Biodistribution and long-term fate of silver nanoparticles functionalized with bovine serum albumin in rats

Abstract

Silver nanocrystals (Ag NCs) hold promising antibiotic and antiviral properties in biological systems. The biodistribution of silver nanostructures injected into animals in vivo is currently unknown, remaining as a fundamental issue for potential therapeutic applications. Here, we injected Ag NCs capped with bovine serum albumin (BSA) in live rats to elucidate their fate in several organs including liver, heart and brain. Very significant accumulations of nanoparticles were confirmed by inductively coupled plasma mass spectroscopy (ICPMS) and transmission electron microscopy (TEM) techniques on the liver and heart. In contrast, the brain tissue did not reveal evidence of particles content. Our results suggest that Ag⁺ permeated across the blood–brain barrier (BBB), and followed swift clearance from the organ.

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Introduction

The massive development of nanotechnology has produced a large number of applications fundamentally based on nanoparticles. However there is still very scarce knowledge of the possible toxicity of nanoparticles. For instance, it is well known that silver nanoparticles in a solution dissolve away producing Ag⁺ (or even Ag⁺⁺).^1–3 However, it has been clearly shown by scanning transmission electron microscopy that nanoparticles are attracted to bacterial membranes and not only stick to it but also penetrate the cell.⁴ This suggests a strong rule for Coulombic interactions. Our ability to develop new products is hampered by the incomplete knowledge of toxic effects. The field of nanotoxicology has experienced an increasing interest in the last few years,^5–8 mainly oriented to advance a responsible use for atomically-designed materials.

In the present paper we investigated the pharmacokinetic effects of silver nanoparticles (NPs) on adult male rats. The intracellular fate of Ag NPs in different organs has been assessed. This research particularly aims at determining whether or not metallic nanoparticles are able to diffuse through the BBB?^9–11 This is a monumental question in nanoparticle bioapplications. Drug delivery to the brain is rigorously controlled by the BBB which represents an insurmountable obstacle for many essential drugs including antibiotics, cytostatics, and central nervous systems (CNS) active drugs. The blood–brain barrier is the homeostatic defense mechanism of the brain against pathogens and toxins. This unique membrane is formed by special endothelial cells sealed with tight junctions, which restrict the entry to the brain from the periphery of many compounds that might be of therapeutic value in the treatment of neurological or psychiatric disorders, and other pathologies.

Our approach to clarify the potential permeability of the BBB membrane to metallic nanocrystals consisted of employing silver NPs passivated with bovine serum albumin , the most abundant protein in the blood plasma. As we reported before,¹² the BSA allows the growth of nanoparticles with a controlled size. In that way, we can avoid a broad distribution size (common in most of the synthesis methods). In order to investigate the transcytosis ability of BSA-silver nanocrystal conjugate and determine whether it was toxic to the integral of BBB tight junction or not, we designed a study model of the rat BBB in vivo.

Different strategies have been proposed to enable NPs to cross the BBB. Many of them are based on the physical adsorption of a hydrophilic surfactant.^13,14 Alternatively, NPs able to cross the BBB were prepared using amphiphilic copolymers,¹⁴ like the block copolymer of polyethylenglycol and n-hexadecylcyanoacrylate (PEG-PHDCA), in which the hydrophobic block itself is able to form a solid phase, while the hydrophilic part remains on the surface.^13,14 It has been already demonstrated that NPs uncoated with surfactants, remained in the blood vessels. Thus the ability to cross the BBB seems to be linked only to the chemico-physical characteristics of the surface of the NP.^13–14,16

Polymeric nanoparticles have demonstrated their utility as drug delivery carriers across the BBB.¹³ Some examples are nanocrystals manufactured with poly-(butyl-cynoacrylate) (PBCA) as the polymer and Tween 80 as the surfactant to promote drugs’ BBB transport. Unfortunately, further research has showed the in vivo toxicity of this carrier, which might be attributed to the toxic compounds produced by the rapid esterase biodegradation of PBCApolymer.¹⁴ On the other hand, BSA exhibits a greater capacity for binding brain microvessels and increased flux into the cerebrospinal fluid of mice.¹⁶ Published reports show that endorphin coupled with cationic bovine serum albumin (CBSA) could be transported into the brain more rapidly than native endorphin.¹⁶ CBSA-polymeric NP preferentially crosses the brain capillary endothelium and accumulates in the brain parenchyma. The process was speculated as absorptive mediated transcytosis (AMT), while its mechanism was not clarified.¹⁷ AMT takes place through interactions of positive charges around the particle surface with negative charges on the surface of the cells. The brain delivery property of CBSA-NP should then be closely related to the surface CBSA density of the particle. The principle behind the brain delivery pathway of CBSA-NP might therefore be AMT across the BBB initiated by the binding of CBSA to negative charges on the brain capillary endothelium.

