Biocompatible FePO4 Nanoparticles: Drug Delivery, RNA Stabilization, and Functional Activity

FePO4 NPs are of special interest in food fortification and biomedical imaging because of their biocompatibility, high bioavailability, magnetic property, and superior sensory performance that do not cause adverse organoleptic effects. These characteristics are desirable in drug delivery as well. Here, we explored the FePO4 nanoparticles as a delivery vehicle for the anticancer drug, doxorubicin, with an optimum drug loading of 26.81% ± 1.0%. This loading further enforces the formation of Fe3+ doxorubicin complex resulting in the formation of FePO4-DOX nanoparticles. FePO4-DOX nanoparticles showed a good size homogeneity and concentration-dependent biocompatibility, with over 70% biocompatibility up to 80 µg/mL concentration. Importantly, cytotoxicity analysis showed that Fe3+ complexation with DOX in FePO4-DOX NPs enhanced the cytotoxicity by around 10 times than free DOX and improved the selectivity toward cancer cells. Furthermore, FePO4 NPs temperature-stabilize RNA and support mRNA translation activity showing promises for RNA stabilizing agents. The results show the biocompatibility of iron-based inorganic nanoparticles, their drug and RNA loading, stabilization, and delivery activity with potential ramifications for food fortification and drug/RNA delivery.


Introduction
Among various inorganic nanoparticles such as gold, silica, and quantum dots, iron-based nanoparticles (Fe-NPs) are widely explored for biomedical applications like contrast agents, drug delivery vehicles, and thermal-based therapeutics [1][2][3]. Owing to the magnetic property, high bio-adaptability, and known endogenous metabolism of iron, Fe-NPs are desirable candidates for biomedical applications. As such, Fe-NPs make the majority of FDA-approved inorganic nanomedicine [1,2]. These include INFeD, DexFerrum, Ferrlecit, Venofer, Feraheme, and Injectafer which are commercially available for their application in iron-deficient anemia and iron deficiency in chronic kidney disease [1]. Similarly, intravenous administration of the chelate iron gluconate is a well-tolerated intervention for anemia [4]. Anemia is one of the most prevalent nutritional deficiency in the world and Fe-based nanoparticles like FePO 4 and FeSO 4 has been used in food fortification to prevent anemia. Food fortification is the process of adding micronutrients to the food with an aim to overcome the nutritional deficiency in a population [5]. FePO 4 NPs are of special interest in food fortification because of their biocompatibility, high bioavailability, and superior sensory performance that do not cause adverse organoleptic effects [6][7][8][9]. Perfecto et al. have demonstrated the FePO 4 NPs internalization in human intestinal cells occurs primarily through divalent metal transporter-1 (DMT-1) and therefore can be readily absorbed [9,10]. Iron-based Feridex ® and Revosit ® are widely used magnetic resonance imaging (MRI) contrast agents for contrast enhanced MRI [11][12][13][14][15][16]. In light of these outstanding reports, FePO 4 NPs present themselves as a good delivery vehicle. Here, we explored FePO 4 as a drug-delivery vehicle by loading an anticancer drug, Doxorubicin (DOX). Ferric ion (Fe 3+ ) can form complex with DOX molecule facilitated by electrostatic interaction between electron deficient Fe in FePO 4 and electron rich -OH group in DOX to form DOX loaded FePO 4 NPs: FePO 4 -DOX NPs. We evaluated the physicochemical properties of FePO 4 and FePO 4 -DOX NPs and assessed their biocompatibility and cytotoxicity profile, respectively, in mouse osteosarcoma K7M2 and fibroblast NIH/3T3 cell-line.
Along with that, the inorganic nanoparticle has shown promises in nucleic acid stabilization and delivery [17][18][19]. In this regard, the gold nanoparticle has been widely studied because of their ability to immobilize oligonucleotides in their surface resulting in the prevention of molecular aggregation and degradation [17,20]. However, gold is not an endogenous element and thereby may limit its translational application. Here, Fe-based nanoparticles like FePO 4 nanoparticles can be of prime interest for RNA stabilization study because of their endogenous nature and established biocompatibility profile. There is two proposed mechanism of interaction of nucleic acid (RNA/DNA) with Fe-NPs for the stabilization-(1) formation of hydrogen bonds and electrostatic interaction between the phosphate group of nucleic acid backbone and Fe-NPs resulting in adsorption of nucleic acid in Fe-NPs, and (2) nucleic acid can adsorb to the Fe-NPs surface via nucleotide base pair interaction [19,21,22]. A study has shown the potential of calcium phosphate nanoparticles for DNA vaccine stabilization and delivery [23]. In this regard, here we have explored the RNA stabilization and functional activity of another phosphate-based nanoparticle, FePO 4 , to investigate the multifunctional potential of FePO 4 based nanoparticles, in the delivery and stabilization of cargo.
With rapid approval of mRNA vaccine against COVID19, mRNA vaccine nanoparticles are of great interest, RNA being subject to rapid hydrolysis and loss of functional expression, it is incumbent upon the nanoparticle to improve these critical characteristics. Here we show FePO 4 NPs stabilize RNA and support functional mRNA translation. Given these excellent characteristics, FePO 4 NPs may merit consideration for food fortification, drug, and RNA delivery, opening up exciting biomedical applications.

