injected BAPc-MNBs and control beads. The quantity of magnetic nanoparticles was different between tumor and non-tumor mice and was not proportional to the dosage of NPs administered. Tumor-bearing mice showed significantly lower levels of BAPc-MNBs in comparison to the non-tumor mice when dosed with high (1×10¹¹) BAPc-MNBs. Kai et al.³⁹ noted that tumors cause global immune changes which results in faster clearance of the NPs from the system. Thus, BAPc-MNBs may clear faster in the tumor bearing mice treated with high dose of the NPs. Control beads were undetectable in the spleen and other tissues analyzed, suggesting clearance occurred faster than BAPc-MNBs. This is consistent with the idea that tumors accelerate the clearance of NPs. With or without tumor, the current method, only detected an average of 25% of the administered NPs in the tissues analyzed. As bones accumulate small amounts of actinium encapsulated BAPCs¹⁸, the remaining NPs could be deposited in other tissues (not analyzed), including bones, brain, lymph nodes, adipose tissue or cleared from the body.

4.5. Effect of surface coating on biodistribution

Control beads with no peptide bilayer coating showed few differences in the tissue distribution in comparison to BAPc-MNBs (Supplementary Figure 2, Figure 6). Significantly higher amounts of control beads were observed in the intestines compared to saline control while significantly fewer were detected in the spleen when compared to BAPc-MNBs in mice with and without tumors. In comparison to BAPc-MNBs, the presence of tumors cleared the control beads faster from most organs (lung, heart, intestine and spleen) at 24 h but only lung and spleen at 48 h (Figure 6). Thus, NPs surface composition and charge but not core size resulted in a different tissue distribution.

5. Conclusion

When injected i.v., BAPc-MNBs distributed widely to various organs and cleared from circulation and the system within 24 h of administration. via the feces. The spleen accumulated the highest amount of BAPc-MNBs in mice administered the NPs i.v. and i.p. and were not absorbed into the system via oral gavage. Tissue distribution depends on the surface chemistry due to in vivo interactions with serum proteins which leads to differences in the retention of magnetic nanoparticles in different organs.^{9, 10, 26, 27} In tumor bearing mice, control beads without the peptide coating contained significantly less Fe than in the absence of tumors but the BAPc-MNBs appeared to target the tumor, lungs, heart, and spleen at 24 h. Thus, the presence of tumors dramatically altered tissue distribution and the peptide coating on the MNBs altered distribution in the presence or absence of tumors. The novel method presented here determines tissue distribution in a simple yet accurate method for quantitative analysis of iron oxide nanoparticle biodistribution administered via different routes. Other qualitative imaging analysis such as MRI⁴⁰, fluorescence live imaging⁴¹ or Prussian blue staining⁹ of tissue sections may complement the simplified method for iron content analysis. This study presents a relatively simple quantification method to determine in vivo biodistribution and demonstrates the use of Branched Amphiphilic Peptides in the form of BAPCs or BAPc-MNBs as a delivery system.

ABBREVIATIONS

BAPCs

Branched Amphiphilic Peptide Capsules

i.v.

intravenously

i.p.

intraperitoneally

NPs

Nanoparticles

BAPc-MNBs

Branched Amphiphilic Peptide bilayer coated Magnetic NanoBeads

IR

Infrared

CT

Computed Tomography

MRI

Magnetic Resonance Imaging

PET

Positron Emission Tomography

AAS

Atomic Absorption Spectroscopy

HEPES

4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

TFE

Trifluoroethanol, Ferene-s,3-(2-Pyridyl)-5,6-di(2-furyl)-1,2,4-triazine-5’, 5”-disulfuric acid disodium salt

PBS

phosphate buffered saline

TBS

tris buffered saline

DLS

Dynamic light scattering

ZP

zeta potential

TEM

Transmission Electron Microscopy