Role of surface charge in bioavailability and biodistribution of tri-block copolymer nanoparticles in rats after oral exposure

Abstract

Tri-block copolymer nanoparticles (TCNP) are increasingly utilized, especially in drug delivery and diagnostics platforms. From in vitro studies, surface charge was observed to influence the transport of TCNP across the Caco-2 monolayers grown on transwell inserts. The objective of this study was to investigate the influence of surface charge on bioavailability and biodistribution of TCNP after oral exposure in vivo and to compare with the in vitro data. To attain the set objectives, monodisperse (45 ± 5 nm), fluorescent and differently charged (positive and negative) TCNP were orally administered to inbred Fischer 344 rats. Blood samples were collected at t = 0, 1/2, 1, 2, 4 and 6 h followed by sacrifice of the animals and collection of the major organs (lungs, liver, kidney, spleen, brain, intestine, and tibia). Quantitative assessments of TCNP in blood and organs were performed by fluorescence measurements. TCNP of both surface charges got absorbed and appeared in the blood within 1/2 h of oral administration. No significant difference in bioavailability and biodistribution could be found between positive and negative TCNP. Both the TCNP, irrespective of charge, showed major accumulation in the liver, kidneys and spleen, were detected in the brain and did not cause any significant increase in the serum alkaline phosphatase levels. Contrary to the in vitro data, surface charge was not found to influence the in vivo bioavailability and biodistribution of TCNP after oral exposure. The obtained results encourage further development of such TCNP, especially for drug delivery purposes.

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Introduction

With the growing use of nanotechnology-based products, it is necessary to characterize their toxic potentials as well as their interactions with biological systems.¹ Unfortunately, most of the investigations performed with that aim were based on in vitro experimental platforms comprising a diverse range of nanoparticles (NP).^2–4 This shortage of in vivo data makes it difficult to understand the bioavailability and biodistribution of NP after introduction to the body milieu. Many of these newly engineered NP, including tri-block copolymer NP (TCNP), were developed keeping their potential use in food⁵ in mind and medicinal applications, like targeted drug delivery.^6,7 This further increases the possibility for human exposure to comparatively larger amounts of different TCNP, especially via the oral route.

Surface charge was identified as an important factor in the transport of TCNP across Caco-2 monolayers, which is a widely accepted in vitro model for human gut enterocytes.⁸ Compared to the anionic NP, the cationic NP demonstrated significantly higher transport as well as greater cellular uptake across such transwell Caco-2 monolayer systems. Hence, the in vitro data indicated a higher bioavailability for cationic TCNP in vivo after oral administration compared to the anionic ones. It is important to investigate this effect of surface charge in vivo, as a better understanding of such influence of surface charge on the bioavailability of TCNP after oral exposure can improve the development of such tailor-made TCNP. Unfortunately, the in vivo data on NP were mostly reported with parenteral routes of exposure (like iv, sc)^9,10 and are difficult to compare with the oral exposure scenario. The intrinsic complexity of the gastrointestinal (GI) tract (like mucosal barrier, pH, associated diseases) can have an effect on the absorption of the TCNP. With very little amounts of such in vivo data after oral exposure of NP available, the need for such an experiment was felt to be justified and timely.

Recently, we reported the synthesis of fluorescent, monodisperse, well characterized tri-block copolymer [PEG₂₀₀₀-polyhexylene adipate-PEG₂₀₀₀] nanoparticles (TCNP) with different surface charges (amine modified positive/TCNP-NH₃⁺ and acid modified negative/TCNP-COO⁻) and sizes (45 and 90 nm).¹¹ These TCNP had a hydrophobic polyester core protected from the outer aqueous environment by hydrophilic PEG tails. Interestingly, with the possibility of encapsulating hydrophobic molecules within these biodegradable TCNP,¹² further applications of these TCNP can be expected to rise in the future. The availability of these TCNP in different surface charges (positive and negative) also fits well with the research question of how surface characteristics (such as surface charge) can influence the bioavailability and biodistribution of these PNP after oral exposure. As an adequate in vivo animal model, the inbred Fischer 344 rats were chosen due to the abundance of bioavailability studies performed on them after oral exposure.^13,14 Due to the high sensitivity, the intrinsic fluorescence of TCNP was used for quantitative assessment.

