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Delivery of FK506-loaded PLGA nanoparticles prolongs cardiac allograft survival
Journal Pre-proofs Delivery of FK506-loaded PLGA nanoparticles prolongs cardiac allograft survival Cheng Deng, Yihan Chen, Li Zhang, Ya Wu, Huiling Li, Yu Wu, Bin Wang, Zhenxing Sun, Yuman Li, Qing Lv, Yali Yang, Jing Wang, Qiaofeng Jin, Mingxing Xie PII: DOI: Reference:
S0378-5173(19)30996-2 https://doi.org/10.1016/j.ijpharm.2019.118951 IJP 118951
To appear in:
International Journal of Pharmaceutics
Received Date: Revised Date: Accepted Date:
3 August 2019 3 December 2019 11 December 2019
Please cite this article as: C. Deng, Y. Chen, L. Zhang, Y. Wu, H. Li, Y. Wu, B. Wang, Z. Sun, Y. Li, Q. Lv, Y. Yang, J. Wang, Q. Jin, M. Xie, Delivery of FK506-loaded PLGA nanoparticles prolongs cardiac allograft survival, International Journal of Pharmaceutics (2019), doi: https://doi.org/10.1016/j.ijpharm.2019.118951
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© 2019 Published by Elsevier B.V.
Delivery of FK506-loaded PLGA nanoparticles prolongs cardiac allograft survival
Cheng Denga,b,1, Yihan Chena,b,1, Li Zhanga,b, Ya Wua,b, Huiling Lia,b, Yu Wua,b, Bin Wanga,b, Zhenxing Suna,b, Yuman Lia,b, Qing Lva,b, Yali Yanga,b, Jing Wanga,b, Qiaofeng Jina,b,*,[email protected], Mingxing Xiea,b,*,[email protected]
aDepartment
of Ultrasound, Union Hospital, Tongji Medical College, Huazhong University
of Science and Technology, Wuhan 430022, China bHubei
Province Key Laboratory of Molecular Imaging
*Corresponding
author.at: Department of Ultrasound, Union Hospital, Tongji Medical College,
Huazhong University of Science and Technology, 1277 Jiefang Avenue, Wuhan 430022, China 1 Equal
contributiors
Graphical abstract
Abstract In this study, FK506-loaded poly(lactide-co-glycolide) nanoparticles (PLGA-FK506-NPs) 1
were
developed
using
an
O/W
emulsion
solvent
evaporation
method.
The
PLGA-FK506-NPs were observed to be monodispersed and spherical by transmission and scanning electron microscopy. The mean size and zeta potential measured by dynamic light scattering were 110 ± 1.3 nm and -20.56 ± 3.65 mV, respectively. The FK506 entrapment and loading efficiency were 94.46 ± 1.88% and 5.38 ± 0.24%, respectively. Moreover, a pharmacokinetics study revealed that the PLGA-FK506-NPs behaved significantly different than free FK506 by exhibiting a higher area under curve (1.69-fold), higher mean residence time (1.29-fold), slower clearance and longer elimination half-life. Notably, the concentrations of FK506 in the spleen and mesenteric lymph nodes of the PLGA-FK506-NP group were 3.1-fold and 2.9-fold higher than those of the free FK506 group. Furthermore, the immunosuppressive efficacy was evaluated in a rat heterotopic heart transplantation model, and the results showed that PLGA-FK506-NP treatment could successfully alleviate acute rejection and prolong allograft survival compared with the free FK506 treatment (mean survival time, 17.1 ± 2.0 versus 13.3 ± 1.7 days). In conclusion, PLGA-FK506-NPs are a promising formulation for spleen and lymph node delivery and have potential use in the treatment of cardiac allograft acute rejection.
