Prussian Blue nanoparticles: An FDA-approved substance that may quickly degrade at physiological pH

Prussian Blue is an FDA-approved drug under the name Radiogardase®, an orally administered medicine for Tl+ poisoning and radioactive Cs+ contamination [1]. It contains “insoluble” PB. This form of PB has the formula FeIII4[FeII(CN)6]3·xH2O (x = 14–16, roman numbers stress oxidation states) [2]. A “soluble” PB form also exists, with formula KFeIII[FeII(CN)6]·xH2O (x = 1–5) [2]. However, it is now clear that the two forms are indeed identical coordination polymers [3], [4]. PB consists in fact of hexacyanoferrate [FeII(CN)6]4- complexes, whose N atoms coordinate Fe3+ ions with an octahedral geometry [3], [4], as sketched in Scheme 1A, creating an infinite tridimensional coordination network. The C-bound Fe2+ centres are d6 low spin and kinetically inert, while the d5 Fe3+ ions are kinetically labile. Fully hexacoordinated Fe3+ centres are found in the soluble form, with K+ cations occupying half of the centres of the lattice cubic cell. In the insoluble form, some Fe3+ ions lack one or more N-donors and their 6-coordination sphere is completed by water molecules [3], [4]. Soluble PB can be easily obtained as nanodimensioned crystals, PB nanoparticles (PBnp). These usually have cubic shape, with side in the 10–200 nm range depending on the synthesis, and form blue colloidal clear aqueous solutions, from which the “soluble” adjective. PB is an FDA-approved drug for oral administration. The pH of the stomach is strongly acidic, it rises to 6 in the duodenum and it further increases gradually in the small intestine, reaching 7.4 in the terminal ileum. Finally, it drops to 5.7 in the caecum and it is 6.7 in the rectum [5]. Thus, except for the terminal ileum tract, orally administered PB experiences acidic pH values. Despite this, in the past decade the FDA approval has been considered as an unconditional pass for using PBnp also under “physiological conditions” different from oral administration, meaning injection in (murine) blood or, more frequently, in-vitro studies with the blood saline concentration and its 7.4 pH. In this context, a number of biomedically-oriented papers has been published in the nanomedicine area [6], [7], [8], suggesting the PBnp use in photothermal therapies, laser-switched drug release, photoacoustic imaging, theranostics [9], [10], [11], [12]. This is related to the intense charge-transfer (CT) absorption band of PBnp, that has λmax ∼ 700 nm [13], [14] and that relaxes thermally when photoexcited. On this basis, PBnp have been proposed for photothermal therapies using Near IR irradiation in the so-called biotransparent window [9], after a first paper on this topic was published in 2012 [15]. Moreover, PBnp have been proposed also for drug delivery [16].

Although a variety of complex synthetic approaches are possible, leading to less symmetric shapes [11], straightforward wet syntheses in water yield cubic PBnp. Three reaction schemes are commonly used: i) 1:1 reaction of Fe3+ with K4[FeII(CN)6] in the presence of citric acid [9], [15], [17]; ii) 1:1 reaction of Fe2+ with K3[FeIII(CN)6], followed by an internal redox [9], [18], [19], [20]; iii) K3[FeIII(CN)6)] as the only iron-containing reactant, in high temperature preparations and strongly acidic conditions, with a polymer acting both as coater and reductant [21]. Thanks to its straightforwardness and to the non-toxic nature of citrate, type (i) synthesis has become more and more commonly used [9]. Moreover, the distinction between “soluble” and “insoluble” PB has almost completely disappeared from nanomedical literature, although the used stoichiometries suggest that KFeIII[FeII(CN)6]·xH2O, i.e., soluble PB, is obtained.

In the course of our research on nanoparticles with photothermal properties [22], [23], [24], [25], we have recently introduced the use of PBnp [9], [19], [20]. In our studies we noticed that often, and apparently in a random and unpredictable fashion, the colloidal solutions of PBnp decolorated after a few hours of contact with various biological media at physiological pH (7.4).