Silver nanoparticles were employed because of their great antibacterial properties which have been exploited in many commercial products as antibiotic-delivery platforms.⁴ Silver nanocrystals have also successfully demonstrated to inhibit the HIV-1 virus infectivity in vitro.¹⁰ These noble metal nanostructures play a key role in near-term applications based on surface-enhanced Raman scattering (SERS) for detection of molecular events in biology.¹⁵Ag NPs display outstanding sensing properties as enhanced image contrast agents and nanoscale probes that can track the displacements of cells and individual molecules as they move about in their environment. The unprecedented ability of noble metal nanocrystals to observe and influence complex systems in vivo and in real time provides detailed information about the fundamental mechanisms and signaling pathways involved in the progression of disease.¹⁵Ag NPs offer alternative therapeutic strategies to probe the central nervous system (CNS), severely limited by the restrictive tight junctions at the endothelial cells of the BBB.

Our study demands intensive use of advanced electron microscopy methods to determine the presence of nanoparticles on organ tissue. In conventional TEM of organic matter a very low contrast has to be increased by the use of stainings. This hampers the identification of nanoparticles. In the present work we use STEM techniques to visualize the tissue (with fixation but no staining) we also perform conventional microscopy to identify features on the samples and compare with STEM images.

Materials and methods

BSA-conjugated Ag nanocrystals synthesis

Protein functionalized silver nanocrystals were synthesized in air by reduction in homogeneous solution at ambient conditions. Silver nitrate (AgNO₃; 0.945 N in water; Aldrich) was reduced in water, in the presence of bovine serum albumin BSA, Fraction V—eat shock treated; Fisher) by the addition of sodium borohydride (NaBH₄; 99%; Aldrich). Desired molarities of ionic silver were obtained by dilution with deionized water to a total solution volume of 25 mL. All materials were used without further treatment. In a typical experiment 1 mL of 0.6857 M AgNO₃ (685.7 μmol Ag) was added to an aqueous solution containing a variable amount of dissolved BSA protein. Then, 2 mL of freshly-prepared 0.6 M NaBH₄ aqueous solution (1200 μmol NaBH₄) was added rapidly and under vigorous stirring, bringing the total reaction volume to 40.0 mL. The introduction of NaBH₄ to the reaction vessel caused rapid protein foaming, and within less than five seconds, the solution acquired a deep brown colour (Ag). The reaction was allowed to proceed under vigorous stirring for 30 min. The product consists of water-soluble silver nanocrystals less than 3 nm in diameter, directly conjugated to bovine serum albumin protein (Fig. 1a).

Fig. 1 (a) Schematic diagram depicting the structure of the BSA-protected silver nanoparticles used in this study (b) UV-visibleabsorption spectra of various silver systems capped with BSA. Note the diminishing peak intensity at 278 nm with increased silver loading, and the increase in peak intensity at 410 nm for the (96 : 1) Ag : BSA system.

The size-limiting capabilities of BSA for silver nanoparticles synthesis are demonstrated here by analyzing colloids with various molar ratios of Ag atoms to protein molecules. The [Ag] : [BSA] molar ratios can also be tuned to engineer the optical properties of Ag nanoparticles as proven in their absorption spectrum (Fig. 1b). The ultraviolet visible (UV-Vis) absorption spectra of silver nanocrystals exhibit a characteristic surface plasmon resonance peak at ∼400 nm. The full-width at half maximum of the surface plasmon resonance is inversely proportional to the nanocrystal diameter, so the absorbance peak becomes broader and less intense as the size of the nanocrystals decrease. For silver nanocrystals ∼2 nm in diameter, the surface plasmon resonance can be blue-shifted due to the onset of quantum size effects. The absorption spectrum for 24 : 1 Ag : BSA exhibits a broad, symmetric peak at ∼395 nm. Both the position and the breadth of the surface plasmon resonance suggest the presence of silver nanocrystals ∼2 nm in diameter. The absorbance maximum for (48 : 1) Ag : BSA is red-shifted to ∼400 nm. This suggests a slightly larger average nanocrystal size than the (24 : 1) Ag : BSA system, but the breadth and symmetry of the maximum still indicates a well-dispersed population of small silver nanocrystals. Compared to the other two systems, (96 : 1) Ag : BSA displays a narrower, much more intense absorbance maximum that is red-shifted to ∼410 nm, indicating the presence of larger silver crystals in this system. Also note that the characteristic absorbance maximum for BSA at 278 nm is progressively quenched with increasing concentrations of silver atoms.