Keywords: FK506; PLGA; Nanoparticle; Heart transplantation; Acute rejection; Lymph node
2
1. Introduction Heart transplantation has become a standard treatment for thousands of patients with irreversible end-stage heart failure (Olymbios et al., 2018; Stehlik et al., 2018). However, cardiac allograft acute rejection remains one of the most important risk factors causing cardiac allograft loss within the first few months after heart transplantation (Stehlik et al., 2010). Fortunately, the incidence of acute rejection has been greatly reduced since the application of efficient immunosuppressive agents, such as FK506, which can inhibit T lymphocyte proliferation and activation efficiently through binding to FK506-binding protein 12 (FKBP12) to block the activation of calcineurin within the T lymphocytes (Schreiber and Crabtree, 1992). Although FK506 has shown a high efficiency, the long-term systemic administration of FK506 inevitably induces side-effects, including nephrotoxicity, neurotoxicity, hypertension and diabetogenic effects. In addition, complications such as opportunistic infections and malignancies often occur after nonspecific entire host immune system suppression following daily FK506 uptake (Lund et al., 2017; Stehlik et al., 2012). Furthermore, current FK506 formulations have various disadvantages including poor bioavailability, high pharmacokinetic variability and a narrow therapeutic window (5-15 ng/mL) (Sikma et al., 2015), which have greatly affected its therapeutic efficacy. Several recent studies have demonstrated that therapeutic efficacy is relevant to the FK506 concentration within the lymphocytes (Bahmany et al., 2019; Capron et al., 2016). Notably, a previous study reported that the effective concentration of FK506 in lymphocytes is only up to 0.26 ng/mL, which is 30 times lower than that in whole blood (Blanchet et al., 2006). Therefore, the targeted delivery of FK506 to the T lymphocytes would have immense 3
potential to promote therapeutic efficacy because T lymphocytes play a dominant role during acute rejection. It is well known that the proliferation and activation of T lymphocytes occurs primarily in the spleen and lymph nodes (LNs) (Lakkis et al., 2000). The targeted delivery of FK506 to the spleen and LNs would be an effective strategy to inhibit T lymphocyte activation.
Recently,
numerous
approaches
have
been
developed
to
deliver
immunosuppressive agents specifically to the lymphatic system (Azzi et al., 2016; Bahmani et al., 2018a; Bahmani et al., 2018b). Formulation modification is one of the most accessible approaches to promote biodistribution and to eventually achieve lymphatic system delivery. Nanoparticle (NP)-based drug delivery systems can deliver drugs to a targeted site, and they release their payload in a sustained manner (Bahmani et al., 2018a). Among various NPs, poly(lactide-co-glycolide) (PLGA) has been widely studied for its biocompatibility, biodegradability, non-toxicity and non-immunogenicity and has been designed to load various immunoregulation agents for the treatment of acute rejection in liver, corneal, and islet transplantation (Azzi et al., 2016; Miyamoto et al., 2004; Pham et al., 2018; Shirali et al., 2011; Tang et al., 2012; Wang et al., 2004; Wu et al., 2019; Xu et al., 2014). Particularly, FK506-loaded PLGA NPs have been reported to have an improved lymphatic-targeting capability in normal rats (Shin et al., 2010). However, their therapeutic efficacy remains to be verified in the transplantation models. In this study, a rat heterotopic heart transplantation model was established to verify the therapeutic efficacy of PLGA NPs loaded with FK506 (PLGA-FK506-NPs), which were prepared by the O/W emulsion solvent evaporation method. We characterized not only their physicochemical properties and the in vitro release patterns, but also the toxicity, 4
biodistribution and pharmacokinetics of PLGA-FK506-NPs. Finally, the therapeutic efficacy of PLGA-FK506-NPs for cardiac allograft was evaluated by measuring the survival rate of allografts and scored by the graft histologic section according to the criteria of the 2005 International Society for Heart and Lung Transplantation (ISHLT). PLGA-FK506-NPs were demonstrated to efficiently deliver FK506 to the spleen and LNs to promote the therapeutic efficacy. 2. Materials and methods 2.1 Materials and animals FK506 was purchased from MCE (New Jersey, USA). PLGA (lactide: glycolide ratio 50:50, molecular weight = 30,000-60,000) was obtained from Sigma-Aldrich (St. Louis, MO, USA).
Pluronic®
F-68
was
purchased
from
Solarbio
(Beijing,
China).