FePO 4 NPs Synthesis, Characterization, and Biocompatibility Analysis
A simple one-step chemical reaction between (NH 4 ) 3 PO 4 and Fe(NO 3 ) 3 gives FePO 4 as precipitate which is dispersed in biocompatible lipid-PEG surfactant that helps to stabilize FePO 4 nanoparticles and prevent aggregation. FePO 4 NPs showed a hydrodynamic size of 175 ± 5 nm with a polydispersity index (PDI) of 0.150 ± 0.01 suggesting good particle homogeneity and narrow size distribution. Zeta potential analysis showed a negative surface charge of FePO 4 NPs with − 19.1 ± 8 mV zeta potential. The negative surface charge further helps to stabilize particles in colloids thereby preventing protein opsonization, a mechanism that prevents cellular targeting and alters pharmacokinetics [24][25][26]. FePO 4 was further characterized by FTIR. Figure 1c shows the spectral characteristic of FePO 4 nanoparticles and their precursor-Fe(NO 3 ) 3 and (NH 4 ) 3 PO 4 . FePO 4 spectra show a distinct sharp peak on 1030 cm −1 which can be attributed to the P-O stretching band, a small peak at 520 cm −1 corresponds to the O-P-O antisymmetric bending, and a broad ranges from 3000 to3500 cm −1 represents water bending and stretching vibrations from adsorbed water molecules [27,28]. The FePO 4 spectra showed the presence of PO 4 3− group and are similar to the FTIR peak reported by other studies thus confirming the formation of FePO 4 nanoparticles [27][28][29]. Fe(NO 3 ) 3 spectra showed characteristic peaks for N-O stretching bands at 1326 and 813 cm −1 [30]. Peak at 1625 can be attributed to -OH bending vibration and a broad peak around 3000 cm −1 can be attributed to water bending and stretching vibrations [30]. Likewise, (NH 4 ) 3 PO 4 showed characteristic peaks for the ammonium group around 1500 cm −1 and phosphate group around 1000 cm −1 [31]. The absence of nitrate and ammonium peaks in FePO 4 nanoparticles suggest the product is free from possible byproducts and confirms the purity of synthesis.
With the assurance of successful synthesis, purity, good size homogeneity, and stable surface charge of FePO 4 NPs, we went on to analyze the biocompatibility of FePO 4 NPs. For this purpose, we used cancer and non-cancer cells: mouse osteosarcoma K7M2 and mouse fibroblast NIH/3T3 and analyzed the biocompatibility of NPs at a varying concentration in terms of cell viability using MTT assay. FePO 4 NPs showed concentrationdependent biocompatibility in both cell lines-K7M2 and NIH/3T3, in the concentration range of 20 to 600 µg/mL (Fig. 1d). FePO 4 NPs showed good biocompatibility up to 80 µg/mL concentration with cell viability greater than 70%. Biocompatibility was relatively higher in non-cancer cell NIH/3T3 compared to cancer cell K7M2.

