Bio-distribution and in vivo/in vitro toxicity profile of PEGylated polymer capsules encapsulating LaVO₄:Tb³⁺ nanoparticles for bioimaging applications

Abstract

Lanthanide-doped nanoparticles are being explored for bioimaging applications owing to their unique optical properties. However, their toxicity profiles have not been explored in detail at the in vivo level which is a pre-requisite for clinical use. To this end, we have investigated a detailed in vivo/in vitro toxicity profiling of PEGylated polymer capsules encapsulating LaVO₄:Tb³⁺ nanoparticles. First, in vitro toxicity studies were carried out in L929 mouse fibroblast cells in which MTT assay, neutral red uptake (NRU) assay, LDH release, cell morphology; quantification of reactive nitrogen species (RNS), reactive oxygen species (ROS), lipid peroxidation level, caspase-3 activity, and apoptosis assay were also performed. Second, in vivo studies were carried out in Swiss Albino mice, which included biodistribution studies (6 h and 24 h), evaluation of hematological parameters, and toxicity marker enzyme levels (inflammation, oxidative stress, tissue infiltration, liver dysfunction, etc.), organ histology, and genotoxocity. The detailed investigation of toxicity at in vitro and in vivo levels indicates the biocompatibility of PEGylated polymer capsules encapsulating LaVO₄:Tb³⁺ nanoparticles proving their suitability for bio-imaging applications.

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1. Introduction

There is a large interest in the development of new bioimaging probes for precise imaging of diseased tissues at the cellular and sub-cellular levels. Various nanomaterials including metal nanoparticles (e.g. gold, silver),¹ quantum dots (e.g. CdSe, PbSe, CdS)² as fluorescence imaging agents, and magnetic nanoparticles (e.g. iron oxide, CoPt, GdVO₄)^3–5 as MRI agents have been explored in bioimaging applications. Though some of the above imaging agents show promising results, they exhibit toxicity and low photostability.⁶ For example, organic dyes possess poor photostability, whereas quantum dots exhibit toxicity.⁶ In this context, lanthanide-doped nanoparticles (e.g. LnF₃:Ln³⁺, NaLnF₄:Ln³⁺, LnVO₄:Ln³⁺, Ln₂O₃:Ln³⁺, LnPO₄:Ln³⁺) are gaining attention as they display unique properties such as large Stoke's shifts,^7–10 long lifetimes,¹¹ sharp emission bands,⁶ higher photostability,⁶ emission ranges from UV to NIR,^12–15 upconversion,¹⁶ Stoke's shift fluorescence and magnetic properties^4,17,18 and are being explored for biological applications.^6,19–24 Lanthanide-ion doped nanoparticles are generally prepared as hydrophobic nanoparticles and they have been converted into hydrophilic ones by various surface modification techniques which quenches their emission properties. This may require administration of large quantities (Y₂O₃:Yb³⁺/Er³⁺ (20 mg per kg of body weight), NaGdF₄:Yb³⁺/Er³⁺ (15 mg Gd per kg of body weight)) of nanoparticles to obtain a good image at in vivo level.^21,22,25 In this regard, our group has reported a strategy²⁶ in which lanthanide doped nanoparticles are encapsulated in polymer capsule using layer-by-layer (LbL) assembly^27–34 for bioimaging applications. The reported strategy confer the following advantages: (a) enhanced emission properties due to the sol–gel synthesis approach, (b) biocompatibility with various cancer cells and macrophages, (c) good stealth nature due to PEGylation, (c) possibility of antibody conjugation to improve targeting ability, (d) prospects for development of theranostic vehicle by loading the drug along with nanoparticles, and (g) single uptake mechanism of nanoparticles-loaded capsules in different cells.

