Assessment of Pharmacokinetics, Toxicity, and Biodistribution of a High Dose of Titanate Nanotubes Following Intravenous Injection in Mice: A Promising Nanosystem of Medical Interest

Abstract

Titanate nanotubes (TiNTs) produced by the static hydrothermal process present a promising nanosystem for nanomedicine. However, the behavior of these nanotubes in vivo is not yet clarified. In this work, for the first time, we investigated the toxicity of these materials, their pharmacokinetic profile, and their biodistribution in mice. A high dose of TiNTs (45 mg/kg) was intravenously injected in mice and monitored from 6 h to 45 days. The histological examination of organs and the analysis of liver and kidney function markers and then the inflammatory response were in agreement with a long-term innocuity of these nanomaterials. The parameters of pharmacokinetics revealed the rapid clarification of TiNTs from the bloodstream after 6 h of the intravenous injection which then mainly accumulated in the liver and spleen, and their degradation and clearance in these tissues were relatively slow (>4 weeks). Interestingly, an important property of these materials is their slow dissolution under the lysosome acid environment, rendering them biodegradable. It is noteworthy that TiNTs were directly eliminated in urine and bile ducts without obvious toxicity in mice. Altogether, all these typical in vivo tests studying the TiNT pharmacokinetics, toxicity, and biodistribution are supporting the use of these biocompatible nanomaterials in the biomedical field, especially as a nanocarrier-based drug delivery system.

1. Introduction

Today, nanotechnologies find applications in the health field from prevention to therapy.¹⁻⁵ There is a substantial interest in developing therapeutic options for cancer treatment based on the use of nanoparticles for drug delivery and theranostics. Indeed, the application of the drug nanocarrier system in nanomedicine can increase the therapeutic efficiency and reduce toxicity. In specific, inorganic nanocarriers are substantial parts in the clinical progress of cancer diagnostic and therapy.⁶⁻⁹ Obviously, a perfect nanocarrier-based drug delivery system should have several characteristics including a stable structure, high drug encapsulation efficiency, high cellular uptake, more desirable biodistribution, and more reasonable pharmacokinetics and selectively accumulate at the tumor site through the enhanced permeability and retention effect.¹⁰⁻¹³ In this regard, titanate nanotubes (TiNTs) are attracting increasing interest, thanks to their tunable shape dotted with a high surface area and large pore volume which provide them a high load capacity of drug storage.¹⁴ Several research studies reported the insertion of TiNTs in the nanomedicine field including radiotherapy treatment,¹⁵ dopamine detection,¹⁶ bone regeneration,¹⁷ and drug delivery.^14,18,19 Recently, we have demonstrated that the tubular shape leads to a higher cellular internalization of TiNTs which can be potentially beneficial in drug delivery to the targeted tissue.^14,20 Indeed, for the first time, we showed that TiNTs could achieve higher genistein drug-loading content (25.2%) and entrapment efficiency (51.2%), leading to a controlled drug release and a higher cellular uptake of genistein-loaded TiNTs.¹⁴ Likewise, the genistein-loaded TiNTs, compared to free genistein, were efficient to induce higher cytotoxicity associated with a significant antimigratory effect on U87-MG human glioblastoma astrocytoma, promising an efficient antitumor activity.¹⁴ Moreover, the safety of TiNTs was assessed in vitro on the U87-MG human glioblastoma cell line and human microvascular endothelial cells (HMEC). No cytotoxic effect was observed under concentrations of up to 100 μg/mL.¹⁴ Although in vitro studies are mainly useful to establish a cytotoxicity ranking or to highlight the mechanism(s) behind eventual nanoparticle toxicity, animal models are needed to investigate whole body toxicokinetics.²¹ Many concerns and debates among the society, industry, and regulatory authorities (i.e., REACH or European Community Regulation on chemicals) are regarding the safety of nanoparticles and their final fate in biological systems.^22,23 Toxicity of novel purely inorganic nanoparticles such as fullerenes²⁴ and carbon nanotubes (CNTs)²⁵ (quantum dots,²⁶ silica,²⁷ and iron oxides²⁸) is not fully understood. Hence, prior to any practical use of nanoparticles particularly for biomedical applications, it is of a high societal relevance to investigate their possible toxic effects. While the toxicity of TiO₂ in its isotropic form (particles with the diameter ranging from few nanometers to micrometers) has been widely studied,²⁹⁻³² the toxic action of TiO₂-based nanotubes, their biodistribution, and pharmacokinetics are essentially unknown. Thus, we report in this work, for the first time, an in-depth evaluation of the acute toxicity of bare TiNTs including pharmacokinetics, biodistribution, and excretion on 45 days in mice after intravenous administration of a relatively high dose. The nanotoxicological tests would be an essential step to evaluate the possibility of future clinical translation of these TiNTs.