1,1 ′
-dioctadecyl-3,3,3′,3′-tetramethylindotricarbocyanine iodide (DiR) was purchased from AAT Bioquest (California, USA). All the other chemicals used in this study were of analytical grade. Male Brown Norway (BN, 200-250 g) and Lewis (200-250 g) rats were purchased from Vital River Laboratory (Beijing, China). All the animals were fasted for 12 h prior to the experiments. Animal experiments were approved by the Animal Care and Use Committee of Huazhong University of Science and Technology and performed in accordance with the experimental animal care guidelines of the Animal Experimentation Ethics Committee of Huazhong University of Science and Technology. 2.2 Preparation of PLGA-FK506-NPs 5
PLGA-FK506-NPs were prepared by the O/W emulsion solvent evaporation method (Fig. 1A) as previously reported with some modifications (Mistry et al., 2015). Briefly, FK506 (1 mg) and PLGA (16 mg) were dissolved in a mixture of ethyl acetate (0.25 mL) and dichloromethane (0.25 mL). This mixture was added to 2 mL of F-68 solution (1.25 % w/v) and was sonicated for 5 min with a probe sonicator (LC1000N, Ultrasonic processor, China) to generate an oil-in-water emulsion. The resultant emulsion was diluted to 10 mL with F-68 solution (0.5% w/v). The solvents were then removed using a rotary vacuum evaporator for approximately 15 min at 35℃. The resultant dispersion was centrifuged for 15 min at 4000 rpm using an Amicon Ultra-4 centrifugal filter (MWCO: 10 k, Millipore, USA), as reported in a previous study (Aryal et al., 2013; Azzi et al., 2010). After the aqueous dispersion was filtered, free FK506 was separated from the nanoparticles, and the residue was collected and lyophilized using trehalose (10% w/v) as cryoprotectant. DiR-labeled PLGA-NPs were prepared in the same method by adding 50 µL of DiR (1 mg/mL) to the initial organic mixture. 2.3 Characterization of PLGA-FK506-NPs 2.3.1 Size distribution, zeta potential and polydispersity The size distribution, zeta potential and polydispersity index (PDI) of the PLGA-FK506-NPs and DiR-labeled PLGA-NPs were determined by dynamic light scattering (DLS) using a zeta potential analyzer (ZetaPALS, Brookhaven Instruments, USA), with a scattering angle of 90° at 25℃. All measurements were repeated in triplicate.
6
2.3.2 Morphological characterization The morphology of the above PLGA-FK506-NPs was evaluated by transmission electron microscopy (TEM, Hitachi HT7700, Japan) and scanning electron microscopy (SEM, Hitachi SU8010, Japan). For TEM, a drop of the PLGA-FK506-NP dispersion was placed on a copper grid coated with carbon film and dried in air. The sample was negatively stained with 1% phosphotungstic acid and dried overnight at room temperature. For SEM, the freeze-dried PLGA-FK506-NPs were placed on tinfoil and coated with platinum via ion sputter (EM ACE 200, Leica, Germany) for 5 min. 2.3.3 FK506 entrapment and loading efficiency The entrapment efficiency (EE) and drug-loading efficiency (LE) were measured by high-performance liquid chromatography (HPLC) with a C18 column (4.6 mm × 250 mm, 5 µm) at 40 ℃. The mobile phase was a mixture of acetonitrile/0.1% phosphoric acid in water (70/30, v/v), and the flow rate was 1 mL/min. The FK506 was detected at a wavelength of 210 nm (Tung Thanh et al., 2018). EE and LE were calculated as follows: entrapment efficiency (%) = (weight of FK506 in nanoparticles/weight of FK506 added) × 100 and drug-loading efficiency (%) = (weight of FK506 in nanoparticles/weight of nanoparticles) × 100. 2.3.4 In-vitro drug release The in-vitro release tests were conducted using a dialysis method and were performed under the sink condition, as previously reported (Mistry et al., 2015; Xu et al., 2014). Briefly, 7
1 mL of PLGA-FK506-NP dispersion (equal to 1.0 mg of FK506) was added to a dialysis bag (MWCO: 8–1.4 kDa), and 1 mL of FK506 in ethanol (1 mg/mL) was used as control. The dialysis bag was submerged in a 50-mL tube that contained 40 mL of phosphate-buffered saline (PBS, pH = 7.4) and 0.5% (v/v) Tween-80 and was shaken at 100 rpm at 37 ℃. At predetermined time intervals (4, 8, 16, 24, 48, 72, 96, 120, 144, and 168 h), 1 mL of the release medium was withdrawn and fresh release medium was added. The released drug was determined by the HPLC method described above. 2.4 Biodistribution Lewis rats were used to study the biodistribution. The rats (n = 6) were intravenously administered 5 mg of DiR-labeled PLGA-NPs via the tail vein. Trafficking of the DiR-labeled PLGA-NPs was evaluated using a small animal imaging system (In-Vivo FX PRO, Bruker, USA) equipped with a 750 nm excitation filter and a 790 nm emission filter. The hearts, livers, spleens, lung, kidneys, mesenteric lymph nodes (MLNs), inguinal lymph nodes (ILNs) and axillary lymph nodes (ALNs) were harvested and analyzed for biodistribution 24 h post injection. 2.5 Pharmacokinetics study 2.5.1 In vivo experiments To
compare
the
