Synthesis Strategies and Biomedical Imaging Application of Iron (III) Hexacyanoferrate (II) Prussian Blue Nanoparticles

The German mineralogist and geologist, Abraham Gottlab Werner discovered a systematized sequence for identifying colors, which was further modified by Scottish painter named Patrick Syme in 19th century. The sequenced number 25 from identified colors is blue color which is known as Prussian Blue (PB). The PB in sequence is specified as ‘Berlin Blue’ which is a significant part velvet black and a little amount of indigo blue. Synthetically PB was first obtained pigment and was discovered by Berlin artist Diesbach during early 18th century.

The PB was discovered many years ago but in the current time also it is as interesting as before for advanced and pioneering research. The chemical formula of PB is Fe4[Fe(CN)6]3 and IUPAC name is Iron(II, III) hexacyanoferrate (II,III). Due to charge transfer transition from iron (II) to iron (III), PB is blue in color. The solubility of PB depends on its stoichiometry and the condition in which it is prepared. The PB has two solubility nature viz. (i) PB insoluble: FeIII4[FeII(CN)6]3.xH2O (where x = 14 to16) and (ii) PB soluble: (AFeIII[FeII(CN)6].xH2O (where x = 1 to 5 and A is single valent cation such as Na+, K+ or NH4+) (Fig. 1). In reality both these solubilities are meaningless as both are insoluble in water. In most cases, the solubility of water is ~10-41 but solubility product of PB having colloidal properties is soluble in water and disperse easily in water.

Soluble of PB containing Na+ can dissociate in aqueous media, giving a crystal surface with net negative ionization consents a stable dispersion to be formed if crystals are small. In soluble PBs to balance charge, alkali metal ions substitute the water molecules in vacant areas(cavities). Whereas insoluble PBs having some defects due to presence of interstitial water molecules, can be classified in two categories based on their coordination viz. water molecules coordinated to Fe(II) sites (coordinated water) and water molecules which are inside cavities which do not coordinate to metal sites (zeolite water). 

Using cyano-bridged ligands PB are assembled and arranged in nano-structures: PBNPs. Nano PB particles and structures have unique properties such as involving solubility, flexible molecular structure, stability, porosity, low density and many more. The chemical and physical properties of PBNPs are adjustable as per desire. Moreover, in current research areas PB plays a very vital role in many scientific, biomedical and industrial uses. Some applications of PBNPs are in hydrogen storage, MRI and NMR, treatment of cancer, bio-sensors, ion sensors, catalysts, etc. Prussian blue nanoparticles (PBNPs) with favourable biocompatibility and unique properties have captured the attention of extensive biomedical researchers. A great progress is made in the application of PBNPs as therapy and diagnostics agents in biomedicine.  

This review begins with basic introduction on crystal structure of PB and mainly focus on the synthetic techniques and strategies of PBNPs. We will also highlight a few applications of PBNPs in field of biomedicines – treatment, therapy and diagnosis.

Crystal Structure

PB and its similar compound PBA are an amalgam of microporous inorganic solids and the unit cell structure is shown in Fig. 2. The formation is done through the coordination of Fe2+ and Fe3+ ions with a cyanide group C-(N-). The PB having Fe3+ and Fe2+ can be reduced to Prussian white to become Fe2+ and Fe2+[Fe2+(CN)6]2- or can be oxidized to form Prussian yellow to become Fe3+ and Fe3+[Fe3+(CN)6]. This is because of the presence of mixed-valent Fe in PB. There are two crystalline forms of PB i.e. soluble and insoluble.

The PBNPs crystalline structure mainly contains Fe3+, Fe2+ and bridging cyano groups. The Fe3+ and Fe2+ interchange systemically with bridging cyanide to form three-dimensional coordination network. In 3D network, the Fe2+ cation is coordinated with carbon atom of cyanide ligand. This acts as bridge for Fe3+ cations that are coordinated with 6 nitrogen atoms to form octahedral structure. The unit cell of PBNB has dimensions of 10.2 ?. The average bond lengths of Fe(II)-C and Fe(III)-N are 1.92 ? and 2.03 ? respectively. Due to this three-dimensional coordination network PB are insoluble and exists in nanoparticle form. Insoluble PB form larger crystal with higher dimensions compared to soluble PBs because they have tendency to aggregate and leading to formation of precipitates which form larger crystals. The Soluble PBs, on the other hand, produces crystals of lower dimension which can reach size of mesophase viz. nanoparticles, forming the blue colloidal solutions.