2. Results

2.1. Characterization of TiNTs

The X-ray diffraction (XRD) pattern of the synthesized TiNTs (Figure 1A) matches well with the standard diffractogram of H₄O₆Ti₂ (JCPDS card: 47-0124), which coincides with the general molecular formula of protonated titanate (H₂TinO_2n+1·H₂O).³³ No layered anatase TiO₂ precursor can be observed in the view field. Results mentioned above indicate that the anatase TiO₂ has converted to titanate with nanotube morphology, entirely in agreement with our previous results.¹⁴ Due to the low solubility of inorganic oxide nanotubular materials in water, TiNT suspensions exhibit a colloidal stability during more than 1 h, as shown in Figure 1 (top left). According to the electron transmission microscopy observation, TiNTs have a typical morphology of multilayer nanotubes with a size of 10 nm in outer diameter and 4 nm in inner diameter, and the mean length is around 80–200 nm (Figure 1B,C). Then, the zeta potential measurement of the aqueous TiNT suspension was negatively charged (−35.3 mV). Moreover, the polydispersity index value (PDI ≈ 0.28) indicated a monodisperse TiNT suspension.

Figure 1.

(A) XRD pattern of TiNTs showing the characteristic of nanotube morphology. (B,C) TEM images of TiNTs and (C) magnification of (B). Yellow arrows show the multilayers of TiNTs. Top left: Image of the TiNT suspension in NaCl 0.9% taken by Baati et al. 2021.

2.2. Mice Behavior and Growth

To check the toxic effects of TiNTs, body weight and behaviors of mice were controlled during the experiment. The growth of all mice increased in the same manner without any significant difference (Figure S1). No lethargy or apathy was observed following the TiNT administration. In addition, the breathing rhythm was similar to the control group, and fully open eyes without secretion were noted. All these parameters are visible in accordance with the lack of toxicity due to TiNTs.

2.3. Pharmacokinetics

According to the Ti blood concentration curve (Figure 2), TiNTs show a fast decrease concentration, followed by more progressive decline. This profile corresponded to a classical bicompartmental pharmacokinetic model which consists of a rapid initial distribution phase followed by a progressive terminal elimination phase. Pharmacokinetic parameters are summarized in Table 1. Indeed, more than 75% of the intravenous administrated dose of TiNTs was eliminated from the bloodstream after 1 h. The plasma half-life of TiNT distribution (T_1/2α = 0.16 ± 0.01 h) is low, and it gets eliminated quickly from the bloodstream. Furthermore, from 1 to 24 h post TiNT injection, the titanium concentration decreases progressively from 283 (±46.2) to 8.9 (±1.5) μg/mL, showing a slow elimination profile which is consistent with the high value of (T_1/2β = 4.24 ± 0.25 h) and the low value of the clearance (0.65 ± 0.05 L h^–1). The V_d reflects the intensity of diffusion of a foreign material in the body (organs and tissues). The value of this parameter (3.98 ± 0.39 L) is higher than the volume of the mice body fluid (≈2 mL), which indicates that TiNTs are eliminated slowly from the central compartment (blood) and then will preferentially accumulate and sequester in the liver and the spleen. Taken together after intravenous injection of a relatively high dose of TiNTs (45 mg/kg), these nanomaterials were eliminated rapidly in plasma in the initial phase, followed by a relatively slow elimination phase.

Figure 2.

Pharmacokinetic profile of TiNTs determined 24 h following the intravenous injection of a single dose of 45 mg/kg (n = 6, data are mean ± SD).

Table 1. Mean Pharmacokinetic Parameters Calculated Using a Bicompartmental Model after Injection of 45 mg/kg TiNTs.

	TiNTs
T1/2α (half-life of the biodistribution phase)	0.16 (±0.01) [h]
T1/2β (elimination half time)	4.24 (±0.25) [h]
Tmax (time it takes for TiNTs to reach Cmax)	0.083 [h]
Cmax (peak of serum concentration)	910.48 (±16.2) [mg/L]
Cl (clearance)	0.65 (±0.05) [L h–1]
Vd (volume of distribution)	3.98 (±0.39) [L]
AUClast (area under the curve)	1730 (±7.95) [mg/h/L]
AUCinf (area under the curve)	1780 (±7.76) [mg/h/L]

In addition, according to the one-way ANOVA, comparing titanate concentration in time intervals to each other, the pharmacokinetics of the TiNTs declined significantly at 5, 15, and 30 min. The difference was no more significant between 30 and 60 min measure (p = 0.569). At 6, 12, and 24 h, the variance was not significant (p = 0.605 and 0.417) (Table S1).