pharmacokinetic
characteristics
between
free
FK506
and
PLGA-FK506-NPs, Lewis rats (n = 6) were divided into two groups and were administrated either free FK506 solution or PLGA-FK506-NP dispersion through the tail vein at a single 1 mg/kg dose of FK506. At predetermined time intervals (2, 4, 8, 12, 24, and 48 h), whole 8
blood (approximately 200 µL) was collected from the jugular vein into a tube with EDTA, as previously described (Wei et al., 2014). To determine the FK506 concentration in spleen and LNs, the rats (n = 4-6) were divided into two groups and administrated each formulation as mentioned above. The spleen, MLNs, ILNs and ALNs were then isolated and weighed 1 h after administration. All the samples were stored at -80 ℃ until assay. 2.5.2 Blood and tissue sample analysis The concentration of FK506 in whole blood and tissues was measured by an HPLC-mass spectroscopy (HPLC-MS) system (UltiMate 3000 RS and TSQ Quantum, Thermo Fisher Scientific Inc., USA) using a previously reported method with some modifications (Shin et al., 2010). Briefly, 100 µL of whole-blood sample and 300 µL of acetonitrile were mixed and vortexed for 5 min, followed by centrifugation at 15000 rpm for 10 min. Next, 10 µL of cyclosporin A (2 µg/mL) was added to the supernatant as an internal standard. The solution was measured on an HPLC-MS system equipped with a C18 column (2.1 mm × 100 mm, 1.9 µm) at 35℃. Methanol and 0.1% aqueous formic acid were used as the mobile phases. The flow rate was 0.4 mL/min, and the injection volume was 5 µL. To evaluate the concentration of FK506 in spleen and LNs, the tissue was homogenized for 2-3 min in 1.0 mL of dichloromethane. After centrifugation for 10 min at 14000 rpm, 100 µL of the organic solvent was transferred to a tube and dried under nitrogen. The residue was then dissolved in 150 µL of methanol and 10 µL of cyclosporin A (2 µg/mL), vortexed for 1 min, and injected onto the HPLC-MS system for analysis. Pharmacokinetic analysis was performed using the DAS2.0 program. 9
2.6 Toxicity assay The in vivo toxicity of PLGA-FK506-NPs was examined by measuring blood biochemistry indicators of liver and kidney function. A total of 18 Lewis rats (n = 6 per group) were intravenously injected with either 400 µL of PBS, or PBS containing PLGA-FK506, or free FK506. All rats were euthanized 24 h after injection, and their blood samples, hearts, livers, spleens, lungs, and kidneys were collected. The organs were fixed in formalin, embedded in paraffin and sectioned at 4 µm. The sections were stained with hematoxylin and eosin (H&E) and observed with an optical microscope (IX73, Olympus, Japan).
2.7 Preparation of BN-to-Lewis rat heterotopic cardiac transplantation model Heterotopic heart transplantation was performed using a microsurgical technique, as previously described (Liu et al., 2018). The rats were anesthetized with 3.5% isoflurane and maintained at 2%. The donor heart was harvested following the ligation of superior and inferior vena cava, and the dissection of the ascending aorta and pulmonary artery. The harvested donor heart was immediately immersed in cold saline solution until transplantation. Following the abdominal incision of the recipient rat, the abdominal aorta and the vena cava were isolated and clamped. The ascending aorta and pulmonary artery of the donor heart were sutured to the abdominal aorta and the vena cava of the recipient rat, respectively. After the transplanted heart began to beat again, the abdomen was closed. BN hearts were transplanted into Lewis recipients as allografts, and Lewis hearts were transplanted into Lewis recipients as isografts. The allograft rats were then randomized into three groups (4-6 10
rats per group). PBS, PLGA-FK506-NPs or free FK506 solution was injected intravenously via the tail vein at a dose of 1 mg/kg on postoperative day (POD) 1 to 5 (Fig. 1B). The isograft rats were used as the negative control. The survival of grafts was determined by daily palpation. The endpoint was defined as complete cessation of heart beats during a follow-up time of 28 days, and the grade of graft rejection was determined by histological examination. 2.8 Histological analysis Allografts were harvested on POD 7 or at the time of complete cessation of heart beats. The tissues were fixed in formalin, embedded in paraffin and sectioned at 4 µm. The sections were stained with hematoxylin and eosin to assess the grade of acute rejection according to the criteria of ISHLT (Stewart et al., 2005). T lymphocyte infiltration of the allografts was evaluated by immunohistochemical staining with anti-CD3 antibody (ab5690, Abcam, Cambridge,
UK). The
secretion
of
inflammatory
cytokines
was
evaluated
by