Doxorubicin Loading in FePO 4 and Cytotoxicity of FePO 4 -DOX
Doxorubicin is loaded in FePO 4 through the co-incubation-precipitation method in which doxorubicin solution is mixed with the precursor of FePO 4 that results in the formation of DOX loaded FePO 4 . Three different formulations to load DOX are employed as discussed in the methods. Formulation 1 showed the best loading efficiency of 26.81% ± 1 whereas formulation 2 showed a loading efficiency of 8.83% ± 2 and formulation 3 did not show any loading (Fig. 2a). For loading, we added DOX solution to the precursor Fe(NO 3 ) 3 in formulation 1 and to (NH 4 ) 3 PO 4 in formulation 2, whereas, in formulation 3, we added DOX solution to FePO 4 NPs directly. The loading data clearly showed that adding DOX to the FePO 4 NPs does not retain the DOX whereas adding DOX to either precursor: Fe(NO 3 ) 3 and (NH 4 ) 3 PO 4 solution helps in the loading and retention of DOX. This can be explained by the fact that Fe 3+ from Fe(NO 3 ) 3 can form a complex with the electron-rich oxygen group present in Doxorubicin [32,33]. The Fe 3+ -DOX complex is then precipitated by the addition of (NH 4 ) 3 PO 4 resulting in FePO 4 -DOX, which is characterized by a change of color from faint yellow to faint brown (Fig. 2b). Despite the color change, there was no change in the emission spectra of FePO 4 -DOX which showed emission maxima at 590 nm similar to that of Free DOX, when excited at 480 nm ( Fig. 2c). FePO 4 -DOX NPs showed a hydrodynamic size of 187 ± 7 nm and PDI of 0.143 ± 0.02, similar to that of FePO 4 (Fig. 2d). However, there was a significant difference in the surface charge of FePO 4 -DOX NPs (-8.89 ± 5 mV), compared to FePO 4 NP (-19.1 ± 8 mV) (Fig. 2e). Change in zeta potential suggests functional changes in the surface property of nanoparticles. Here, the reduction of zeta potential from − 19.1 to − 8.89 mV can be attributed to the DOX complexation which adds cationic property in the complex.
Following the physicochemical characterization, the cytotoxicity of FePO 4 -DOX was analyzed in K7M2 and NIH/3T3 cells and compared with free DOX (Fig. 3).   an enhanced cytotoxicity profile of FePO 4 -DOX NPs. The equivalent FePO 4 concentration in the IC50 concentration range of FePO 4 -DOX is 40 µg/mL (0.107 µM in K7M2 cells) and 100 µg/mL (0.248 µM in NIH/3T3 cells), which are both within the biocompatible range of FePO 4 concentration, with more than 70% cell viability. Hence, the elevation of FePO 4 -DOX cytotoxicity can be attributed to the Fe 3+ -DOX complex formation and not to the individual contribution of FePO 4 and DOX. Literature has shown the elevated cytotoxic effect of anthracycline like doxorubicin in presence of iron [34][35][36][37]. These reports are further supported by the alleviation of Fe-DOX cytotoxicity by the use of iron chelators [35][36][37]. One proposed mechanism is Fe-DOX complex potentiates the toxicity of DOX-derived reactive oxygen species (ROS) transforming relatively safe ROS (O 2· -and H 2 O 2 ) into much more toxic ROS leading to elevated DNA damage and cell death [34,36]. Another proposed mechanism is the interaction of DOX with the function of iron regulatory proteins and ferritin in presence of excess Fe thereby affecting iron homeostasis leading to ROS-dependent and independent damage and apoptotic cell death [36,38].
Along with the elevated cytotoxicity, FePO 4 -DOX showed selectivity toward cancer cells with higher cytotoxicity behavior similar to that of Free DOX. Figure 3c shows 0.1 µM DOX equivalent FePO 4 -DOX showed 53% cell viability for cancer cell K7M2 compared to 72% cell viability for non-cancer NIH/3T3. Likewise, Free DOX also showed higher cytotoxicity behavior toward cancer cells, with 54% cell viability in K7M2 cells compared to 66% in NIH/3T3. However, the differences have increased in the case of FePO 4 -DOX, with 19% differences in cell viability among cancer and non-cancer cells compared to 12% in Free DOX. Cytotoxicity analysis has shown that Fe complexation with DOX in FePO 4 -DOX NPs has significantly enhanced the cytotoxicity and improved the selectivity toward cancer cells.

Cellular Internalization of FePO 4 -DOX NPs
The internalization behavior of FePO 4 -DOX NPs was analyzed using confocal microscopy following a timedependent internalization study (Fig. 4). Free DOX was used as a positive control. Both FePO 4 -DOX NPs and Free DOX did not show significant internalization in the initial 0.5 and 1 h incubation time points. However, at 3 h incubation, both showed internalization as depicted by red DOX fluorescence in the confocal image. The blue color comes from nucleus staining by DAPI. The analysis shows that within 3 h, FePO 4 -DOX NPs internalize to cells following similar internalization behavior as that of Free DOX. It is important to note that, due to the change of color of FePO 4 -DOX, which is brownish compared to the red color of Free DOX, we may not quantitatively compare the relative internalization profile of FePO 4 -DOX. Nonetheless, the internalization assay confirmed that FePO 4 -DOX is uptake by the cells within 3 h. Given the well-understood mechanism of handling iron by our body, proposed NPs could hold promises in the development of iron-based anticancer therapeutics with an ability to monitor therapeutic response in a single therapy session.