2.4. TiNT Biodistribution

Investigations dealing with biodistribution of the TiNTs have been undertaken by the determination of the titanium level in organs (liver, spleen, lungs, heart, brain, and kidneys), serum, urine, and feces. The results of the TiNT biodistribution profile are summarized in Figure 3. The titanium concentrations represented in the graphic are calculated as the difference between the titanium level in the matrices of the treated and control animals. As shown in Figure 3, the titanium level was different from one matrix to another, indicating a heterogeneous TiNT biodistribution. Indeed, the titanium level increased significantly in the liver at 6 h (370 μg/g ± 75) following the TiNT administration to achieve a maximum of 600 μg (±160) after 24 h and then decreased progressively after 7 and 30 days to reach 460 μg/g (±150) and 90.7 μg/g (±25), respectively. In addition, 45 days after TiNT injection, the titanium content in the liver continues to decrease slowly to achieve a minimum value of 25 μg/g (±6) which represents 2% (±0.5) of the total titanium contained in the injected dose. Similarly, in the spleen of treated mice, the titanium content increases rapidly at 6 h following the TiNT injection of 150 μg/g (±26) to achieve a maximum of 220 μg/g (±30) after 7 days and then declined progressively down to 10.2 μg/g (±4) at 45 days post injection. Regarding the serum TiNT levels, the titanium concentration decreases rapidly from 90.38 μg/mL (±22.12) at 6 h to achieve 8.9 μg/mL (±1.4) after 24 h following the intravenous injection, indicating the rapid sequestration of TiNTs from the bloodstream by the reticuloendothelial system (RES), particularly the liver and the spleen, as confirmed by the pharmacokinetics profile. Besides the liver, spleen, and serum matrices, the assay of titanium in the kidneys, urine, and feces shows several variations depending on time. Indeed, the concentration of titanium increased progressively in the kidneys from 7 μg/g (±3) after 3 days following the TiNT injection to achieve 170 μg/g (±30) at 15 days post injection and then declined after 45 days to achieve 24.12 μg/mL (±7). Likewise, the titanium concentration increases in urine and feces slowly from 3 days (41.1 and 71.3 μg/mL and μg/g, respectively) to achieve a maximum 30 days post injection of TiNTs (214.2 μg/mL and 322.12 μg/g, respectively). These results sum up the rapid elimination of TiNTs from the bloodstream to accumulate in the RES, notably the liver and spleen, where they are degraded and progressively eliminated from the body by kidney excretion and feces. In addition, a period longer than 45 days is needed to achieve the total elimination of TiNTs. Finally, a slight increase in titanium within the lungs (67.4 μg/g ± 9) was noticed 24 h following the TiNT injection and related to the aggregation of these nanomaterials within the smallest capillaries of this highly irrigated organ. This phenomenon disappears progressively after 7 days following the TiNT administration without any tissue damage, as confirmed by the histopathological examination (Figure S3). On the other hand, no significant changes were observed in titanium concentration within the heart after the administration of TiNTs. However, a slight but significant increase in titanium cerebral levels was noted after 1, 3, and 7 days of the administration of TiNTs. The cerebral titanium concentration decreased progressively down to 1% after 45 days of the TiNT injection.

Figure 3.

Concentration of titanium (μg/g of tissue or mL of urine or serum) determined by ICP-OES in organs, serum, urine, and feces, 6 h and 1, 3, 7, 15, 30, and 45 days following the intravenous administration of TiNTs (45 mg/kg). Data are mean ± SE (n = 6). The titanium concentrations represented in the graphic are calculated as the difference between the titanium level in the matrices of the treated and control animals.

2.5. Histopathological Examination

After isoflurane anesthesia and abdomen incision, the liver, spleen, kidneys, lungs, heart, and brain were collected following 6 h and 1, 3, 7, 15, 30, and 45 days of the intravenous administration of TiNTs. Macroscopic examination of all the organs revealed a normal aspect and color, without hyperplasia or necrosis. Moreover, all mice livers showed a normal morphology without hypertrophy or adherent lobes (data not shown). The comparison of the organ weights between treated and control groups did not show any statistically significant difference as confirmed by Student’s test (Figure S2). Histopathological examination of livers of treated mice with TiNTs for 6 h showed transitional hepatic sinusoidal dilatation characterized by the enlargement of the capillaries in the centrilobular zone in the liver, as confirmed by the microscopic observation of hepatic sections stained with hematoxylin–eosin (Figure 4A, yellow arrows). The normal hepatic parenchyma architecture was observed relative to the corresponding control without apparent changes in the hepatocyte structure notably after TiNT administration without any sign of hypertrophy or hyperplasia of perisinusoidal cells or necrosis. However, some hepatic neutrophil infiltrations were observed in livers (Figure 4A, green arrows) from 6 h to 7 days post injection of TiNTs, suggesting an acute inflammation due to the activation of Kupffer cells by the rapid accumulation of TiNTs inside, as observed by transmission electron microscopy (TEM) (Figure 4B). In contrast with Kupffer cells, hepatocytes and endothelial cells were devoid of TiNT accumulation. Despite the high level of TiNTs accumulated in the liver, Kupffer cells retained their integrity expressed by the spherical or oval morphology of mitochondria and the well-distributed nuclear chromatin, indicating the absence of nucleus degeneration. Moreover, an aggregate of TiNTs was located inside Kupffer cell lysosomes, showing degradation of the electron-dense material with different sizes (Figures 4B,C(1–3)). Magnified views of the square area show that TiNTs were degraded under the effect of lysosomal degradative enzymes and acidic conditions (pH = 2) to generate short tubular nanotubes (5–20 nm, C(3) green arrows) and amorphous TiO₂ with different irregular sizes (4–10 nm, C(3) yellow arrows). Interestingly, more than half of the lysosome surface was filled with amorphous TiO₂ released from TiNT degradation, as shown in C(2). Seven days post injection of TiNTs, the dilated liver capillaries come back progressively to normal, proportional to the decrease in TiNT concentration in the liver.

