Effects of paclitaxel and methotrexate associated with cholesterol-rich nanoemulsions on ischemia-reperfusion injury after unilateral lung transplantation in rats

Abstract

Currently, the barrier to successful lung transplantation is ischemia and reperfusion injury, which can lead to the development of bronchiolitis obliterans. Paclitaxel and methotrexate are drugs known to inhibit cell proliferation and have anti-inflammatory effects, and the association of these drugs with cholesterol-rich nanoparticles has been shown to be beneficial in the treatment of other transplanted organs. Thirty-three male Sprague Dawley rats were divided into 3 groups: Basal group, no intervention; Control group, received only nanoparticles; Drug group, paclitaxel and methotrexate treatment. Donors and recipients were treated with nanoparticle-paclitaxel and nanoparticle-methotrexate, respectively, 24 h before surgery. The donor lungs from the Drug group were perfused with a preservation solution supplemented with nanoparticles-paclitaxel. After 12 h, the left lung was implanted and reperfused for 1 h. Recipients had an increase in erythrocytes, neutrophils and hemoglobin and a decrease in lymphocytes, and an increase in oxygenation and lactate and a decrease in carbon dioxide. These animals showed an increase in urea and creatinine. The grafts showed perivascular edema and hemorrhage, as well as elevated values of airway resistance, tissue resistance and tissue elastance under mechanical ventilation. The tested drugs were not effective in attenuating the effects of ischemia and reperfusion injury.

Introduction

Ischemia and reperfusion injury (IRI) is one of the main causes of morbidity and mortality after lung transplantation (LTx). IRI leads to increased vascular resistance and permeability of the blood-air barrier, causing perivascular and alveolar edema, and decreased gas exchange^1–3. Furthermore, patients with perioperative IRI are at increased risk of developing bronchiolitis obliterans syndrome (BOS)^4,5.

Ischemia during transplants causes a decrease in the synthesis and resynthesis of adenosine triphosphate (ATP), which leads to the accumulation of xanthine oxidase and hypoxanthine. When reperfusion occurs, the enzyme xanthine oxidase comes into contact with oxygen, transforming hypoxanthine into reactive oxygen species (ROS)^1,6.

Approximately 15% of transplant patients experience complications, which can lead to severe primary graft failure in 20% of transplant patients, and is a significant cause of morbidity and mortality in the first 90 days after transplantation. Impaired gas exchange can lead to primary graft dysfunction, for which ECMO can be used as a measure for allograft salvage and treatment of associated comorbidities, and can also be used as a bridge to retransplantation¹.

Evidences show that cellular and humoral immune responses are exacerbated by inflammatory processes associated with IRI and play a key role in the development of BOS^4,5,7. Due to the complex and multifactorial pathophysiology of BOS, its prevention has become a major challenge for increasing the success rate of LTx⁸.

Paclitaxel (PTX) and methotrexate (MTX) are drugs used to treat cancer and autoimmune diseases^9–11. PTX may also have beneficial effects in the treatment of other diseases such as kidney/liver fibrosis and inflammation¹².

One study showed that PTX was able to attenuate lung injury in mice by suppressing the TLR-4/NF-Kβ pathway and reducing inflammatory cytokines¹³. Lourenço et al.¹⁴ reported that PTX reduced macrophage infiltration in transplanted hearts, which presented an arterial lumen 3 times wider than that of untreated animals.

Fiorelli et al.⁹ reported that MTX attenuated the expression of TNF-α and VCAM1, and stimulated the expression of anti-inflammatory molecules such as IL-10. Other studies reported that MTX-treated mice exhibited attenuated TLR4 expression¹⁵. Studies have shown that TLR4 influences IRI in mouse livers, in addition to contributing to the development of BOS in LTx^12,16,17.

To minimize the toxic effects of these drugs and increase their pharmacological effects, they were associated with cholesterol-rich nanoparticles called LDE, which have affinity for cells with high metabolic activity due to cell division^18–21.

On the basis of these previous data, we hypothesized that PTX and MTX attenuate IRI after LTx by reducing the perivascular inflammatory process. Our objective was to assess whether PTX and MTX attenuate IRI in a rodent model of LTx.

Materials and methods

This study was approved by the Ethics Committee for the Use of Animals of the Faculty of Medicine of the University of São Paulo—CEUA-FMUSP—085/15. Thirty-three male Sprague Dawley rats weighing 350/400 g were provided by the Central Animal Facility of FMUSP and divided into three groups:

• Basal (n = 5): Not subjected to transplantation, only anesthetized and euthanized for data collection.