In order to employ them as imaging agent for clinical applications, it is essential to investigate their biodistribution with clearance and toxicology profile under in vivo conditions.³⁵ We note that several reports are available for in vivo biodistribution of lanthanide-doped nanoparticles; however, their in vivo toxicity has not been studied in detail.^17,36–38 In addition, limited reports are available on biodistribution of polymer capsules including a couple of reports by our group.^39,40 To this end, we report detailed toxicological study of PEGylated polymer capsules encapsulating LaVO₄:Tb³⁺ nanoparticles at both in vitro and in vivo levels. In vitro toxicity studies were carried out in L929 mouse fibroblast cells which include MTT assay, cell morphology, NRU assay, LDH release, and quantification of reactive oxygen species (ROS), reactive nitrogen species (RNS), lipid peroxidation level, caspase-3 activity, and apoptosis measurement. Erythrocyte lysis was checked to analyze blood compatibility. In addition, in vivo studies were performed in Swiss Albino mice which include biodistribution studies (6 h and 24 h), evaluation of hematological parameters, and activity of toxicity marker enzymes for inflammation, oxidative stress, tissue infiltration, liver dysfunction, etc. Furthermore, myeloperoxidase (MPO) assay and organ histology were carried out in kidney and liver tissues in which the major uptake has taken place. Finally, comet assay was performed to evaluate genotoxicity in peripheral blood (PB). All the above studies clearly suggest that PEGylated polymer capsules encapsulating LaVO₄:Tb³⁺ nanoparticles possess biocompatibility with minimal toxicity which substantiates them as potential bioimaging agents.

2. Materials and methods

2.1. Materials

All the reagents used were of analytical grade. Lanthanum nitrate hydrate (LaNO₃·xH₂O), terbium nitrate pentahydrate (Tb(NO₃)₃·5H₂O), polyethylene glycol (M_w 10 000), poly(sodium 4-styrene-sulfonate) M_w 70 000, poly(allylamine hydrochloride) M_w 56 000, bisamine (polyethylene glycol) M_w 6000 were purchased from Sigma-aldrich. All the lanthanide salts were 99.9% pure and were used without further purification. Polyethyleneimine (PEI, M_w 70 000 branched), Alfa Aesar; citric acid (Qualigens), liquor ammonia (about 24%, Merck), ammonium fluoride (Merck), sodium metavanadate lobachemie; hydrofluoric acid (40% w/v) sd-fine chem., tetraethylorthosilicate (Fluka) were the other chemicals procured.

Dulbecco's modified eagle's medium (DMEM), fetal bovine serum (FBS) and antibiotic–antimycotic solutions were procured from Gibco, USA. Biochemicals and caspase-3 assay kit were purchased from Sigma Chemical Company, USA. LDH cytotoxicity kit was purchased from Cayman Chemical Company and all chemicals of reputed makes were procured locally. Enzyme based assays for detection of AST, ALT, ALP, CK and LDH at in vivo were carried out using ERBA Mannheim, India. L929 mouse fibroblast cell line was obtained from National Centre for Cell Science Pune, which is a national repository of cell lines in India.

2.2. Synthesis of polymer encapsulated LaVO₄:Ln³⁺ nanoparticles

Synthesis of silica particles (∼500 nm) were carried out using modified Stöber's process.^41,42 It was coated with LaVO₄:Tb³⁺ nanoparticles using sol–gel method reported elsewhere.⁴³ The core–shell particles were annealed at 500 °C for 3 h. Layer-by-Layer (LbL) assembly was carried out with PEI as first layer, followed by PSS/PAH polyelectrolytes alternatively to form 8 layers. Finally, PEGylation was carried out and removal of silica core was performed using buffer oxide etchant (pH 5) to form PEGylated polymer capsules entrapping LaVO₄:Tb³⁺ nanoparticles.