Figure 4.

(A) Histological sections of the liver after 6 h and 1, 3, 7, 15, 30, and 45 days of the intravenous administration of TiNTs (45 mg/kg) in comparison with the control group. All sections were stained with hematoxylin–eosin. (B) TEM images of the liver of mice sacrificed 1 day after the intravenous administration of TiNTs compared to the control group (N = nucleus). [C(1–3)] TEM images of Kupffer cell cytoplasm showing aggregation and degradation of TiNTs inside lysosomes. Magnified views of the square area show that TiNTs were degraded to generate short tubular nanotubes (5–20 nm, C(3) green arrows) and amorphous TiO₂ with different irregular sizes (4–10 nm, (C3) yellow arrows).

As confirmed by the histopathological examination, an increased macrophagic activity was observed in the spleen tissues from 6 h up to 15 days following TiNT administration (Figure 5A,C). Indeed, the enlargement of the marginal zone in the red pulp as a consequence of the infiltration of macrophages (containing TiNTs) indicated a transitory acute inflammation due to the accumulation of a large amount of TiNTs. Similar to the hepatic tissue, TiNTs were exclusively located into the splenic macrophages (Figure 5B,D, yellow arrows) observed as brown cluster and did not induce any granuloma formation, a typical acute inflammatory response. This was also confirmed by dosing the interleukin-6 (IL-6) serum levels, an usual biochemical marker of inflammation (Figure 7C). IL-6 values were comparable to those obtained from the control group, supporting the absence of inflammatory reaction issued from the administration of TiNTs. However, 15 days after TiNT administration, the splenic infiltration has been reduced in a similar manner to the control spleen, proportional to the decrease in TiNT concentration. TEM of spleen sections revealed the aggregation of TiNTs, which are observed as tubular nanomaterials in macrophage lysosomes of treated mice (Figure 5E, arrows). Note that the amount of TiNTs in the liver and spleen decreases progressively from 7 days post injection to reach a residual level after 45 days. Conversely, the titanium content increased in the kidneys, urine, and feces, as confirmed by the biodistribution profile which indicated a progressive elimination of TiNTs in urine and bile ducts.

Figure 5.

(A–D) Histological sections of mice spleen treated with TiNTs (45 mg/kg) after 6 h and 1, 3, 7, 15, 30, and 45 days of the intravenous administration compared to the control group. All sections were stained with hematoxylin–eosin. (E) Representative TEM micrographs of spleen macrophages of mice scarified 1 day after intravenous injection of TiNTs compared to the control group. Magnified views of the square area in treated animals show the endolysosomal compartment containing TiNT aggregates (yellow arrows).

Figure 7.

ALAT (A) and ASAT (B) values in mouse plasma 6 h and 1, 3, 7, 15, 30, and 45 days post TiNT intravenous injection of 45 mg/kg or vehicle injection for the control group. Data represent the mean ± SEM of six mice, and statistical significance was determined by Student’s t-test (*p < 0.05, **p < 0.01).

The histological study of the kidneys after treatment with TiNTs 6 h and 1, 3, 7, 15, 30, and 45 days postinjection shows a normal tissue architecture consisting of unchanged nephrons (glomeruli and tubules) similar to that of the control groups (Figure 6A). No cytoplasmic vacuolization or proliferative glomerulonephritis was observed. Thanks to TEM, a normal brush border beside the basement membrane and intact mitochondria and nuclei were observed, thus providing the best evidence of the absence of nephrotoxicity (Figure 6B). Moreover, numerous normal long mitochondria situated between the extensive infoldings of the basolateral plasma membrane that create the lateral cell processes and a prominent lysosomal compartment that includes lysosomes containing degraded TiNTs as the electron condensed nanomaterial were visualized (Figure 6B, arrows). Indeed, high magnification of the kidney tissue of TiNT-treated animals (Figure 6B, square area) shows accumulation of amorphous TiO₂ with irregular size (4–6 nm) inside lysosomes located around the proximal renal tubules prior to urine excretion without inducing any sign of nephrotoxicity. This amorphous nanomaterials were probably released from TiNT degradation in macrophages. Note that in the control kidney, lysosomes are devoid of electron condensed nanomaterials (arrows).

Figure 6.

(A) Histological sections of mice kidneys treated with TiNTs (45 mg/kg) after 6 h and 1, 3, 7, 15, 30, and 45 days of the intravenous administration compared to the control group. All sections were stained with hematoxylin–eosin. (B) Representative TEM micrographs of kidney tissue of mice 1 day after intravenous injection of TiNTs compared to the control group (N= nucleus and Mi = mitochondria). Magnified views of the square area in the kidneys of treated animals show endolysosomal compartments (yellow arrows) containing amorphous TiO₂ released from TiNT degradation. These released nanomaterials have an irregular size (4–6 nm), as confirmed by high magnification.