• Control (n = 4 Tx, 8 animals): Donor and recipient animals received only LDE. The donor lungs were flushed with only preservation solution, in the same proportions as those in the Drug group.

• Drug (n = 10 Tx, 20 animals): Donor and recipients received 4 mg/kg LDE-PTX and 1 mg/kg of LDE-MTX intraperitoneally, respectively, 24 h before surgery. After 12 h of drug administration, the donor lungs were flushed with 20 mL of preservation solution supplemented with 0.5 mL of LDE-PTX (4 mg/kg) and subjected to cold ischemia during 12 h.

All animals received human care in accordance with the Principles of Laboratory Animal Care (National Society for Medical Research) and the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources). Authors complied with the ARRIVE guidelines. Figure 1 shows study design and timeline.

Fig. 1.

Study design and timeline.

LDE preparation and association with PTX and MTX

The preparation of LDE nanoemulsions and their association with PTX and MTX were performed as described by Maranhão et al.^21,22 Lipid emulsification was initiated by adding Tris-HCl buffer solution (0.001 M and pH 8.05) and the surfactant Tween80 (Merck, Germany) in a high-pressure homogenizer (Microfluidizer-M110S, Microfluidics™, USA). Homogenization was performed for 30 to 40 min at 55–60 °C, and the diameter of the lipid particles was monitored by a ZetaSizer Nano ZS90 instrument (Malvern, UK) throughout the production. The drug was added to the lipid mixture at a 1:10 drug: lipid mass ratio. The preparations were sterilized on a Milipore 0.22 μm filter and stored at 4 °C. LDE-associated drugs were quantified via high-performance liquid chromatography (HPLC) (Shimadzu LC10A, Shimadzu, Japan).

LDE-MTX was prepared from a lipid mixture of cholesteryl oleate, egg phosphatidylcholine (Lipoid GMBH, Ludwigshafen, Germany), triglycerides, cholesterol, and MTX²³. The aqueous phase, consisting of 100 mg of polysorbate 80 (Tween 80, Merck, Hohenbrum, Germany) and 10 mL of Tris-HCl pH 8.05 buffer, was kept at room temperature. The preemulsion was obtained by adding the hydrophilic phase to the oil phase via ultrasonic radiation until complete dissolution of the drug was achieved. Emulsification of all lipids, MTX and the aqueous phase was achieved via high pressure homogenization via an Emulsiflex C5 homogenizer (Avestin Inc, Ottawa, Canada). After 30–40 min of constant temperature homogenization, the nanoemulsion was centrifuged at 1800 x g for 15 min to separate unbound MTX, which precipitates after centrifugation. The nanoemulsion was sterilized by passing through a polycarbonate filter with 0.22 μm pores (EMD Millipore Corporation, Billerica, MA, USA) and kept at 4 °C until use. The incorporation of MTX into LDE was measured before its administration to the animals by HPLC. The mean drug load was 4 mg/mL. As previously described, the average diameter of the LDE-MTX particles was 60 nm, which was measured via the laser light scattering method, via a zeta potential analyzer (ZetaPALS, Brookhaven Instruments Corporation, Holtsville, New York, USA)²⁴.

Surgical procedure

The lung graft preparation and implantation procedures were performed as described by Almeida et al.^25. Briefly, 12 h after LDE-PTX administration, the donor animals were anesthetized in a closed chamber with 5% isoflurane and orotracheally intubated with a polyethylene cannula, which was connected to a mechanical ventilator (FlexiVent, SCIREQ, Montreal, HERE), where anesthesia was maintained at 2%. After laparotomy and median sternotomy, the animals received 50 IU of heparin via the abdominal vena cava and the lungs were washed through the pulmonary artery with 20 mL of cold preservation solution (Perfadex, Vitrolife, Sweden) at a pressure of 20 cm of H₂O. Then, the trachea was occluded at the end of inspiration to keep the lungs inflated and the heart-lung block was extracted and kept in a Petri dish containing gauze soaked in preservation solution. The cuffs (16G) were fixed in the vein, artery and bronchus of the left lung with 7.0 polypropylene thread and kept in cold ischemia (4 °C) until implantation. Twenty-four hours after LDE-MTX administration, the recipients were anesthetized and intubated in the same way as the donors, and kept under mechanical ventilation (10 mL/kg and 90 cycles/min) until euthanasia. After left thoracotomy, the pulmonary hilum was dissected and the graft was implanted via the insertion and fixation of the cuffs into the vena cava, artery and bronchus of the recipient. Graft reperfusion occurred for one hour. The rats were anesthetized until euthanasia.

Ventilatory mechanics

The airway resistance (RAW), tissue elastance (HTIS), and tissue damping (GTIS) parameters were calculated via the forced oscillation model²⁶ immediately after the reestablishment of airflow and blood circulation and after 1h of reperfusion.