2.3. In vitro cytotoxicity assays and cell uptake

The cyto-compatibility of polymer capsules encapsulating LaVO₄:Tb³⁺ nanoparticles were initially performed in L929 cells and PBMNCs. 5 × 10³ numbers of cells were treated with different concentrations of polymer capsules (P1 − 3 × 10⁶/mL, P2 − 4 × 10⁶/mL, P3 − 5 × 10⁶/mL capsules) for 24, 48 and 72 h in 96-well plates. Cell viability was determined using MTT assay. Further cytotoxicity evaluation was carried out in L929 cells by NRU assay, LDH release, ROS and RNS levels, lipid peroxidation, caspase-3 activity and apoptosis assay after exposing 5 × 10³ cells to the different concentrations of polymer capsules for 24 h. We wanted to ensure that these capsules are non-toxic to healthy cells and does not initiate any toxic response. Thus, in vitro toxicity assays were performed in L929 fibroblast cells which are adherent non-cancerous or normal cells isolated from mouse. Following are the assays carried out: (a) for MTT assay, culture media was removed after 24 h incubation; MTT (0.5 mg mL⁻¹ prepared in fresh basal media) was added and incubated for 4 h. After incubation, DMSO was added to the wells and absorbance was measured at 570 nm wavelength,²⁶ (b) NRU assay was carried out using neutral red dye (3-amino-m-dimethylamino-2-methylphenazine hydrochloride) which is actively transported to lysosomes in live cells. Absorbance was recorded at 540 nm wavelength,⁴⁴ (c) LDH release has been carried out as per instructions in Cayman's LDH cytotoxicity assay kit, (d) ROS generation has been evaluated by quantification of green fluorescence of dichlorofluorescein (DCF) formed from dichlorofluorescein diacetate (DCFDA) by fluorescence measurement⁴⁵ at 502 nm (excitation) and 523 nm (emission) and by subtracting the fluorescence intensity of polymer capsules. Cells were also observed under fluorescence microscope to compare the intensities of fluorescence, (e) RNS generation is measured by quantifying nitrite production using Griess diazotization assay,⁴⁶ (f) thiobarbituric acid-reactive substances (TBARS) were measured to assess the lipid peroxidation level, (g) activity of caspase-3 was determined by colorimetric assay kit from Sigma-Aldrich Co, USA, (h) apoptosis measurement was carried out by FACS (fluorescent activated cell sorting) after propidium iodide/annexin V staining (Muse kit). All results were compared with PBS treated control and corresponding positive controls. Cytotoxic drug cisplatin (10 μM) treated cells served as positive control in all the assays except LDH release assay and ROS generation where 1% triton X-100 and 100 μM H₂O₂ were respectively used. Cell uptake experiments were carried out to study the internalization of polymer capsules encapsulating nanoparticles in L929 cells. To brief the protocol, 10⁵ cells were seeded in a 24-well plate, with gelatin coated 13 mm glass coverslips. Once the cells got adhered to the coverslips, polymer capsules were added and incubated for 6 h. It was followed by washing with PBS (7.0) to remove excess capsules attached in the coverslip. Finally, the cells were fixed with formaldehyde and stained for the cell membrane with red emitting dye (cy 5.0). Imaging (including z-stacks) was carried out using confocal laser scanning microscopy.²⁶

2.4. Blood compatibility studies

Blood (2–3 mL) drawn from healthy volunteers was collected into citrate tubes. Informed consent was obtained from the subjects for collection of blood. Erythrocytes were separated, washed thrice with n-saline and diluted in the ratio 1 : 5 with saline. Hemolysis assay was carried out by treating 50 μL of erythrocyte suspension with different concentrations of polymer capsules (P1 – 3 × 10⁵, P2 – 4 × 10⁵, P3 – 5 × 10⁵ capsules) and incubated at 37 °C for 1 h, centrifuged the suspension and absorbance of supernatant was measured at 542 nm. 0.1% triton X-100 treated group has been taken as positive control.

2.5. In vivo studies

Female Swiss Albino mice (2–3 months) weighing between 20 and 25 g were reared in the department animal house and were fed with standard laboratory pellet diet. They were housed in an environment of controlled temperature, humidity and light/dark cycle and drinking water was provided ad libitum. The animals were handled as per Committee for Purpose of Control and Supervision of Experiments on Animals (CPCSEA) rules. Animal experiments were done in absolute compliance with the approval and guidelines of Institutional Animal Ethical Committee; ethical sanction no. IAEC-KU-5/2010-'11-BC-AA. They were weighed weekly and the food and water consumption was observed. In order to check the lethal toxicity of polymer capsules, different concentrations of the capsules (1 × 10⁹ capsules, 2 × 10⁹ capsules and 3 × 10⁹ capsules per kg body weight) were intravenously administered to mice as described in the protocol. The animals were kept away from intense light thereafter. Body weight, animal behavior and food & water intake was observed for a period of three months. All the mice of the different groups survived till the end of the experimental period and did not exhibit any toxicity or behavioral changes. Therefore, we chose the highest dose (3 × 10⁹ capsules equivalent to 0.4 mg LaVO₄ per kg body weight) for further experiments.