The aim of this study was to investigate the role of surface charge in bioavailability and biodistribution of TCNP in rats after oral exposure and to compare the findings with the available in vitro data. Keeping in mind the probable uses of these TCNP in food-based formulations and drug delivery systems in the future, the obtained data on bioavailability and biodistribution can be helpful in realizing the true potential of the TCNP for such applications.

Experimental details

TCNP synthesis and characterization

The positive TCNP-NH₃⁺ and negative TCNP-COO⁻, of 45 ± 5 nm sizes, were synthesized and characterized as described before.¹¹ A summary of the characterization data of these TCNP is provided in Table S2.† For the in vivo study, aqueous suspensions of TCNP (0.05, 0.1 and 0.4%) (w/v) were used.

Animal experiment

The animal experiment proposal was approved by the ethical committee on animal experimentation in the Wageningen University, the Netherlands and complied with the Dutch law on animal experimentation (Stb. 1977, 67; Stb. 1996, 565), revised on February 5, 1997. Eight weeks old male Fischer 344 rats, with an average body weight of ∼200 g, were obtained from Harlan (Horst, the Netherlands). The rats were divided into groups and housed together under standard conditions (22 °C, relative humidity 30–70%, 12 h light/dark cycle) with ad libitum access to food and water. Before oral gavage, rats were fasted for 4 h.

First, a pilot experiment was done with 4 rats (2 rats for both positive or negative TCNP) for each concentration (0.05, 0.1 and 0.4% w/v) of TCNP (a total of 12 rats). A single dose of a 1.5 ml aqueous suspension of TCNP was administered through oral gavage at the three above mentioned concentrations of 0.05, 0.1 and 0.4% w/v resulting in dose levels amounting to 0.75 mg, 1.5 mg and 6.0 mg of TCNP per rat, respectively. Blood was collected (150 μl) from tail veins of the rats in heparinized tubes at t = 0, 1/2, 1, 2, 4 and 6 h. After 6 h, rats were sacrificed under isoflurane anaesthesia and blood from the aorta (7–8 ml) was collected. The following organs were collected en masse: lungs (both), liver, kidneys (both), brain, spleen, stomach, small intestine, caecum and tibia (both). The organs were weighed and preserved in a 10% formalin solution before dissecting a portion with known weight for further homogenization and study with fluorescence or histopathology.

The pilot experiment was followed by a main experiment with 14 rats (2 rats as the control where only 1.5 ml of water was administered and 6 rats each for both the positive and negative TCNP that were each given a 1.5 ml oral dose of only 0.4% (w/v) amounting to 6.0 mg of TCNP per rat of both the TCNP). Blood and organs were collected and analysed like in the pilot phase.

Quantitative assessment of TCNP by fluorescence measurements

The blood samples (150 μl), collected from the tail veins at different time points as well as the aorta after sacrifice, were analysed by fluorescence (λ_ex = 488 nm; λ_em = 543 nm). The amount of TCNP was estimated by a pre-determined calibration curve obtained by mixing Fischer 344 rat blood with serial dilutions of TCNP (Fig. S1†). The obtained data were adjusted to the total volume of blood present in the rat to measure the bioavailability. The dissected pieces of the organs were homogenized and a 150 μl aliquot of the homogenized tissue sample was diluted in ice cold nanopure water to 1 ml before analysing it by fluorescence. The blood contents of the organs were determined as described before.¹⁵

Estimation of serum ALP levels

The levels of serum ALP were measured by a commercial kit available from Sigma Aldrich Chemie BV (catalogue number: APF) and measured with fluorescence (λ_ex = 360 nm; λ_em = 440 nm). The ALP levels in serum obtained from control rats were taken as the negative control.