Blood gas analysis

After the last pulmonary mechanics data collection, 0.5 mL of arterial blood was collected from the abdominal aorta artery for blood gas analysis.

Chemical analysis of blood

Then, 5.0 mL of blood from the inferior vena cava was collected to test the degree of toxicity of the drugs through the hematological profile and plasma levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine, gamma glutamyl transferase (GGT), and urea. The hematological profile was evaluated by counting the total number of red blood cells and leukocytes, and the percentages of lymphocytes, monocytes, neutrophils and eosinophils.

Euthanasia

The animals were euthanized by exsanguination, through the total section of the abdominal aorta artery.

Histology

Five sections of the transplanted lung tissue were fixed in 10% formalin, embedded in paraffin and stained with hematoxylin-eosin. The slides were analyzed via the point counting technique with a reticulated eyepiece of 100 points and 50 lines. The vessel and alveolar edema index and the hemorrhage index were determined. In addition, mono- and polymorphonuclear cells in the lung parenchyma were counted.

Statistical analysis

The normality of the data was verified by the Shapiro-Wilk test and comparisons between the groups were performed via ANOVA with Bonferroni and Dunn’s correction for parametric and nonparametric data, respectively. The data are expressed as the means ± standard deviations (SD) or medians and interquartile ranges. Differences were considered significant at a value of p < 0.05. The software used for statistical analysis was SigmaPlot.

Results

There was no difference between groups concerning cold ischemia times (Control 12.15 ± 0.20, Drug 12.12 ± 0.23 h, p > 0.05).

Blood gases

Compared with the Basal group, the Tx groups presented increased lactate concentrations (Basal 18.60 ± 4.39, Control 83.50 ± 23.82, Drug 68.67 ± 13.94 mmol/L, p < 0.001), pH (Basal 7.38 ± 0.37, Control 7.50 ± 0.65, Drug 7.46 ± 0.65, p = 0.015) and pO2 [Basal 46.60 (43.60-65.05), Control 74.75 (65.88–107.10), Drug 230.00 (146.50–361.00) mmHg, p = 0.001] and decreased pCO2 values (Basal 40.22 ± 7.04, Control 16.43 ± 3.59, Drug 19.31 ± 7.89 mmHg, p < 0.001) (Fig. 2).

Fig. 2.

Blood gas analysis. Lactate concentration (cLac): *p < 0.001; partial pressure of carbon dioxide (pCO²): *p < 0.001; hydrogen ion potential (pH): *p = 0.021; pO²: *p ≤ 0.005.

Chemical analysis of blood

There was an increase in creatinine (Basal 0.23 ± 0.41, Control 0.85 ± 0.049, Drug 0.73 ± 0.15 mg/dL, p < 0.001) and urea (Basal 36.00 ± 5.14, Control 102.60 ± 16.03, Drug 86.82 ± 12.38 mg/dL, p < 0.001) in all Tx animals in comparison with the Basal group (Fig. 3). The other parameters did not differ (ALT: Basal 72.20 ± 5.40, Control 93.25 ± 9.00, Drug 73.00 ± 18.52 U/L, p = 0.073; AST: Basal 129.40 ± 39.20, Control 130.25 ± 14.80, Drug 136.20 ± 46.71 U/L, p = 0.942; GGT: Basal 3.26 ± 1.51, Control 2.33 ± 1.54, Drug 3.22 ± 1.20 U/L, p = 0.513).

Fig. 3.

Blood chemical analysis. Urea: *p < 0.001; Creatinine: *p < 0.001.

Blood cell count

Compared with those in the Basal group, there was an increase in erythrocytes (Basal 7.18 ± 0.38, Control 10.03 ± 0.86, Drug 9.47 ± 0.62 × 10⁶/mm³, p < 0.001) and hemoglobin (Basal 15.24 ± 0.94, Control 19.53 ± 1.99, Drug 18.39 ± 1.27 g/dL, p < 0.001) in the Tx groups. However, the number of lymphocytes was lower in the surgical groups (Basal 4.43 ± 0.64, Control 2.19 ± 0.50, Drug 2.53 ± 0.46 × 10³/mm³, p < 0.001). Finally, the Control group presented a greater number of neutrophils than did the Basal group (Basal 0.82 ± 0.15, Control 2.07 ± 0.68, Drug 1.48 ± 0.31 × 10³/mm³, p = 0.015) (Fig. 4). There was no difference concerning the number of monocytes (Basal 0.07 ± 0.02, Control 0.16 ± 0.14, Drug 0.17 ± 0.08 × 10³/mm³, p = 0.210).