2.5.1. Biodistribution by elemental analysis of lanthanide by ICP-MS. Polymer capsules encapsulating LaVO₄:Tb³⁺ nanoparticles were intravenously administered at a dose of 3 × 10⁹ capsules per kg body weight. The mice were sacrificed at 6 h and 24 h post injection. Blood was drawn and whole organs such as liver, spleen, kidney and lungs were excised, urine was also collected. The organs were thoroughly washed with PBS and blotted with filter paper. The whole organ samples, blood and urine were digested with 70% nitric acid at a temperature of 110–120 °C for 4–5 h. The remaining undissolved solids were removed by filtering through a membrane filter and the clear solution obtained was diluted with distilled water for ICP-MS analysis.⁴⁷

2.5.2. Biocompatibility studies. Swiss Albino mice were randomized into three groups. Group I – control mice (PBS treated), group II & III – 3 × 10⁹ capsules per kg body weight were administered intravenously through tail vein according to the body weight (4 μL g⁻¹) per week. Animal behavior, survival, and animal mass were monitored and evaluated over one week (group II) and one month (group III), after which animals were euthanized. Blood was collected for evaluating toxicity marker enzymes including aspartate transaminase (AST),⁴⁸ alanine transaminase (ALT),⁴⁸ lactate dehydrogenase (LDH), alkaline phosphatase (ALP), and creatinine kinase (CK) in serum, 5-lipoxygenase (5-LOX)⁴⁹ activity was measured in peripheral blood mononuclear cells (PBMCs) and myeloperoxidase (MPO) activity in liver and kidney tissues. Antioxidant levels have been determined by estimating activity of super oxide dismutase (SOD),⁵⁰ catalase,⁵¹ amount of thiobarbituric acid reactive substance (TBARS)⁵² and reduced glutathione (GSH).⁵³ Hematologic parameters such as hemoglobin content, total white blood cell count (WBC), red blood cell count (RBC) and blood urea nitrogen (BUN) were analyzed using a semi-auto analyzer. Liver and kidney were collected after 1 month of administration of polymer capsules to study organ histology. Genotoxicity was determined in peripheral blood by Comet assay.⁵⁴ Comet scoring was performed using TriTek CometScore™ v1.5.

2.6. Statistical analysis

All statistical calculations were carried out using statistical package for social sciences (SPSS) software program (version 17 for Windows). The values were expressed as the mean ± standard deviation. The data were analyzed using one-way analysis of variance (ANOVA) and significant difference of means between groups was determined using Duncan's multiple range tests at the level of P < 0.05. Same alphabet on error bars indicates no significant change between groups, whereas different alphabets indicate significant difference of values between groups at P < 0.05.

2.7. Characterization

FEI Technai Twin microscope (TEM) and SUPRA® Series ultra high resolution field emission-scanning electron microscope FE-SEM (SEM) have been used for acquiring images for confirming morphology and presence of nanoparticles in capsules. Photoluminescence spectrum was obtained in Edinburgh Instruments Fluorescence spectrometer (FLSP 920) equipped with 450 W Xe arc lamp as source. DLS measurement for determining hydrodynamic size was obtained using Malvern instruments. Internalization of polymer encapsulated nanoparticles in L929 cells was confirmed through images from laser scanning confocal microscope Carl Zeiss LSM 710. Olympus CKX41 with optika pro5 CCD camera was used to capture fluorescent images in ROS determination and phase contrast images for morphology studies. Muse flow cytometer Millipore USA was used for FACS analysis. ICP-MS was done using Thermo Scientific XSERIES2 ICP-MS. Absorbance measurements have been performed using JASCO-L-670 spectrophotometer. Leica DM 1000 LED, Camera DFC295 was used to capture images of organ histology.