Histological examination of the lungs of TiNT-treated mice showed a normal parenchyma with the appearance of fine lace composed of thin-walled alveoli, comparable with that of the control group (Figure S3), supporting the lack of toxicity for this tissue. However, some occasional emboli were detected due to the aggregation of TiNTs clearly visible as brown cluster in some pulmonary capillaries of treated animals from 6 h up to 7 days post injection (Figure S3B,D, arrows) compared to the equivalent control groups. Then, as shown in Figure S3, only sections of the control of 6 h post injection of NaCl 9% were represented because histological sections of all groups were similar. Noteworthy, after 7 days of TiNT administration, these emboli disappeared, suggesting the removal of the nanotubes, as evidenced by biodistribution results which show the decrease in the titanium level in the lungs.

Finally, a classical architecture of myocardial and brain tissues was revealed, as shown in Figures S4 and S5, respectively, without any histological alteration due to the TiNT administration.

2.6. Biochemical Parameters

The accumulation and clearance of TiNTs in organs can potentially cause their damage. To control these processes, we monitored activities of typical biochemical markers of hepatic cytolysis [serum alanine aminotransferase (ALAT) and aspartate aminotransferase (ASAT)] and the kidney function (serum creatinine level). For liver function, ALAT and ASAT activities, Figure 7A,B, increased significantly from 6 h up to 7 days after TiNT administration to reach 90 and 270 IU/L, respectively, and then decreased after 15 days similar to the control groups pointing out a latency time for the regulation of titanium hepatic content and its elimination. In addition, according to the ANOVA test, a significant difference was confirmed in the rate of ALAT and ASAT in the treated group among time (Table S2). When comparing time intervals with one another, the mean rate of ALAT varied non significantly at 6 h and 1, 3, and 7 days (p = 0.878, 0.974, and 0.251). The difference was significant between 7 days and 15 days (p = 0.000). Between 15, 30, and 45 days, the variance was not significant (p = 0.925 and 1.000) (Table S3). For the ASAT, the difference was not significant between 6 h and 1, 3, and 7 days. Between 7 and 15 days, the variance was not significant (p = 0.095) (Table S4). Moreover the increase in these serum markers did not exceed 1.5 times compared to the control group, showing a transient phenomenon due to the Kupffer cells activated by the accumulation of TiNTs. Concerning the kidney function, no significant changes in the serum creatinine level (Figure 8A) were observed in TiNT-treated animals compared to the control groups, indicating the elimination of TiNTs without inducing any nephrotoxicity. Finally, the amount of interleukin-6 (IL-6), as a specific marker for inflammation, was determined in all animal groups. As shown in Figure 8B, no significant difference in the serum IL-6 level was observed in control and TiNT-treated animals, suggesting the absence of chronic inflammation. Note that the accumulation of TiNTs inside Kupffer and their biodegradation were not accompanied by any granuloma formation in the liver or spleen, as confirmed below by histopathological examination. Taken together, these results discarded the fact that TiNTs are an infectious pathogen agent.

Figure 8.

Creatinine level (A) and IL-6 concentration (B) in mouse plasma 6 h and 1, 3, 7, 15, 30, and 45 days post TiNT intravenous injection (45 mg/kg) or vehicle injection for the control group. Data represent the mean ± SEM of six mice. No statistical significance was observed between control and TiNT-treated groups after application of Student’s t-test (*p < 0.05 and **p < 0.01).