Fig. 4.

Blood count. Erythrocytes: *p < 0.001; Hemoglobin: *p = 0.002; Lymphocytes: *p = 0.009; Neutrophils: *p = 0.015.

Ventilatory mechanics

Immediately after Tx, the Basal group presented lower Gtis values (Basal 0.29 ± 0.03, Control 0.44 ± 0.03, Drug 0.39 ± 0.04 cmH₂O.s/mL, p < 0.001). There was no difference between groups in Raw (Basal 0.10 ± 0.03, Control 0.15 ± 0.02, Drug 0.12 ± 0.04 cmH₂O.s/mL, p = 0.157) and Htis (Basal 1.46 ± 0.14, Control 1.59 ± 0.31, Drug 1.70 ± 0.31 cmH₂O.s/mL, p = 0.228).

After 1 h of reperfusion, the Basal group continued to have lower values of Gtis [Basal 0.29 (0.26–0.30), Control 0.43 (0.38–0.51), Drug 0.44 (0.38–0.46) cmH₂O.s/mL, p = 0.003], but also presented the lowest value of Htis compared with the Drug group (Basal 1.46 ± 0.14, Control 1.76 ± 0.13, Drug 1.98 ± 0.36 cmH₂O.s/mL, p = 0.005), and Raw, compared with the Control group (Basal 0.10 ± 0.03, Control 0.17 ± 0.03, Drug 0.12 ± 0.04 cmH₂O.s/mL, p = 0.016) (Fig. 5).

Fig. 5.

Ventilatory mechanics. Tissue resistance (Gtis): *p < 0.001; airway resistance 1 h (Raw 1 h): *p = 0.015; Tissue resistance 1 h (Gtis 1 h): *p < 0.05; tissue elastance 1 h (Htis 1 h): *p = 0.004.

Histology

Compared with those in the Basal group, the perivascular edema index (Basal 0.28 ± 0.23, Control 2.17 ± 0.08, Drug 2.17 ± 0.45, p < 0.001) and hemorrhage index (Basal 1.07 ± 0.03, Control 1.52 ± 0.04, Drug 1.76 ± 0.38, p = 0.004) were greater in the grafts from recipient animals (Fig. 6). There was no difference in alveolar edema (Basal 21.02 ± 5.39, Control 17.88 ± 11.77, Drug 19.04 ± 9.96%, p = 0.896), polymorphonuclear cells (Basal 88.71 ± 25.28, Control 97.81 ± 89.25, Drug 43.31 19.78 mm², p = 0.363) or mononuclear cells (Basal 121. 24 ± 42.28, Control 110.42 ± 101.21, Drug 59.40 ± 54.88 mm², p = 0.215).

Fig. 6.

Histology. Perivascular edema: *p < 0.001; Hemorrhage: *p = 0.003. Representative photomicrographs of grafts (HE, magnification ×200).

Discussion

This study aimed to verify the effects of PTX and MTX associated with LDE on IRI. We used a well-established model of left unilateral LTx in rats^6,25. The model used was also based on another previous study, in which PTX improved the state of the transplant. With the intention of already having the drug circulating in the animals, to enhance the results, we decided to administer PTX to the donor animals²⁷.

The incorporation of drugs into LDE is a known model used in our institution^28,29. The ischemia time and the use of PTX with preservation solution were based on external work²⁷. Several studies have attempted to reduce the toxic side effects of PTX and MTX by their association with LDE. One of these studies showed that this association was able to reduce atherosclerosis in rabbits²¹. Another study demonstrated that PTX oleate does not dissociate from particles in the plasma circulation of breast cancer patients; there was also an improvement in the pharmacokinetic parameters³⁰. In the present study, this association did not cause damage to the kidneys of the animals, since the increase in urea and creatinine values observed can be attributed to the surgical procedure.

The results of the blood count suggest that the drugs did not cause toxic damage, since there was no neutropenia or leukopenia, two toxic effects reported in the literature^31–33. We can infer that the decrease in the number of lymphocytes observed, is a condition reported in the literature as surgically induced immune dysfunction. In fact, a study revealed that after an intestinal lesion in a murine model, lymphocytopenia occurred in relation to animals that underwent only laparotomy³⁴. All the groups undergoing transplantation presented increases in erythrocytes and hemoglobin. We believe that this was caused by an increase in the concentration of these cells, as a result of the loss of water by evaporation, since the thoracic cavity remained open throughout the transplant and reperfusion.