3. Results and discussion

3.1. Synthesis and characterization of polymer capsules

Polymer capsules encapsulating lanthanide doped nanoparticles were prepared by following unique approach which is divided into five steps: (a) synthesis of monodisperse silica core using modified Stöber's process, (b) coating of silica with LaVO₄:Tb³⁺ particles followed by calcination, (c) layer-by-layer assembly of oppositely charged polyelectrolytes (PSS/PAH) over core shell particles, (d) PEGylation, and (e) removal of silica core leading to encapsulation of nanoparticles inside polymer capsules. TEM image (Fig. 1A) confirms entrapment of LaVO₄:Tb³⁺ particles within the polymer capsules and high resolution TEM (Fig. 1B) shows lattice fringes indicating crystalline nature of sample. SEM image indicates the formation of monodispersed polymer capsules (∼500 nm size) and absence of nanoparticles on surface (Fig. 1C). In addition, fluorescence spectra of polymer capsules (dispersed in water) at 488 nm excitation shows emission bands of Tb³⁺ ions at 545 nm, 585 nm and 620 nm, as seen in Fig. 1D. Energy-dispersive X-ray spectrum (ED) furthermore confirms the presence of lanthanum and vanadium (Fig. S1†). DLS measurements suggest that the hydrodynamic radius of polymer capsules is ∼560 nm which matches with the TEM results (Fig. S2†). In order to check the stability of capsules, TEM images were acquired for samples after six months from preparation. Fig. S3† shows that polymer capsules encapsulating nanoparticles are stable (aggregation in images are attributed to drying effect during sample preparation for TEM).

Fig. 1 PEGylated polymer capsules encapsulating LaVO₄:Tb³⁺ nanoparticles (A) TEM, (B) HR-TEM, (C) SEM, and (D) emission spectrum (λ_ex = 488 nm).

3.2. In vitro toxicology studies

Internalization of polymer capsules encapsulating LaVO₄:Tb³⁺ has been studied by incubating them with L929 cells for 6 h. Confocal microscopy image (Fig. 2) demonstrates green fluorescence exhibited by Tb³⁺ ion in cells, indicating the internalization of capsules (cell membrane has been stained red using cy5 dye). From z-stack images at 0.5 μm interval, it can be stated that capsules are internalized in cells which is confirmed with the green emission arising from nanoparticles obtained at different intervals (Fig. S4†). Cytotoxicity of polymer capsules encapsulating LaVO₄:Tb³⁺ was assessed in L929 cells using MTT assay and NRU uptake (Fig. 3). The former measures reduction of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a tetrazole) to purple formazan in the mitochondria of living cells and the latter measures the incorporation and binding of the supravital dye, neutral red (3-amino-m-dimethylamino-2-methylphenazine hydrochloride) in the lysosomes. Results indicate more than 90% cell viability at all the concentrations of polymer capsules when compared to positive control (cisplatin). It was observed that cisplatin (10 μM) treated cells showed around 40% cell death (Fig. 3A). Similarly MTT assay in L929 and PBMNCs for 24, 48 and 72 h also exhibits biocompatibility of polymer capsules (Fig. S5†). Damage to cell membrane was checked using lactate dehydrogenase (LDH) assay. LDH is an enzyme present in the cytosol of cells and therefore, its leakage in medium is used to assess disruption of cell membrane. It has been found that membrane integrity is not affected by polymer capsules as comparable LDH release is observed to that in control cells (Fig. 3B). Positive control cells treated with 1% triton X-100 showed an increased LDH release indicating membrane damage.

Fig. 2 CLSM images showing PEGylated polymer capsules LaVO₄:Tb³⁺ nanoparticles internalized in L929 cells: (A) cell membrane stained with red emitting dye, (B) green emission arising from Tb³⁺ ions, (C) merged image of cell membrane and Tb³⁺ emission, (D) differential interference contrast (DIC) image showing cell, and (E) merged image of DIC with all channels.

Fig. 3 Graphs show biocompatibility of L929 cells treated with different concentration of PEGylated polymer capsules encapsulating LaVO₄:Tb³⁺ nanoparticles (P1 – 3 × 10⁶/ml, P2 – 4 × 10⁶/ml, and P3 – 5 × 10⁶/ml) for (A) MTT assay, neutral red uptake (NRU) assay, and (B) LDH release. Different alphabets indicate significant difference at p < 0.05.