RNA Stabilization and mRNA Expression
As can be seen in Fig. 5a, whereas copper nanoparticles (Cu NP) and carbon nanotubes (CNTs) accelerate RNA hydrolytic degradation (lower band intensity than control), the FePO 4 and control silver (Ag) nanoparticle stabilize the RNA as shown by relatively strong band intensity in RNA agarose gel electrophoresis (RAGE). The FePO 4 and control zinc oxide nanoparticle (ZnO NP) also impart some resistance to degradation in serum, as depicted by the band intensity which is slightly higher than controls (Fig. 5b). Importantly the functional activity, mRNA expression is higher than non-nanoparticle controls, whereas the RNA-degrading Cu NP causes loss in mRNA expression as measure by relative light units (Fig. 5c)  mesoporous silica nanoparticle (MSN), carbon-based polymers, composites, and others [39][40][41][42][43][44][45]. For example our group had reported nanoparticle complexation to macromolecular RNA can cause it to resist degradation by RNase, or nucleases present in serum and tissues. The COVID-19 mRNA vaccine has renewed interest in such macromolecular RNA therapies extending beyond vaccines, where it is incumbent upon the nanoparticle to not only protect RNA from hydrolysis and nucleasemediated digestion, but complexation to the NP must preserve RNA function, for example, mRNA expression. Previously we had seen copper nanoparticle complexation macromolecular RNA causes RNA denaturation [46] Thus we investigated the effects of NP complexation to

Biocompatibility of FePO 4 NPs and Cytotoxicity of FePO 4 -DOX NPs
Biocompatibility of FePO 4 NPs and cytotoxicity of FePO 4 -DOX NPs were assayed in mouse osteosarcoma K7M2 and mouse fibroblast NIH/3T3 using MTT assay following established protocol [48,49]. Briefly, 10,000 cells were seeded in 96 well plates and incubated for 24 h in a 37 °C 5% CO 2 incubator. Then, media was removed and fresh media with varying concentrations of nanoparticles were treated to cells and left for incubation for 48 h. Control cells were maintained with media only. FePO 4 NPs concentration ranges from 20 to 600 µg/mL and DOX concentration ranges from 0.05 to 5 µM. After NP incubation, media was removed and cells were incubated with MTT solution (0.5 mg/ml) in serum-free media for 2 h to allow for the formation of formazan crystal. MTT solution was removed and formazan crystal was dissolved in DMSO and left for 15 min at room temperature for proper mixing. Then the absorbance of DMSO solution was measured at 550 nm using a microplate reader (BioTek, Synergy H1 Hybrid Reader) and percentage cell viability was calculated.

Cellular Internalization via Confocal Microscopy
Cellular internalization of FePO 4 -DOX NPs was analyzed in mouse osteosarcoma K7M2 cells using confocal microscopy [49][50][51]. Briefly, 12,000 cells were seeded in 8-well plates and incubated for 24 h in 37 °C 5% CO 2 incubator. Then, 200 µL of 5 µg/mL DOX concentration in media were treated for 3 h, and cells were fixed with 4% Paraformaldehyde for imaging. The nucleus was stained by DAPI and cells were observed under a Confocal Laser Scanning Microscope (Carl Zeiss, LSM-700). Here, the emission maximum of DOX at 560 nm can be exploited to track its internalization which gives red color in confocal microscopy. Using the same protocol, a time-dependent internalization assay was performed by incubating FePO 4 -DOX NPs and Free DOX for 0.5, 1, and 3 h respectively.

RNA Stability and Expression
Torula yeast RNA (Sigma-Aldrich) was dissolved at 1 mg/ ml in sterile deionized water and 2 µg aliquots exposed to 20 ug/mL nanoparticle (CNT, Cu, Ag, ZnO NP or FePO 4 ) incubated at 37 deg C and assayed over time by RNA agarose gel electrophoresis as we have previously reported [42,52]. Timepoint shown in Fig. 5 is overnight. Similarly, the RNA with/without nanoparticles was exposed to 10% FBS/DMEM and again assayed by RAGE as above. mRNA fLuc was obtained from Trilink Biotechnologies, 2 µl were incubated in rabbit reticuloysate supplemented with Methinine, Cysteine and Leucine (ProMega Corp) for 30 degrees for 1.5 h with or without nanoparticle at 20 μg/ml, standard Luciferin reagent added, and luminescence measurement taken on a Biotek Synergy H1 plate reader under standard conditions.

Statistical Analysis
All data represents at least three independent replicates and expressed as mean ± s.d. whenever possible. Cell viability data includes six replicates.