Inflammation and loss of lung function are the main events involved in IRI. Its mechanisms can result in alveolar edema, increasing endothelial permeability, causing poor gas exchange, and a decrease in pO² values³⁵. A study tested PTX in this unilateral LTx model in rats, and reported an attenuation of acute lung injury, which significantly reduced edema and improved oxygenation performance²⁷. In our study, we observed that the drugs were not able to prevent or attenuate edema. However, the gas exchange was greater in the treated groups. If the improvement in oxygenation occurred only in the treated group, we could hypothesize that it was due to the effects of MTX, which promoted the release of adenosine. A study revealed that, when stimulated, adenosine A2A receptors can promote muscle relaxation and vascular dilation³⁶. Another study involving infarction in dogs revealed that MTX can improve IRI through its adenosine-related action³⁷.

Maranhão et al.²² used a model of myocardial infarction in rats, and revealed that LDE-MTX was able to decrease hypoxia, cell death, oxidative stress, the number of inflammatory cells, and increase antioxidant enzymes and angiogenesis. However, we believe that this increase in oxygenation observed in our study was due to the greater number of red blood cells and hemoglobin, in addition to the mechanical ventilation to which they were subjected at the time of collection of blood samples, unlike the animals from the Basal group.

Ventilatory mechanics were used to verify any change in lung function. However, an expected increase in values was noted only in these two groups compared with the Basal group because of the transplant.

Considering that BOS is characterized by an increase in mesenchymal cells in the airways and an increase in the deposition of extracellular matrix in the lumen of respiratory bronchioles³⁸, it was speculated that the drugs used could attenuate these effects, since both of them have anti-proliferative effects. Lourenço et al.¹⁴ reported that PTX associated with LDE was able to maintain the arterial lumen of transplanted hearts 3 times wider, and reduce macrophage infiltration.

We used histology to count polymorphonuclear and mononuclear cells. Despite the combined use of these drugs, it was not possible to observe significant differences. One of the factors that could explain this result is that the treatment method was ineffective due to the long period of ischemia. Suzuki et al.²⁷ used the same ischemia time, but the organ remained immersed in Perfadex with PTX. In our work, we kept the organ soaked only in Perfadex. Suzuki et al.²⁷ reported that reperfusion can dilute the drug in the circulation, decreasing its effectiveness.

Barbieri et al.²⁸ noted that both drugs, when combined, did not cause the expected positive effects on graft vascular disease. He considered the possibility that drugs are competing for LDE receptors. To avoid this, in our work, donor animals received LDE-PTX, while recipients received LDE-MTX. However, the final results also revealed no improvement. Yüceyar et al.³⁹ reported that PTX can reduce hydroxyproline levels and new vessel formation in intestinal anastomoses when it is administered intraperitoneally. In contrast, MTX increased angiogenesis in hearts subjected to infarction²². Additional studies are needed to determine whether this effect remains even after LDE association, and whether these events can cause problems in the success of LTx.

Another important point is that the drug vehicle LDE uses LDL receptors to enter cells. Our study used cold ischemia, which decreases cellular metabolism. This may lead us to hypothesize that the exposure time to the drug was shorter than necessary.

We believe that the results obtained are important because the drugs had no effect on this model, and that the combination of two well-established animal models does not always work exactly as observed in each one separately. With respect to future clinical applications in lung Tx, several changes in the drug administration protocol should be tested.

Author contributions

A.S.B.: Investigation, Data Curation, Formal Analysis, Writing—Original Draft, Writing—Review & Editing. E.R.T.: Investigation, Formal Analysis, Writing—Original Draft, Methodology. A.T.C.: Data Curation, Formal Analysis. F.M.A.: Investigation, Data Curation, Formal Analysis. P.O.C.: Investigation, Formal Analysis. M.C.G.: Investigation, Formal Analysis. P.M.P.: Conceptualization, Review—Original Draft. R.C.M.: Conceptualization, Review—Original Draft. R.P.: Investigation, Data Curation, Formal Analysis, Writing—Review & Editing, Methodology, Supervision, Project Administration, Funding Acquisition.

Data availability

The data that support the findings of this study are available from the corresponding author, Rogerio Pazetti, upon reasonable request.

Declarations

Competing interests

Elaine Rufo Tavares, Aristides Tadeu Correia, Francine Maria de Almeida, Priscila Oliveira Carvalho, Maria Carolina Guido, Paulo Manuel Pêgo-Fernandes, Raul Cavalcante Maranhão declare to have no conflict of interest directly or indirectly related to the manuscript contents.Rogerio Pazetti declare this work was supported by the São Paulo Research Foundation (FAPESP 2015/21556-2) for material acquisition only.Angela da Silva Battochio received a scholarship for PhD program from Coordination for the Improvement of Higher Education Personnel - CAPES.