The phenomenon was unexpected, given the many papers describing the use of PBnp under similar “physiological conditions”. However, literature indeed reports some cases in which PBnp or other PB materials (films, macroparticles) rapidly decompose in neutral or slightly basic conditions, i.e. at pH ≥ 7.0, including the physiological pH value 7.4. The CT absorption band of colloidal PBnp completely disappeared in 30 min at pH 8.0, while when PBnp were synthetized inside the apoferritin cavity, they resisted unchanged for 1 h at the same pH [26]. Films of PB formed on the inner walls of a cuvette were stable at pH < 6 but quickly lost color at pH ≥ 7.0 [27]. PB formed in situ in a silica sol-gel matrix was stable at acidic pH while showed a fast decay at pH ≥ 7.0 [28]. Moreover, in situ reaction of Fe2+ and [Fe(CN)6]3- was found to give stable PBnp solutions only at pH < 6.8, while PBnp decomposed at pH ≥ 7.0, and this observation was used to carry out pH-sensitive photoacoustic imaging and photothermal therapy on murine models [29]. On the other hand, only minor and slow degradation at pH 7.4 was reported for cubic PBnp (side ∼ 200 nm) prepared with excess polyvinylpyrrolidone (PVP) as coater [21]. Cuboidal PBnp (side >100 nm) prepared in a 10-fold mass excess of PVP (vs Fe precursor) were reported to last intact at pH 7.0 or 9.0 for weeks [30]. Cubic PBnp coated with polyethyleneglycol amine (PEG-NH2, mw 5000) were stable for one week in cell culture media buffered at pH 7.4 [31]. Cubic PBnp (side ∼ 60 nm) featuring a cross-linked polyallylamine hydrochloride (PAH)-polyacrylic acid (PAA) coating terminated with PEG-NH2 (mw 5000), were used for in-vivo antitumor photothermal therapy [32]. Judging from this picture, a pH value ≥ 7 rapidly compromises PBnp stability, unless a significantly thick coating is used to protect them, allowing their use in-vivo or in in-vivo-like conditions (pH 7.4). However, as a contradiction, examples of uncoated, citrate-stabilized PBnp have been published, claiming their stability under the same conditions: 42 nm PBnp in cell culture media buffered at pH 7.4 [15]; small citrate-coated cubic PBnp (side 13 nm) in human blood serum and in cell culture medium (pH 7.4) [25]; citrate-coated PBnp (side 17 nm) used in-vivo in mice [33].

As PBnp-mediated drug delivery, photothermal therapies or photoacoustic imaging require circulation, extravasation, and accumulation in specific sites (e.g., a tumor), leading to a chain of processes that need nanoparticles to be stable in the circulatory systems for hours or even days [34], [35], it is key for the PBnp safe and reliable use in medical therapies/diagnostics to make definitely clear the conditions for their stability at the physiological pH 7.4. This is what we have done with this paper. We prepared citrate-stabilized PBnp (c-PBnp) and used them as a reference to determine the intrinsic stability of PB vs pH. Decomposition kinetic studies were carried out on PBnp in two typical cell-culture media, DMEM (Dulbecco’s Modified Eagle’s Medium) and PBS (Phosphate Saline buffer), both buffered at pH 7.4. The same kinetic studies were repeated with the H2PO4-/HPO42- and acetic acid/acetate buffers, exploring the 4–8 pH range. We demonstrate here that while c-PBnp are stable at pH < 6.0 they rapidly degrade with decoloration at pH ≥ 7.0, with a rate directly proportional to pH. In particular, a pH 7.4 environment, either in DMEM, PBS or phosphate buffer, decomposes PBnp in few hours. PVP was then introduced as a coating agent for PBnp, showing that only a large excess of this widely used stabilizing polymer allows for a significant slow-down of the decomposition process. Moreover, we also investigated PBnp stability at pH 7.4 in DMEM, PBS and H2PO4-/HPO42- buffer with the addition of 10% fetal bovine serum (FBS), a common additive for plated cell culture that contains a rich pool of plasma proteins. In the presence of FBS, the c-PBnp became stable for over 24 h, thanks to the fast formation of a protein corona. The mechanism of decomposition of c-PBnp at pH 7.4 was also investigated, combining TEM (transmission electron microscopy), dialysis, quantitative iron analysis by ICP-OES (inductive coupled plasma optical emission spectroscopy), and DLS (dynamic light scattering). The formation of hydroxo complexes of the labile Fe3+ cation is proposed as the driving force for the disruption of the PB coordination network, as sketched in Scheme 1B, and leading to PBnp erosion and disappearance of the CT absorption band. TGA and DLS experiments were also carried out to determine the amount and thickness of the coating layers on PBnp, both with PVP and with protein corona. Finally, in order to understand if PBnp decomposition could lead to detrimental effects in-vivo, the cytotoxicity of c-PBnp was examined in DMEM (pH 7.4) on three cell lines, NCI-H1299, A549 and EA.hy926, both with and without the protein corona-forming FBS supplement, and then compared to the independent effect of PB decomposition products, i.e., [Fe(CN)6]4- and Fe3+ hydroxo complexes.

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