Statistical analysis

The data were plotted using Origin Pro (version 8.0) software. Results are shown as means ± standard error of mean (SEM). A Student's t-test was performed and data points were marked with “*” symbol when the readings for positive and negative TCNP were significantly different (p < 0.05).

Results

Bioavailability and blood-clearance study

A quantitative assessment of TCNP in rat blood at t = 0, 1/2, 1, 2, 4 and 6 h was done by fluorescence and the results are shown in Fig. 1. Both the positive and negative TCNP got absorbed and were detected in blood at t = 1/2 h. The amount of TCNP in blood kept on increasing in a dose-dependent way for 2–4 h and then started declining which was more visible for the negative TCNP-COO⁻ compared to the positive TCNP-NH₃⁺.

Fig. 1 Amount of positive (▲) and negative (■) TCNP, expressed as % of administered oral dose in rat blood after t = 0, 1/2, 1, 2, 4 and 6 h at three different concentrations (0.05, 0.1 and 0.4%). Results are shown as mean ± SEM. The “*” symbol signifies p < 0.05 between the TCNP-NH₃⁺ and TCNP-COO⁻.

Organ distribution of TCNP

As the TCNP contained fluorescent probe, the organs were illuminated under UV-light to search for TCNP. The results are shown in Fig. 2. The TCNP were absorbed from the gut after oral administration irrespective of the surface charge and were mainly found to be deposited in liver and kidneys. Interestingly, some fluorescence could also be detected from the brain. The detection from the caecum showed that the TCNP moved with peristalsis.

Fig. 2 Different organs (L = liver, K = kidney, S = spleen, Br = brain, Lu = lungs, St = stomach, SI = small intestine, C = caecum, LI = large intestine) collected from the rats treated with only water (control), positive and negative TCNP under UV-light illumination showing that the TCNP are present mostly in the liver followed by kidney and brain. Both the positive and negative TCNP could be detected from the large intestine as well.

Quantitative assessment of organ distribution of TCNP

The quantitative assessment of the TCNP in different collected organs is shown in Fig. 3. The highest amounts of TCNP, irrespective of surface charge, were detected in the hepatic tissue followed by the kidneys and spleen. Some amount of TCNP could also be detected in the lungs, although the amounts were much less compared to that of hepatic or renal tissues. Interestingly, both the TCNP were detected to some extent in the brain.

Fig. 3 Organ distribution of positive TCNP-NH₃⁺ (black columns) and negative TCNP-COO⁻ (shaded columns) in Fischer 344 rats after 6 h of oral administration. Three concentrations of TCNP were applied: 0.05% (top histogram), 0.1% (middle histogram) and 0.4% (bottom histogram). Results are shown as mean % of administered dose ± SEM.

To understand the efficiency of different organs in accumulating the TCNP from blood circulation, the content of the TCNP in the organs were re-plotted as the amount of TCNP per g of different organ tissues. The results are shown in Fig. 4. It could be seen that when the amount of TCNP was expressed as per unit mass of tissue, the amounts in spleen and liver were almost the same with small but detectable amounts in brain. The rest of the organs also showed the presence of TCNP, although the rate of accumulation was much lower.

Fig. 4 Relative organ distribution of TCNP expressed as per g of tissue and % of liver content (100%) after 6 h of exposure to three different concentrations: 0.05% (top histogram), 0.1% (middle histogram) and 0.4% (bottom histogram). Results are shown as means ± SEM.

In order to investigate the presence of any dose-dependence, the data were expressed keeping the TCNP-NH₃⁺ content of the organs at 0.05% concentration exposure as 100%. The results are shown in Fig. 5. A clear dose-dependence could be observed in the organ accumulation of various TCNP irrespective of the surface charge. Hence, with increased concentration of TCNP, higher accumulation of PNP was observed in different organs without any significant surface charge-dependence.

Fig. 5 Dose-dependent organ distribution after 6 h exposure to positive TCNP-NH₃⁺ (upper histogram) and negative TCNP-COO⁻ (bottom histogram) at three concentrations: 0.05% (dark columns), 0.1% (grey columns) and 0.4% (shaded columns). The data were normalized by setting the readings of 0.05% concentration as 100%.