Fig: 1 Scheme of unit cell of (a) insoluble {fe^{III 4} [Fe ^II ( CN)6]³ X H2O}, and (b) Soluble soluble {KFe^III [ Fe^II (CN)6]}

Synthesis Method of PBNPs

Development in synthesis technology leads to preparation of PB with specific composition, different particle size, different structural properties and different morphology have become more and more thorough and advanced. Currently, various methods for preparation of PBNPs are determined. This includes single and double precursor method, co-precipitation method, hydro-thermal method, reversed phase microemulsion method, chemical etching and so on. All these different methods have their own advantages and disadvantages. Most of these methods are able to form stable single crystal and uniform PBNPs. The synthesis of PBNPs involves the use of polymers, acids or aliphatic amines as shielding agents to generate the solubility of PBNPs in water or organic media. The morphology of PBNPs depends on the preparation method and synthesis strategy. Commonly the shape of PBNPs is characterized as cubic or spherical.

Fig.2 Schematic diagram representing various PBNP synthesis techniques. 

Single Precursor Method

The single precursor synthesis method was first developed in 1998 by Yang et al. Usually, the ferricyanide complex of K3[Fe(CN)6] or K4[Fe(CN)6] ions are used as precursor. The Fe3+(ferric) and Fe2+(ferrous) ions are slowly released in acidic medium wherein they are either reduced or oxidized to form ferrous or ferric ions, respectively. The undecomposed ions and the above formed ferrous and ferric ions react to form PBNPs. This is slower reaction method hence, the NPs formed have high mono-dispersity or isodispersity. Thus, this method is said to be best strategy for production of PBNPs. The reaction mechanism steps are shown below.

When iron oxide NPs obtained by using hydro-thermal process are dispersed in an aqueous solution of pH 2 and quickly mixed with a potassium hexacyanoferrate (II) solution of pH 2, after 1-hour magnetic PBNP’s were formed. Thus, using single precursor method magnetic PBNPs are formed via shellgrowing procedure. However, the drawbacks of this method as discussed earlier is that it takes longer time (mostly more than an hour) along with it traces of hydrogen cyanide can be released as toxics contaminating the PBNP because cyanide is 35-40% of the molecular composition of PB. 

Coprecipitation Method

The coprecipitation method comprises of a one-step process in which reaction of a solution of iron (III) salt (i.e. FeCl3) and a solution of alkali hexacyanoferrate (II) salt [i.e. K4Fe(CN)6] gives formation of voluminous blue precipitate or colloidal suspension of PB. Depending on proportion of iron salt i.e. whether iron salt is in excess or if both salts viz. iron salt and alkali hexacyanoferrate salt are mixed in a 1:1 molar ratio. The other way of production of PB is using an indirect method involving two-step reaction process based on reaction between an iron (II) salt and hexacyanoferrate (II) salt. The product of this reaction is a precipitate, Berlin White. The chemical formula for Berlin White is Fe2[Fe(CN)6] namely, ferrous ferrocyanide. After formation of Berlin White, in the second step, this precipitate is oxidized using an oxidizing agent, like hydrogen peroxide to form PB. On the other hand, when the oxidized states of the precursor are interchanged, a blue pigment is also obtained. On mixing solution of iron (II) salt (i.e. FeCl2) and solution of hexacyanoferrate (III) salt [i.e., K3Fe(CN)6] blue solid called Turnballs Blue is produced. On revelation by spectroscopic techniques like Mossbauer spectroscopy and X-ray diffraction, it is observed that Turnballs Blue is same product as PB. Thus, regardless of the precursor used, the blue compound formed is inevitably ferric ferrocyanide having characteristic structural element FeIII-N-C-FeII. Also, it is known that the formation of solid precipitate is accompanied by an electron transfer reaction. Usually, the temperature of solution is between room temperature and 60ºC in coprecipitation synthesis method.

The reaction mechanism explaining the formation of soluble and insoluble PB is as follows: 

As a result of slow assembly of three molecules of soluble PB and ferric ion into face-centered cubic (FCC) structure, compound of insoluble PB is formed.

Whereas small and stable soluble dispersion is formed due to dissociation of monovalent metal ion inclusion complex present in soluble for PB in aqueous media and net negative ionization occurring on crystal surface.

Many a times, it is required or desired to synthesis the of PBNPs in which the NPs form by a core-shell structure of magnetite. To carry out this synthesis, magnetite nano particles are suspended in a solution of alkali hexacyanoferrate (II) [i.e. K 4 Fe(CN) 6 ] . Then ferric solution is added and thereby PBNPs are formed. Here, the nano particles comprise of a core-shell of magnetite which is coated with layer of PB. Thus, here the magnetic properties are retained by magnetite. Researchers have prepared nano particles by this method wherein instead of magnetite, iron (Fe 0 ) is used. As result, it was observed that the saturation magnetization of NP having iron (Fe 0 ) core was higher as compared to magnetite of same size. PBNPs formed are usually unstable and hence tend to aggregate with each other.