It is reported by some groups that cellular internalization of nanoparticles has been shown to activate immune cells including macrophages and neutrophils, contributing to ROS/RNS generation.^35,55 Therefore, oxidative stress levels were also measured in cells incubated with polymer capsules by analyzing ROS and RNS generation. In general, small amounts of reactive oxygen species (ROS) and reactive nitrogen species (RNS) are naturally generated during body's metabolic reactions. These include hydroxyl radicals (OH˙), hydroperoxyl radicals (HOO˙), peroxy nitrite (ONOO⁻), nitric oxide radicals (NO˙), oxygen radicals, and superoxide radical anions (O₂˙⁻). When there is an imbalance between ROS generation and detoxification of the reactive intermediates, it results in excess ROS that can initiate damaging biological responses leading to oxidative stress phenomenon.⁵⁶ Inside the cell, DCFDA is deacetylated by cellular esterase and then oxidized by ROS to DCF, a highly fluorescent compound. Measurement of this fluorescence intensity per mg of protein is employed to quantify ROS generation. Fig. 4 demonstrates higher fluorescence intensity in H₂O₂ (100 μM) treated positive control cells indicating elevated ROS generation compared to cells incubated with capsules. Polymer capsules at all the concentrations do not make any significant change to the levels of ROS and are comparable to control cells (Fig. S6A†). The RNS generation has been determined by spectral measurement of a colored compound formed as a result of reaction between nitrite and Griess reagent. Results of RNS were compared to cisplatin treated positive control cell in which there was increased generation of RNS (Fig. S6B†). Further, oxidation of lipids in cell membrane has been checked by quantifying TBARS or malondialdehyde (MDA) (a by-product of lipid peroxidation) which is also an indicator of peroxide level. MDA levels in cells treated with polymer capsules were found to be comparable to the control group thus indicating that the capsules do not produce oxidative stress (Fig. S6C†).

Fig. 4 Fluorescence microscopy images indicating ROS generation in L929 cells incubated with: (A) PBS (control), (B) PEGylated polymer capsules encapsulating LaVO₄:Tb³⁺ nanoparticles and (C) 100 μM H₂O₂ as positive control (intense green fluorescence arises from dichlorofluorescein (DCF) as a result of higher ROS generation).

We analyzed caspase-3 enzyme activity and FACS to find whether the polymer capsules encapsulating LaVO₄:Tb³⁺ nanoparticles are inducing apoptosis (a programmed cell death phenomena). Caspases are group of proteolytic enzymes that are activated at early stages of apoptosis and therefore considered as biomarker for apoptosis.⁵⁷ The activity of caspase-3 enzyme is observed by its ability to cleave acetyl-Asp-Glu-Val-Asp-p-nitroanilide which leads to formation of p-nitroaniline which is detected at 405 nm. There is no significant increase in activity of caspase-3 in cells treated with polymer capsules encapsulating LaVO₄:Tb³⁺ nanoparticles (Fig. S7A†). In addition, one of the earliest apoptotic features is the translocation of phosphatidyl serine (PS) from inner to the outer layer of plasma membrane. The PS exposure was detected by FACS after propidium iodide/annexin V FITC staining wherein; the binding of annexin V with PS is analyzed. Viable cells are annexin V and PI negative, while early apoptotic cells are annexin V positive and PI negative, dead cells and cells that are in late apoptosis are both annexin V and PI positive. Results exhibit 90% live cells and very low percentage of apoptotic cells in control (Fig. S7B†) as well as polymer capsule treated cells (Fig. S7C†). Therefore it is confirmed that polymer capsules do not induce apoptotic response in L929 cells. The cytocompatibility of the polymer capsules is supported by the morphology of L929 cells that has remained unaffected by polymer capsules, whereas cells treated with cisplatin have disrupted morphology and have rounded up (Fig. 5).

Fig. 5 Optical microscopy images representing morphology of L929 cells incubated with (A) PBS (control), (B) PEGylated polymer capsules encapsulating LaVO₄:Tb³⁺ nanoparticles (5 × 10⁶/ml capsules), and (C) cisplatin (10 μM) as positive control.

Erythrocyte membrane stability is analysed by hemolysis assay which is also an important parameter to consider, as its disruption can result in life-threatening conditions such as anemia, hypertension, arrhythmia, and renal failure. As seen in Fig. S8,† polymer capsules even at higher concentration shows lower than 5% hemolysis indicating that it is suitable for intravenous administration.