3. Discussion

This study aimed to develop and determine the pharmacokinetic parameters, toxicity aspects, and biodistribution profile of TiNTs following an intravenous administration of a relatively high dose (45 mg/kg) to mice. Prior to the animal’s treatment, TiNTs were characterized for their physicochemical proprieties. Typical tunable multilayer nanomaterials were obtained after hydrothermal synthesis as we reported previously.¹⁴ The healthy behavior of animals, as well as the absence of acute and chronic toxicity in the kidneys, spleen, and liver, confirms the safety of TiNTs as described in vitro in our previous study.¹⁴ Indeed, cytotoxicity of TiNTs was tested on healthy HMEC cells and U87-MG human glioblastoma. A cellular survival inhibition below to 20% was observed for both of the cell lines incubated with relatively high concentrations (100 μg/mL), suggesting a satisfactorily low toxicity effect.¹⁴ According to the pharmacokinetic study, TiNTs were promptly cleared from the bloodstream 6 h following the intravenous injection as a result of the phagocytic process. Indeed, circulating opsonin reacts with the TiNT surface to make them more susceptible to ingestion by phagocytes, as described for other types of nanoparticles.³⁴⁻³⁸ Likewise, the negative charge of TiNTs, due to the hydroxyl groups presented on their surface, plays a crucial role in the elimination process since negatively charged nanoparticles are generally considered to have faster blood clearance than neutral particles.^14,30 Meanwhile, it was not surprising that TiNTs, irrespective of size and charge, mainly accumulated in the RES including the liver and spleen where most macrophages reside. The TiNT pharmacokinetic profile can be compared to some other types of bare inorganic nanoparticles described in the literature such as spherical TiO₂ and CNTs.^33,39,40 The blood and tissue distribution of TiO₂ nanoparticles in rat after intravenous injection of 5 mg/kg was studied.⁴¹ The authors reported a rapid plasmatic clearance of TiO₂ nanoparticles as a result of the undetectable levels of TiO₂ nanoparticles in blood at 1, 14, and 28 days postexposure.⁴¹ Then, TiO₂ nanoparticles were found to be accumulated in the liver, spleen, kidneys, and lungs, causing the damage of the endothelial barrier integrity.⁴¹ Compared to spherical TiO₂ nanoparticles, TiNTs were rapidly eliminated from the blood stream to concentrate not only in the liver, spleen, kidney, and lungs but also in the brain without inducing tissue damage during 45 days. Furthermore, in contrast to TiO₂ nanoparticles, TiNTs were degraded under lysosomal conditions and eliminated via urine and biliary excretion without inducing toxicity despite the injection of a relatively high dose. Regarding the CNTs, the pharmacokinetics, toxicity, and biodistribution of these nanomaterials showed controversial results in the literature related to the physicochemical proprieties (size, surface charge...) of CNTs and the experimental conditions.⁴²⁻⁴⁵ Singh et al. have intravenously injected into mice the radiolabeled (111In-DTPA) SWNTs in order to investigate CNT biodistribution and pharmacokinetics. In contrast with TiNTs, the majority of CNTs (>95%) was cleared out within 3 h, without accumulating in the liver and spleen.⁴³ Another study reported that ultrapure single-walled CNTs (SWCNTs) intravenously injected to mice were accumulated mostly in the lungs and then in the spleen and liver without apoptosis in the main organs except inflammation of lung tissue.⁴⁴ The prolonged circulation time of nanoparticles is one of the most important parameters which must be improved to obtain efficient drug delivery. In this regard, several studies were focused on surface functionalization of nanoparticles by stabilizing agents such as polyethyleneglycol (PEG), dextran, or chitosan. Obviously, PEG, dextran, and chitosan coating dramatically prevented the aggregation of nanoparticles, showing much better colloidal stability under the physiological conditions and long-time residence in the bloodstream circulation.⁴⁶⁻⁴⁸ Liu et al. have functionalized the SWCNTs by PEG to study their pharmacokinetics and biodistribution after intravenous injection into BALB/c mice or to female athymic mice. Compared to bare TiNTs, PEGlated-SWCNTs show a significant long-time blood circulation up to 1 day and low uptake in the RES.^48,49 Considering the large volume of TiNT distribution, one can note that nanotubes distribute extensively into body tissues and fluids as known for hydrophobic drugs.⁵⁰ Regarding the slow kinetics of TiNT degradation in the liver and the spleen and also their slowly elimination in urine and feces, we can suggest that the level of released Ti over time is so low to be detected in blood by the ICP-AES method. However, functionalization of the TiNT surface would be an essential step to increase their circulation time in the bloodstream. According to the biodistribution data, 24 h after the intravenously administration of TiNTs, nanotubes were accumulated intensively in the liver, spleen, and lungs until 7 days. Obviously, the liver is known to be the major target organ for nanoparticle accumulation (mainly inside Kupffer cells) and biodegradation before elimination.^51,52 The high amount of TiNTs accumulated in the liver induces hepatic vein enlargement in the centrilobular zone associated with a transient congestion of the vessels and dilatation of the sinusoids as reported for hepatic disease.^53,54 One can note that hepatic enlargement comes back to the normal after 3 days of TiNT injection without inducing pericardial disease, heart failure, compression, or thrombosis of the hepatic veins or inferior vena cava. In contrast to TiNTs, the exposition of rats to spherical TiO₂ for 30 days (40 mg/kg) showed histological damage of the liver characterized by edema associated with swelling of the cells per lobular with cytoplasmic vacuolation and hydropic degeneration.⁵⁵ In addition, enzymatic markers of liver functions (ASAT and ALAT) were disrupted. Likewise, the authors reported a decrease in the activity of cardiac enzymes (troponins) due to the production of inflammatory cytokines as a result of the production of reactive oxygen species, but without accumulation of nanoparticles in the heart.⁵⁵ Moreover, the high accumulation of TiNTs in the liver was not associated with morphology and alteration in the cardiovascular system. The cytotoxicity of TiNTs with regard to neonatal rat cardiomyocyte cells has been previously reported and shown to be noncytotoxic after a 24 h period of incubation.¹⁹ After 7 days of TiNT administration, the level of nanotubes decreases slowly over time in the liver, spleen, and lungs and then conversely increased in the kidneys, urine, and feces, indicating the biodegradation of TiNTs. The increase in titanium in urine can be explicated not only by the presence of ionized titanium but also by the biodegraded nanotubes smaller than 6 nm which were able to cross the glomerular filtration membrane as confirmed for other nanoparticles.³⁶ We believe also that unfiltered TiNTs (>6 nm) were captured by kidney resident macrophages to be either degraded into smaller nanoparticles or ionized in acidic lysosome digestion. Moreover, the particle size and shape play a vital role in TiNT toxicity and tissue distribution.⁵⁷⁻⁵⁹ The progressive increase in titanium in feces suggested clearing efficacy through the bile ducts. Such a slow elimination could be due to the longer size and resistant shape of TiNTs.^57,58 Clearly, a period longer than 45 days is required to achieve total elimination of TiNTs. Until 45 days post injection of TiNTs, the nanotubes are progressively biodegraded and eliminated via urine and feces but without any toxicity. With regard to the shape of nanoparticles, CNTs are the main competitors of TiNTs for drug delivery.^60,61 However, they have several drawbacks: they are insoluble in most solvents,⁴² and in contrast to TiNTs, they have closed ends.⁶³ Several nanomaterials compared to TiNTs demonstrate a similar kinetic of clearance but accompanied with high dramatical sings of toxicity and perturbation of the liver, heart, and kidney functions.^19,64,65 Interestingly, the determination of biomarkers, related to the liver, kidney, and spleen functions, evidenced the safety/toxicity risk of TiNTs. Indeed, activities of typical biomarkers of hepatic cytolysis including circulating ALAT and ASAT activities increased 1.5 times compared to the control group from 6 h until 7 days. This probably results from the accumulation of a high level of TiNTs inside Kupffer cells. However, cell damage and necrosis were excluded, in agreement with hepato-histological examinations, and normal values of ALAT and ASAT were observed after 7 days of TiNT administration, pointing out a latency time for the regulation of titanium hepatic homeostasis. Bearing in mind that liver inflammation injury is normally associated with an increase of more than twice the ALAT activity compared to the basal level⁶⁶ and the absence of histological damage, one can consider that transitory liver hypertrophy is not associated with severe hepatic toxicity. Moreover, the serum creatinine levels have varied in a similar manner without any significant differences during the experiments, confirming that TiNT elimination is not associated with any renal toxicity. Furthermore, we detect no sign of histological damage such as fibrosis or inflammation in kidney tissue. Likewise, the serum IL-6 level varied homogeneously to the control group, discarding thus the chronic inflammation due to TiNT administration for 45 days. Indeed, it has been reported that macrophages are highly tolerant to the cytotoxicity of anticancer drugs and nanoparticles.⁶⁷ Finally, a slight but significant increase in titanium cerebral levels was observed from 6 h until 30 days following the TiNT administration. Note that the bypass of the blood brain barrier (BBB) depends on the polarity and size of the particles: the smaller their size and the higher their surface hydrophobicity, the better their BBB crossing.⁶⁸ Due to their small external diameter (≈10 nm) and their hydrophobic tubular shape, TiNTs may have crossed the BBB through the 20 nm space between the astrocytic end-feet basement membrane and capillary endothelium.⁶⁹ Affinity of hydrophobic tubular TiNTs to circulating lipoproteins could have further facilitated to cross the brain barrier, suggesting that TiNTs can be useful for brain tumor treatment as a drug delivery vector or photothermotherapy after optimizing brain uptake. Finally, no genotoxic effects (metaphase cells and micronuclei) due to TiNTs were observed, according to the histopathological examination of all organ tissues. In the literature, several studies reported the genotoxic effects of spherical TiO₂ particles, but the data available are very heterogeneous.⁷⁰⁻⁷² The results are difficult to interpret because they are strongly influenced by the experimental conditions, the type of cell line, and the type of particles (size, surface, crystalline form, etc.). A few in vitro studies show DNA damage through the generation of reactive oxygen species.⁷³ An increase in the number of gene mutations of pulmonary epithelial cells and in the frequency of micronuclei were also evident in the bone marrow and lymphocytes and in human bronchial epithelial cells after exposition to TiO₂ (40–200 mg/kg) for 7 days.⁷⁴ Additional careful genotoxicity tests would be a considered step to evaluate the possibility of future clinical translation of these TiNTs.