Results and discussions

Transmission electron microscopy characterization

TEM analysis of silver nanocrystals was performed using a JEOL 2010F transmission electron microscope equipped with Schottky-type field emission gun, ultra-high resolution pole piece (Cs = 0.5 mm), an Energy Dispersive X-ray spectrometer (EDS), and a scanning transmission electron microscope (STEM) unit with high angle annular dark field (HAADF) detector operating at 200 kV. Onto a lacey carbon-coated copper TEM grid a drop of the as-synthesized aqueous product was allowed to slowly evaporate. As the water content evaporates, neighboring protein molecules interact to form a thin film ; so, by focusing within the holes of the lacey carbon, TEM and HAADF images of the protein-functionalized nanocrystals were obtained without the interference of the lacey carbon support layer. In brief, a sub-nanometre electron probe is rastered over the sample, and incoherently scattered electrons are collected by means of a high-angle annular detector. Fig. 2 presents a visual comparison of the nanocrystal size distributions observed in the (24 : 1) (Fig. 2a) and (48 : 1) Ag : BSA (Fig. 2b), respectively. The silver nanocrystals formed in the (24 : 1) Ag : BSA system were well-dispersed, with a measured size distribution of 2.094 ± 0.287 nm, or a standard deviation of ∼13.7%. A bimodal distribution is clearly observed in the (96 : 1) Ag : BSA system, as summarized later in Fig. 2b, with ∼30% of the nanometric-scale crystals greater than 2.50 nm in diameter. The size distribution for this system was measured as 2.289 ± 0.460 nm, or a standard deviation of ∼20.1%. Although this represents an increase of only ∼10% in the mean particle diameter as compared with the (24 : 1) Ag : BSA system, the percentage standard deviation in the (96 : 1) Ag : BSA system is more than 45% greater. Colloids with molar ratios of 24 : 1 Ag : BSA were employed for the in vivo test in order to assure critical experimental conditions (small diameter and narrow size-distribution) under which the nanocrystals could permeate the BBB.

Fig. 2 High Angle Annular Dark Field images and corresponding size distribution for silver nanocrystals protected with different Ag : BSA molar ratios: 24 : 1 (a) and 96 : 1 (b).

High-resolution transmission electron microscopy (HRTEM) analysis was performed to determine the structures of silver nanocrystals produced in the (24 : 1) Ag : BSA system (Fig. 3). About one-third of the structures were FCC single crystals with either truncated octahedral (Fig. 3a,b) or cuboctahedral (Fig. 3c,d) morphologies. Both of these structures can be considered as resulting from truncation of a cube, resulting in the formation of {111}-type surfaces, and they are among the most stable morphologies for nanocrystals in this size range. Another one-third of the observed nanocrystal structures were single twinned cuboctahedral (Fig. 3e–g). The formation of structures where a twin plane forms a boundary between subunits with mirror symmetry to each other is quite common in FCC nanocrystals, and especially in silver. The final one-third of the structures consisted of multiple-twinned crystals with fivefold symmetry (Fig. 3h–i). Silver possesses the lowest twinning energy for FCC metals, so it is not surprising that many single-twinned and multiple-twinned nanocrystals of silver are observed.

Fig. 3 High Resolution Transmission Electron Microscopy images for 24 : 1 Ag : BSA nanocrystals: (a)–(b) FCC truncated octahedral; (c)–(d) FCC cuboctahedral; (e)–(g) single twinned cuboctahedral; (h) Marks’ decahedron; (i) pentagonal bipyramidal.

In vivo tests. Female Wistar rats (150–200 g) were maintained prior to death at room temperature (22 ± 2 °C) on 12 h/12 h light/dark cycle having access to food and water ad libitum during the course of study. All animal experiments were conducted according to the protocols of the Ethical Committee of the School of Medicine of the Autonomous University of Nuevo Leon in accordance with the Public Health Service (PHS) Policy on Humane Care and Use of Laboratory Animals (Department of Health and Human Services, HHS).

For in vivopharmacokinetic studies, the rats were killed after 24, 96 and 168 h after intraperitoneal (IP) injection, and the brain, heart, kidney, liver, lung and spleen were collected. The tissues were weighed, digested and then analyzed for silver content using ICPMS. TEM images of the aforementioned organ tissues were acquired and examined by a pathologist.