Measurement of alkaline phosphatase enzyme activity

Serum alkaline phosphatase (ALP) levels were measured in the blood samples and the results are shown in Fig. 6. Compared to the negative TCNP-COO⁻, the positive TCNP-NH₃⁺ caused some increase in serum ALP, only at the highest concentration (0.04%) and after 6 h exposure. For the other concentrations and time points, no effect of surface charge could be observed.

Fig. 6 Serum ALP levels in Fischer 344 rat blood samples after oral exposure to positive (▲) and negative (■) TCNP at t = 0, 1/2, 1, 2, 4 and 6 h at three different concentrations (0.05, 0.1 and 0.4%). Results are shown as means ± SEM. The “*” symbol signifies significant difference (p < 0.05) between the TCNP-NH₃⁺ and TCNP-COO⁻.

Discussion

The mechanism of the absorbance of TCNP from the gut is still unclear. A few groups have proposed a role of intercellular juncture and fenestrations of the endothelia¹⁶ apart from a role of dendritic cells¹⁷ in the absorption of NP. Another explanation may be the role of M-cells located in Peyer's patches.^18–20 After being absorbed and thus becoming detectable in the blood, both the positive and negative TCNP showed an initial increase in the amounts present in blood, followed by a decline. This decline could be due to excretion of TCNP from the body or accumulation in different organs. Contrary to the in vitro data, surface charge overall seemed not to have any significant (p < 0.05) effect on the bioavailability of TCNP. One possible explanation for this discrepancy may be other factors (such as mucus coating over TCNP, pH and local gut pathology) which are present only in vivo. Due to the scarcity of in vivo studies on NP after oral exposure, it is hard to compare our data. Although a few reports tried to shed light on the role of specific conditions of gut in the absorbance of NP,^21,22 a true understanding of this issue is yet to be achieved. Very recently, an experiment with oral exposure with positive and negatively charged gold NP of different sizes was reported.²³ Interestingly, in line with our data, no significant surface charge-dependence of bioavailability or biodistribution of gold NP was observed. Similarly, no significant effect of surface charge was found in a report by Yamamoto et al.,²⁴ when neutral tyrosine and negative tyrosine–glutamine coated PEG/PDLLA nano-micelles were investigated for bioavailability in mice after iv infusion. As the primary focus of this study was to investigate the bioavailability of the TCNP in rats after oral exposure, the excretion of the TCNP in the faeces and urine was not included.