4. Conclusions

For the first time, we investigated pharmacokinetics, biodistribution, target organs, and potential adverse effects of TiNTs after intravenous injection of a relatively high dose (45 mg/kg) in mice. We have demonstrated that TiNTs were rapidly eliminated from the blood stream and then accumulated mostly in the liver, spleen, and kidneys without any residual hepatic or renal toxicity, as confirmed by the biochemical parameters and organ microscopic examination. An important property of these TiNTs is their slow dissolution in an acid environment, rendering them biodegradable. These materials present a promising nanocarrier-based drug delivery system. Meanwhile, appropriate surface coating is essential for biomedical applications of TiNTs to prevent their aggregation and opsonization, to reduce their macrophage uptake, and finally to increase their blood circulation time.

5. Experimental Section

TiNTs were prepared by a classical hydrothermal method and characterized as reported previously.¹⁴ The particle surface charge and PDI were determined using Malvern Mastersizer 2000 (Zetasizer Nano ZS90, Malvern Instruments Ltd., UK). Before measurement, the freshly prepared TiNTs were appropriately suspended in pure physiological saline solution (0.1 mg/mL) and then ultrasonicated during 5 min. All measurements were performed at room temperature after equilibration for 10 min. The data were obtained with the average of three measurements. For size and shape propriety, a sample of TiNT suspension was observed by TEM as reported previously.¹⁴ For the crystalline phase, X-ray powder diffraction was performed at room temperature with an X-ray diffractometer (X’Pert PRO MPD, PANalytical Co., Holland). Indeed, monochromatic Cu Kα-radiation (λ = 1.5418 Å) was obtained with Ni filtration and a system of diverging and receiving slides of 0.5° and 0.1 mm, respectively. The diffraction pattern was measured with a voltage of 40 kV and a current of 30 mA over a 2θ range of 3–40° using a step size of 0.02° at a scan speed of 1 s per step.