Animals were divided into 6 groups (including 10 rats in each group). Groups designated as 1, 2 and 3 received an aqueous suspension of BSA-capped Ag nanoparticles (50 mg Kg⁻¹) by IP injection and control group (designated 4, 5 and 6) received distilled water. The animals from groups 1, 2 and 3 and their corresponding control groups (4, 5 and 6) were anesthetized with etilic ether inhalation and subsequently sacrificed. The liver, kidney, heart, lungs, spleen and brain were removed. The organs were weighed and a section was fixed in glutaraldehyde for electronic microscopy studies and the rest of the organs were used to determine the silver concentration.

The biodistribution of the silver nanocrystals was assessed by ICPMS, revealing significant accumulation of Ag in the reticuloendothelial system (RES) (liver, spleen) (Fig. 4a). This result is expected, as many other nanomaterials commonly present high uptake in RES organs as a result of the efficient renal clearance of the organism, aimed at mitigating potential toxic effects. The remarkable silver concentrations in the liver and spleen after 96 (Fig. 4b) and 168 h. (Fig. 5a) suggest slow renal excretion. The existence of silver in the kidney also proposes possible urinary and biliary excretion routes^18,19 for nanostructured silver. The clearance mechanism for silver in the brain proceeds in much faster fashion (Fig. 4a,b and 5a) The observed biodegradation of silver nanocrystals enable us to think that the first 24 h could be a time constraint for these nanomaterials to carry out therapeutic functions.

Fig. 4 In vivo biodistribution of BSA-capped Ag nanocrystals over periods of 24 (a) and 96 (b) hours in rats. Aliquots of Ag nanocrystals were intraperitoneal administered into the rats. The silver concentration in the organs was determined at different time points after injection using ICPMS. N = 10, P < 0.05.

Fig. 5 (a) In vivo biodistribution of BSA-capped Ag nanocrystals over a period of 168 h in rats. (b) Comparison of Ag nanocrystals biodegradation for organs of reticuloendothelial (spleen), cardiovascular (heart) and central nervous (brain) systems. N = 10, P < 0.05.

However, this fast self-destruction rate could also be advantageous to design effective antibiotics and antivirals that degrade in vivo into benign soluble compounds that clear renally within specific timescales.^18–20 The prompt clearance mechanism of silver in the brain clearly differs from the one observed in organs of the reticuloendothelial and cardiovascular systems (Fig. 5b). Additional statistical information is included as ESI.† The next key-questions to address are (a) what is the physico-chemical structure of the silver loading confirmed in the organs? and (b) what are their consequences in the different organ cells? These queries can only be answered by thorough electron microscopy analysis.

We focused on the brain, liver, kidney, and heart for TEM examination. The brain and other organs were removed, and after several subsequent rinses with 0.1 M phosphate buffer solution (PBS), they were harvested with 4.0% formaldehyde and kept at 4 °C. After washing the tissue samples several times with 0.1 M PBS, these were sliced and randomly selected for post-fixed treatment with 2.5% glutaraldehyde in 0.1 M PBS for 24 h, washed again with 0.1 M PBS, and exposed to 1% osmium tetraoxide in 0.1 M PBS buffer (pH 7.3) for 1 h. After rinsing the tissue several times, it was dehydrated in stepped absolute ethanol solutions and flat embedded in Spur’s embedding media as previously described earlier. In other cases, the organs were fixated with glutaraldehyde and thin sections were microtomed.

Observation of particles on the liver, heart and kidney

We examined images of liver tissue using TEM and STEM analysis of unstained samples, both fixated with Os and without fixation.

The images clearly demonstrate the presence of nanoparticles as shown in Fig. 6a,b. The observed bright spots correspond to silver nanoparticles. The size of the nanoparticles differed of their original dimensions on the original synthesis suggesting that coalescence or aggregation events occurred in the blood serum or in the tissue.

Fig. 6 Liver TEM (a) and STEM (b) histology. Silver nanocrystals can be clearly observed. The organ was collected after intraperitoneal injection of BSA-capped Ag nanocrystals. STEM-EDX (c) analysis confirmed content of silver.

STEM-EDX analysis in mapping mode revealed relatively weak silver signal attributed to the presence of the embedding media employed for microtoming the tissue. STEM images of the heart and kidney also confirmed an evident content of silver nanoparticles (Fig. 7a,b). The concentration of silver in the urine was monitored in order to determine the clearance mechanism of the studied nanoparticles (Fig. 7c).