An interesting observation was the relatively rapid clearance of negative TCNP-COO⁻ from the blood compared to the positive TCNP-NH₃⁺. It was reported by Bhattacharjee et al.²⁵ that acid terminated anionic silicon NP (1.6 ± 0.2 nm) induced phagocytosis in rat alveolar macrophage NR8383 cells to a greater extent than positive amine terminated ones. It is often hypothesized that the negative NP easily acquires a coating of opsonin proteins in the blood which induces phagocytosis by macrophage cells. Another explanation may be that after reaching blood, the NP get coated with serum proteins which render the NP to acquire a protein corona^26–28 that triggers phagocytosis by the macrophages.^29–31 The high hepatic accumulation of TCNP of both surface charges was expected due to the high blood perfusion of liver. It is suggested that due to the macrophage cells, such as Kupffer cells of liver,³² the reticulo-endothelial system (RES) has a higher capability of phagocytosing the TCNP from blood circulation. Hence, organs like spleen also showed good accumulation of TCNP. Previously, Bhattacharjee et al.²⁵ showed that these TCNP can actively enter the rat macrophage NR8383 cells in vitro. So, it can be expected that the macrophage cells present in the RES can phagocytose the TCNP from blood circulation causing a deposition of the TCNP. A similar accumulation of different NP in RES organs, such as liver and spleen, has also been reported before. Bhattacharjee et al.²⁵ showed that the fluorescent probe was embedded in the middle block of the copolymer and, hence, was an integral part of the TCNP. Therefore the fluorescence signal detected was obtained from the TCNP and not from fluorescent probe leaching out or fallen apart from the TCNP. This was also proven by control experiments where the fluorescence spectrum from the isolated fluorescent probe was different from the fluorescent TCNP. Choi et al.³³ reported also a high accumulation of CdSe/ZnS quantum dots in the liver and spleen of CD-1 mice after iv exposure. A similar involvement of macrophage cells in clearance of NP from blood circulation has been reported before for other cells, such as hepatocytes³⁴ and hepatic endothelial cells.^35,36 For splenic tissue, a role for macrophage cells (dendritic cells) has also been proposed.³⁷ Interestingly, osseous tissue was often omitted in previous in vivo studies with NP. In our case, we noticed some bio-accumulation in tibia bone. A possible explanation for this may be that the bone marrow is also rich in macrophage cells. The quick filtering of the TCNP from the bloodstream by macrophage cells of the RES can pose a challenge for targeted drug delivery by decreasing the concentrations of nanoparticulate drug formulations in blood. A detailed discussion on the ongoing research on preventing this problem is beyond the scope of this article. However, it can be said that reducing the phagocytosis of NP via surface modifications, like attaching hydrophilic PEG,³⁸ looks like a promising option. Interestingly, the stomach showed some accumulation of TCNP as well. This may be caused by the TCNP being embedded in the mucous lining of the stomach wall. The organ distribution of both the TCNP showed an overall dose-dependence with bioaccumulation of the TCNP in each organ increasing with higher concentrations. Interestingly, both the TCNP could also be detected in the brain. In the only comparable study with oral exposure of NP,²³ in contrast to the negative ones, positive gold nanoparticles (2.8 nm) reached the brain. Especially for NP-based drug delivery in brain, where crossing the BBB is crucial, this finding can be of importance. Surface charge seemed not to influence the penetration of TCNP across the BBB. Serum ALP activity is a reliable and widely used biomarker for hepatic toxicity and hence was included in the investigation. Our data on ALP concentration hinted towards a hepatic stress caused by these TCNP. By in vitro experiments, polystyrene NP were reported to be toxic for HepG2 cell lines,³⁹ although in vivo data on such hepatic stress are rare. In a 13 weeks exposure study in F344 rats with 56 nm silver NP, a mild increase in serum ALP was observed.⁴⁰ However, due to the different NP with varied exposure times, it is hard to compare with our data. Still, it can be stated that the obtained results on ALP showed an overall match with this previous study and indicated signs of hepatic stress.

Conclusion

Contrary to the in vitro data, surface charge does not influence the bioavailability and biodistribution of TCNP after oral exposure. The interaction of TCNP with gut mucous can be an explanation for this discrepancy since mucous components may adsorb to the TCNP masking their surface charge. With high accumulation of TCNP occurring in liver and spleen, macrophage cells in the RES seemed to play an important role in deposition of the TCNP. Other organs (such as kidney, lungs, and tibia) also showed deposition of TCNP. The TCNP, irrespective of surface charge, crossed the BBB which encourages their further development as drug delivery systems in cerebral tissues. The obtained data can be pivotal in the further development of newly engineered TCNP meant for oral administration while targeting specific purposes like food-based formulations and therapeutics.

Acknowledgements

The authors would like to thank Graduate School VLAG and Wageningen UR Strategic Research Programme Bionanotechnology for funding. Rene Bakker, Wilma Blauw, Lisette van Druten, Bert Weijers, Judith Hulsman and Romy van Ingenand of the animal housing facility (CKP) in Wageningen University, the Netherlands are acknowledged for their indispensable assistance while performing the animal experiments.

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Footnote

† Electronic supplementary information (ESI) available: Fluorescence spectrum of the TCNP in water and blood at λ_ex = 488 nm along with summary of characterization data of the used TCNP are provided in supplementary information. See DOI: 10.1039/c3tx20072h

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