5.1. Animals and Treatment

All experimental protocols and animal analyses were conducted in accordance with the guidelines of the Tunisian Government and the Regional Committee for Ethics on Animal Experiments in the Faculty of veterinary medicine of Sidi Thabet in accordance with related EU directives (authorization number: E 19-013-4) as described in our previous studies.³⁶ All animals used in this work were obtained from SIPHAT, Tunisia. Seven groups of female swiss mice (n = 6) were injected with a single dose of 45 mg/kg TiNTs. At the end, the injection site was closed with one stitch. Animals were then observed 6 h and 1, 3, 7, 15, 30, and 45 days post injection for survival and behavior. Animal weights were controlled for any body weight loss due to TiNTs. In the same time, seven control groups of female mice (n = 6) were injected with saline solution (NaCl 0.9%). Twenty-four hours before sacrifice, animals were housed individually in metabolic cages to recover urine and feces. A dose of 45 mg/kg TiNTs was selected considering the drug-loading content efficacy conditions obtained in our previous experiments on genistein.¹⁴ After anesthesia under isoflurane, animals were sacrificed and the blood samples (≈800 μL) were collected by intracardiac puncture and then inoculated in heparin tubes and centrifuged at 3600 rpm to separate serum. Serum samples were aliquoted and then stored at −20 °C until analysis. Moreover, organs were collected and washed with NaCl 9% prior to storing at −20 °C until analysis as reported previously.³⁶

5.2. Pharmacokinetics

For the pharmacokinetic study, 48 female Swiss mice with a body weight of 20 (±2) g were randomly divided in eight groups (n = 6) depending on time, 5 min, 15 min, 30 min, 60 min, 3 h, 6 h, 12 h, and 24 h post injection of TiNTs. Following the intravenous administration of TiNTs (45 mg/kg), mice of each group were sacrificed under isoflurane anesthesia. Blood samples (≈800 μL) were collected by intracardiac puncture and then inoculated in heparin tubes and centrifuged at 3600 rpm to separate serum. Serum samples were aliquoted and then stored at −20 °C until analysis. In order to determine the pharmacokinetic profile of TiNTs, the titanium level was determined in 100 μL of each serum sample by inductively coupled plasma optical emission spectrometry (ICP-OES) after mineralization in nitric acid solution, as described previously.¹⁴ Finally, the pharmacokinetic parameters were calculated with R studio software Package PKNCA version 0.9.4.

5.3. TEM of Organs

After animal sacrifice, fragments of the liver, spleen, and kidneys were prepared for TEM, as described previously.³⁶ Ultrathin sections of 90 nm were cut from dried blocks with a diamond knife on an LBK ultramicrotome Leica UCT and stained with 0.5% aqueous uranyl acetate followed by Reynold’s lead citrate. The transmission electron microscope used for grid observation was a Tecnai G2 at 200 kV (FEI, Netherlands), and images were acquired with Veleta camera (Olympus, Japan).

5.4. Histological Analysis

For histopathological examination, organ fixation, section preparation, and hematoxylin–eosin coloration were performed as described previously.³⁶

5.5. Titanium Determination for the Biodistribution Profile

In order to determine the TiNT biodistribution, serum, urine, feces, and organ matrices were mineralized with nitric acid for titanium level determination by ICP-OES, as reported previously.¹⁴ (Quantification threshold was fixed at 0.01 ng/mg for tissues and feces and 0.06 ng/μL for urine and serum.)

5.6. Biochemical Parameters

Serum levels of ALAT and ASAT (biomarkers for liver function), creatinine (biomarker of kidney function), and IL-6 (biomarkers of inflammation) were determined according to the protocols described previously in our studies.³⁶

5.7. Statistics

Data are shown as the mean ± standard deviation. Comparisons with the control were performed for quantitative variables using Student’s test. For the ANOVA and Tukey HSD test, we have used SPSS 12 software. A value of p < 0.05 was considered statistically significant.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.1c01733.

• Mice weight evolution after 45 days of the intravenous administration of TiNTs; organ weight of different mice groups; histological sections of the lungs after 6 h and 1, 3, 7, 15, 30, and 45 days of the intravenous administration of TiNTs; histological sections of the heart after 6 h and 1, 3, 7, 15, 30, and 45 days of the intravenous administration of TiNTs, histological sections of the brain after 6 h and 1, 3, 7, 15, 30, and 45 days of the intravenous administration of TiNTs; and one-way ANOVA of the pharmacokinetics of the TiNTs (PDF)

The authors declare no competing financial interest.