3.3. In vivo biodistribution and toxicology studies

The bio-distribution of intravenously administered polymer capsules in Swiss Albino mice is measured after 6 h and 24 h by quantifying amount of lanthanum in tissue extracts by ICP-MS. Fig. 6 demonstrates that maximum percentage of polymer capsules corresponding to concentration of lanthanum (in ppb) (Fig. S9†) observed in liver followed by kidney, lungs and spleen indicating bioavailability of polymer capsules in these tissues in 6 h. Furthermore, bio-distribution after 24 h showed decrease in the amount of lanthanum concentration in liver compared to 6 h sample. In addition, urine and spleen also show higher levels of lanthanum concentration indicating the clearance of particles from the body in 24 h. Since, it was observed that liver and kidney has maximum concentration of polymer capsules, hepatoxicity and renal toxicity was evaluated by measuring toxicity marker enzymes as well as histopathological examination. In general, injury in hepatocytes leads to leakage of various enzymes into blood circulation.^36,58 For example, increase in ALP activity occurs mainly in liver dysfunction and biliary tract diseases. Measurements of the serum activity of CK seem to be an excellent marker for acute damages of liver, heart and skeletal muscle. Stress related elevation of serum AST and ALT has been found in various reports.⁵⁹ It has been observed that activities of toxicity marker enzymes like AST, ALT, LDH, ALP and CK in serum have not been altered by treating the animals with polymer capsules (Table S1†). Hematology parameters (Table S2†) were also not affected by administration of polymer capsules substantiating the non-toxic nature of polymer capsules encapsulating LaVO₄:Tb³⁺ nanoparticles at in vivo conditions.

Fig. 6 Plot representing ICP-MS data of percentage of lanthanum ion in various tissues (6 and 24 h) and urine of mice after intravenous administration of PEGylated polymer capsules encapsulating LaVO₄:Tb³⁺ nanoparticles (3 × 10⁹ capsules per kg body weight).

Tissue infiltration and inflammation are accompanied by an elevation of MPO that resides in neutrophils and are employed to tissues when there is an inflammatory stimulus. As seen in Fig. 7, activity of myeloperoxidase was found to be normal in liver and kidney of animals treated with polymer capsules, which shows that the capsules do not induce inflammation. Activity of 5-LOX in PBMNCs, which is a key enzyme of inflammation processes, is also found to be comparable to that in control animals. Antioxidant enzymes were extensively used as a biochemical indicator of pathological states associated with oxidative stress.⁵⁷ Superoxide dismutase (SOD) dismounts superoxide radical and catalase converts H₂O₂ to H₂O. During H₂O₂ scavenging, glutathione is oxidized by glutathione peroxidase. A decrease in activity of free radical scavenging enzymes and reduced glutathione (GSH) indicates hepatic injury. In animals treated with polymer capsules encapsulating LaVO₄:Tb³⁺ nanoparticles, there is no significant change in activities of SOD and catalase and levels of TBARS (measures peroxidation level) and GSH (Fig. 8). Therefore, administration of the polymer capsules does not induce oxidative stress in mice.

Fig. 7 Graphs representing enzyme activity after administration of PEGylated polymer capsules encapsulating LaVO₄:Tb³⁺ nanoparticles (3 × 10⁹ capsules per kg body weight) in mice sacrificed at 1 week and 1 month post injection: (A) myeloperoxidase in tissues and (B) 5-lipoxygenase in peripheral blood mononuclear cells. Different alphabets indicate significant difference at p < 0.05.

Fig. 8 Graphs representing evaluation of oxidative stress after administration of PEGylated polymer capsules encapsulating LaVO₄:Tb³⁺ nanoparticles (3 × 10⁹ capsules per kg body weight) in mice tissues sacrificed at 1 week and 1 month post injection: (A) activity of superoxide dismutase (SOD), (B) catalase, (C) thiobarbituric acid-reactive substances (TBARS) and (D) reduced glutathione. Different alphabets indicate significant difference at p < 0.05.

Genotoxicity evaluation is essential to examine as it can give insight on DNA level changes. Comet assay (single-cell gel electrophoresis) has been employed for measuring DNA strand breaks,⁶⁰ if any, in whole blood of animals administered with polymer capsules encapsulating LaVO₄:Tb³⁺ nanoparticles. After electrophoresis, slides were observed under fluorescent microscope for presence of comets. For positive control, cyclophosphamide (DNA alkylating agent) was administered to animals (40 mg per kg body wt i.p.). Fig. 9 demonstrates comet tail in peripheral blood of animals treated with polymer capsules which is comparable to that of control. It illustrates that capsules do not induce any DNA damage, where as positive control group displayed intense comet tails. Fig. S10† shows the corresponding comet scores. Further, histological examination of liver and kidney has been carried out by hematoxylin–eosin staining of tissue sections. Fig. 10 shows optical microscopy images which exhibit normal histology further confirming the biocompatibility of polymer capsules.