Fig. 7 Heart (a) and kidney (b) STEM histology. Ag nanocrystals presence can be visualized in these organs, extracted after intraperitoneal injection. (c) Evolution of the concentration of silver in the urine over a three-week period. Inset shows weight observation of rat.

The size and charge of most nanoparticles prevent their prompt secretion from the body as intact nanocrystals. The lack of such renal excretion or their biodegradation into biologically benevolent components could potentially amplify their toxicity, while also hindering diseases biosensing and imaging. Renal filtration and urinary excretion of silver was verified over a three week period (Fig. 7c).

Silver on the brain tissue

We extensively examined tissue from the brain corresponding to animals injected with and without silver nanoparticles exploiting TEM and STEM modes (Fig. 8). No nanoparticles were found in the brain tissue corresponding to the animal injected with BSA-capped Ag nanoparticles (Fig. 8a,b). Comparison with control tissue (Fig. 8c,d) revealed signs of silver-induced damage, evidenced in abnormal swelling of neuritic processes with loss of internal organelles and abnormal membranes with vacuolated cytoplasma. EDX mapping analysis of a single neuron shown in Fig. 9a,b demonstrates the presence of silver on the tissue. The absence of nanoparticles in the brain contrasts with the silver loading confirmed by ICPMS (Fig. 4,5).

Fig. 8 Brain histology of silver loaded analyzed by TEM (a) and STEM (b) modes. No nanocrystals were observed. Control tissue was also analyzed by TEM (c) and STEM (d) modes.

Fig. 9c shows an STEM-EDX mapping of Ag-loaded tissue. As the figure shows there is a silver distribution which is rather uneven. No well defined nanoparticles were observed. We also acquired Confocal Microscopy images of the brain tissue with Ag-loading and without (Fig. 10) and did not find fundamental differences for different excitation wavelengths (415, 573 and 719 nm). Therefore, light and electron microscopy analysis conducted in this work also seem to indicate that analyzed silver particles were not able to cross the BBB.

However it is most likely that Ag⁺ ions crossed the BBB and interacted with the brain cells. We have shown that after injecting adult rats with fixed doses of aqueous suspension of BSA-capped Ag nanoparticles and conducting histology analysis at 24, 96 and 168 h, the nanostructures readily situate in the reticuloendothelial and cardiovascular systems. On the other hand, silver loadings were actually confirmed in the brain but no particles were detected.

Fig. 9 STEM histology of Ag-loaded brain tissue at low- (a) and high-magnification (b). EDX mapping (c) and spectrum (d) revealed silver content, likely present in ionic form.

Fig. 10 Ex vivo Confocal Microscopy images of Ag-loaded and control brain tissues for different wavelengths and transmitted light. No evidence of silver nanoparticles was found. Scale bar indicates 2 μm.

Conclusions

After a very careful analysis using advanced TEM techniques such as HAADF-STEM, EDS and HRTEM we found no evidence that silver nanoparticles, with sizes between 1–2 nm, can cross the BBB. No evidence of silver nanoparticles in the brain was collected. The only way to explain this is assuming that silver ions were released from the nanoparticles. Silver ions can apparently penetrate the BBB.

However the mechanism is not clear and further research is needed. Nevertheless our analytical spectroscopy results did detect the presence of silver dispersed along the brain. Silver (soft acid ) has a greater tendency to react with sulfur- or phosphorus-containing proteins in the membrane or inside the cells and phosphorus-containing elements like DNA are likely to be the preferential sites for silver nanoparticle binding.^12,21 It has been suggested that disruption of the membrane morphology may cause a significant increase in permeability, leading to uncontrolled transport through the plasma membrane and, finally, cell death.

According to recent work on the cytotoxicity of mammalian cells^21,22 it has been suggested that the mitochondria are very sensitive to Ag⁺ ions. It has also been suggested that oxidative stress might be a mediation mechanism for the cytotoxicity of silver ions.²² There is however growing evidence that the damage in mitochondria is due to the interaction of Ag⁺ with thiol groups of the mitochondrial inner membrane. This mechanism has been suggested to be responsible for the bactericidal and antiviral effects of silver nanoparticles.

Acknowledgements

The Authors are indebted to the Welch foundation financial assistance and to the ICNAM for supporting exchange between UTSA and UANL. We thank Dr Angela Bardo, affiliated to the Institute of Cell and Molecular Biology for her assistance in the Confocal Microscopy acquisition and analysis. Dr Gilberto Arevalo kindly provided assistance for the experiments with animals.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: Additional statistical information. See DOI: 10.1039/b916107d

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