Hydrothermal Method

In hydrothermal method, the components dispersed in water are introduced in autoclave reactor. The autoclaves provide high temperature and high pressure than the ambient temperature and pressure to carry out the reaction. Hydrothermal method is variant of coprecipitation method which is carried out at higher values of temperature (generally >100ºC) and pressure. At higher temperatures, the formation of PBNP increases. This leads to quick accumulation of PBNP and overly rapid crystallization. Thus, this rapid crystallization makes this method unsuitable for PBNP formation since single crystal growth of PBNP is not induced several times. So, several hydrothermal techniques are developed and modified to induce single crystal growth. 

To overcome this problem and control accumulation rate of PBNP, protective layer of various polymers such as poly vinylpyrrolidone (PVP), polyethyleneimine (PEI), polyethylene glycol (PEG) and polyaniline (PANI) have been used. In this method, the amide groups of the polymers react with the Fe ions and forms weak bond coordinate bond. This, the rapidly grown PBNP gets fixed into polymer chain leading to formation of new crystal seed which is distributed uniformly and increases in size gradually. Using hydrothermal method, when potassium hexacyanoferrate (III) and PEG 4000 are mixed in acidic medium and maintained at 120ºC for 20 hours PB meso-crystals are prepared. This synthesis was done by Wu et al. and the PBNP formed was a pseudo-truncated cube. In the above process, when PEG 4000 was replaced by PEG 2000, the product meso-crystals were truncated cube having both smooth faces and curved surfaces. When Teflon-sealed autoclave is heated to 180ºC holding the mixture of ferric and hexacyanoferrate (III) ions, ethylene glycol, sodium dodecyl sulfonate (SDS), sodium acetate, hydrochloric acid and PEG 2000 for 6 hours nanoparticles of PB were formed. This synthesis was obtained by Qian et al. Similarly, by making different modifications and using different polymers as protective coating various types of PBNPs are obtained such as PBNP coated with polyaniline, PBNP having hollow interior controlled by chemical etching, etc. Thus, PBNPs formed having different morphology are produced.

However, hydrothermal reaction is very simple and has high potential of yield nanosized or micro-sized crystal structure. Although hydrothermal reaction method has various advantages it is not feasible to carry out synthesis of all applications such as fabrication of bio-sensors.

Biomedical applications of PBNPs

PBNPs have several applications due to their bio-compatibility and safety. PBNPs have been widely used for different biomedical applications such as targeted drug delivery, used in cancer treatment in chemotherapy, different types of imagining such as MRI, photoacoustic tomography (PAT), photoacoustic imaging (PAI) or the multimodal imaging which is combination of MRI and PA. The physiochemical properties are adjustable and promising. Hence, this results in use of PBNPs in biomedical applications. The physiochemical properties of PB particles leading to biomedical applications are:

Fig. 3 Schematic representation of various biomedical applications of PBNP

Biomedical Imaging

1. PB possess large cavities within its single crystal structure. This makes it a nanoporous material and it can host small molecules and ions within this cavity.
2. PB has strong absorption in near-infrared region (NIR) of EM spectrum.
3. PB can be easily oxidized or reduced to its different forms like Prussian White, Prussian Yellow or Berlin Green.

Medical imaging enables clinicians to visualize and understand intricacies of human body. This technology helps to improve patient care by enabling more accurate and timely diagnosis of disease. This helps in early diagnosis, image-guided therapy, research, etc. Moreover, it is a non-invasive examination technique. Biomedical imaging uses variety of technologies including X-ray, radiography, magnetic resonance imaging, ultrasound, thermography and tomography.

Contrast agents (Contrast media) are used in biomedical imaging to improve and enhance the quality of images by making it easier to distinguish between different tissues and structures on the image. PBNPs because of their unique properties is used as external contrast agents for photoacoustic imaging and magnetic resonance imaging.

Photoacoustic Imaging

When pulsed laser irradiates light on material, that light absorbed causing the material to heat up and expand. The expansion creates ultrasonic pressure waves that travel through medium. An ultrasonic transducer detects the pressure waves as sound signals and there by reconstructs the image of light energy absorption distribution in the tissue.

Photoacoustic Imaging (PAI) and Photoacoustic Tomography (PAT) are two noninvasive imaging techniques based on the effect of photoacoustic effect. Non-ionizing laser pulses are used to irradiate the samples used in PAI. This technique has high penetration depth and high temporal and spatial resolution. Some nanoparticles which are NIR absorbers used in PAI are gold nanostructures, carbon nanotubes , polypyrrole , and copper sulfide nanoparticles which are expensive also deep tissues imaging by PAT is not feasible. Moreover, these compounds being non-biodegradable raises concerns regarding long term toxicity towards body. Hence, raises concerns about their potential clinical applications. Liang et al. used NIR laser pulses of wavelength 765 nm on PBNPs an explored use of PBNPs as a contrast agent that enhances PAT in vitro and vivo. Cai et al. added gadolinium ion into PBNPs to improve the efficiency of PAI. This integration changed the electronic transition since Fe3+ ions were replaced by Gd3+ ions leading to deeper penetration into tissues at longer wavelength. 

Fig. 4 Schematic representation of PBNP used for biomedical application