Fig. 9 Fluorescence microscopy images of comet assay for genotoxicity assessment in peripheral blood after administration of PEGylated polymer capsules encapsulating LaVO₄:Tb³⁺ nanoparticles (3 × 10⁹ capsules per kg body weight): (A) control mice, (B) mice sacrificed 1 week post injection, (C) mice sacrificed 1 month post injection, and (D) mice administered cyclophosphamide (40 mg per kg body wt). Insets show image of nucleus of a single cell.

Fig. 10 Optical microscopy images representing tissue histology in mice administered with PEGylated polymer capsules encapsulating LaVO₄:Tb³⁺ nanoparticles (3 × 10⁹ capsules per kg body weight), one month post-injection: (A) liver, and (B) kidney. Left panel shows control.

4. Conclusions

A detailed toxicity profiling of PEGylated polymer capsules encapsulating LaVO₄:Tb³⁺ nanoparticles has been performed at in vitro and in vivo levels. In vitro studies carried out in L929 fibroblast cells indicates cyto-compatibility by evaluating MTT assay, cell morphology, NRU assay, and LDH release. In addition, no, oxidative stress and inflammatory response were observed by analyzing ROS, RNS and lipid peroxidation levels. Caspase-3 activity, and apoptosis assay also indicates in vitro level biocompatibility. Hemolysis assay indicates intact morphology of RBCs. In vivo studies performed in Swiss Albino mice indicates bioavailability of lanthanum ion in liver and clearance through urine samples 24 h post-injection. Evaluation of hematological parameters and toxicity marker enzymes show non-toxic nature of the capsules. Histopathological examinations represent normal architecture of organs. Studies also indicated that the polymer capsules do not induce inflammation, oxidation stress and genotoxicity. All the above studies show a detailed set of experiment-cum-analysis proving the biocompatibility of PEGylated polymer capsules encapsulating LaVO₄:Tb³⁺ nanoparticles which can find potential application in bioimaging.

Conflict of interest

The authors declare no competing financial interest.

Notes

All authors have given approval to the final version of the manuscript.

Abbreviations

LbL	Layer by layer
ICPMS	Inductively coupled plasma-mass spectrometry
SEM	Scanning electron microscope
TEM	Transmission electron microscope
EDS	Energy-dispersive X-ray spectroscopy
PBS	Phosphate buffered saline
DMEM	Dulbecco's modified eagle's medium
FBS	Fetal bovine serum
FACS	Fluorescent activated cell sorting
PS	Phosphatidyl serine
PI	Propidium iodide
AST	Aspartate transaminase
ALT	Alanine transaminase
LDH	Lactate dehydrogenase
ALP	Alkaline phosphatase
CK	Creatinine kinase
MPO	Myeloperoxidase
5-LOX	Lipoxygenase
SOD	Super oxide dismutase
TBARS	Thiobarbituric acid reactive substance
GSH	Reduced glutathione
PBMNC	Peripheral blood mononuclear cells
PB	Peripheral blood
WBC	White blood cell count
RBC	Red blood cell count
BUN	Blood urea nitrogen

Acknowledgements

The authors greatly acknowledge the grants from DST Nanomission, Department of Science and Technology (DST), UK-India Education and Research Initiative (UKIERI) and Department of Biotechnology (DBT). Authors also express appreciation to Dr Anantha Lekshmi, Veterinary Doctor, Department of Biochemistry, University of Kerala, Kariavattom, India for assisting in animal experiments.

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Footnotes

† Electronic supplementary information (ESI) available: EDX spectrum of polymer capsules encapsulating LaVO₄ nanoparticles, TEM image showing stability of capsules, z-stacking of L929 cells internalized with capsules, assessment of oxidative stress in vitro (ROS, RNS and TBARS), caspase-3 assay, apoptosis measurement, hemolysis assay and ICP-MS data. It also includes tables showing toxicity marker enzymes and hematological parameters in mice and data representing comet assay scores for genotoxicity in vivo. See DOI: 10.1039/c6ra06719k

‡ These authors contributed equally.

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