immunofluorescence with anti-IFN-γ (ab216642, Abcam) and anti-IL-2 antibody (ZI091710A, R&D). 2.9 Statistical analysis Statistical analysis was performed by Student’s t-test (α = 0.05) or one-way analysis of variance (ANOVA) using SPSS version 19.0 (IBM, Armonk, NY, USA). All data are presented as the mean value ± standard deviation (SD). P-values less than 0.05 are considered to be statistically significant. 3.Results and discussion 11
3.1 Characterization of PLGA-FK506-NPs and DiR-labeled PLGA-NPs The PLGA-FK506-NPs were prepared by the O/W emulsion solvent evaporation method with FK506 encapsulated into the core of the NPs. The morphology of the PLGA-FK506-NPs examined by SEM and TEM is shown in Fig. 2A and 2B, respectively. The PLGA-FK506-NPs were monodispersed and spherical in shape under SEM and TEM, with a narrow size distribution (PDI = 0.14 ± 0.01). These findings conformed well with the mean size of the NPs (110 ± 1.3 nm) measured by DLS, as shown in Fig. 2C. In addition, the zeta potential of the NPs, which predicts their stability, was -20.56 ± 3.65 mV. The mean diameter and zeta potential of the DiR-labeled PLGA-NPs were 117.6 ± 1.3 nm and -26.83 ± 2.56 mV, respectively. According to HPLC analysis, the FK506 entrapment and loading efficiencies of the PLGA-FK506-NPs were 94.46 ± 1.88% and 5.38 ± 0.24%, respectively, suggesting that most of the added FK506 was effectively encapsulated into the PLGA NPs. These results demonstrated that the PLGA-FK506-NPs were successfully fabricated for use in subsequent studies. It has been reported that NPs will passively be delivered to the LNs in a size-dependent manner and that NPs with a size of 20 to 200 nm are easily taken up by dendritic cells or macrophages and migrate to the LNs (Manolova et al., 2008). Furthermore, a negative zeta potential of NPs can not only influence the stability of the NPs but also contributes to the enhanced mononuclear phagocyte system (MPS) uptake (Reis et al., 2006), which may trigger NP uptake by macrophages, followed by drainage into the regional LNs and the spleen (Rao et al., 2010). Therefore, the prepared PLGA-FK506-NPs may have the advantage of delivering FK506 into the lymphatic system and improving the concentration of FK506 in this system to inhibit the activation and proliferation of T lymphocytes. 12
3.2 In vitro release The in vitro release of FK506 from PLGA-FK506-NPs was investigated by a dialysis method at preset times. As shown in Fig. 3, free FK506 solution exhibited a rapid release behavior; more than 90% of FK506 was released within 16 h, and almost 100% was released within 24 h. In contrast, the PLGA-FK506-NPs exhibited a nearly linear FK506 release profile; approximately 22.68 ± 2.65% of the FK506 was released in the initial 2 days at a relatively fast speed, and approximately 50.99 ± 1.87% was released within the following 5 days in a sustained manner. This drug release profile showed no distinct burst release behavior but rather a relatively smooth process, suggesting that FK506 was mainly released by diffusing from the NPs, while little was released from the surface of the NPs. The release process was sustained for more than 7 days, and approximately half of the residual drug might become further released with the degradation of the PLGA matrix. Generally, the FK506 release could be explained by a triphasic release pattern (Busatto et al., 2018; Hu et al., 2011b). These release profiles indicate that PLGA-FK506-NPs are promising carriers for controlling the release of FK506. 3.3 Biodistribution To study biodistribution, the major organs (hearts, livers, spleens, lung, kidneys) and lymph nodes (MLNs, ILNs, ALNs) were harvested 24 h post injection of the DiR-labeled PLGA-NPs and detected by a small animal imaging system. As shown in Fig. 4A, the NPs were mainly accumulated in liver and spleen, which was consistent with a previous report (Hu et al., 2011a). Except for liver and spleen, the fluorescence signal in LNs, especially in MLNs, was higher than that in other organs. Furthermore, the quantitative results shown in 13
Fig. 4B further confirmed that the fluorescence intensities of the spleen and MLNs were approximately 10.3 times and 2.6 times higher than that of the kidney. This phenomenon may have been caused by mononuclear phagocytic uptake and regional LN drainage. It is well known that the spleen and LNs, as the secondary lymphatic organs, are critical sites for the immune response and play an important role in regulating the immune response and allograft rejection (Burrell et al., 2011; Lakkis et al., 2000). Thus, if the DiR-labeled PLGA-NPs are substituted with PLGA-FK506-NPs, higher accumulation of FK506 in lymphatic organs and stronger immunosuppression can be expected. In a previous study, Azzi et al. demonstrated that longer graft survival can be achieved by active targeting of the LNs through modifying FK506-loaded microparticles so that they will bind high endothelial venules in LNs (Azzi et al., 2016). 3.4 Pharmacokinetic analysis After demonstrating that DiR-labeled PLGA-NPs can be passively targeted to the spleen and LNs, the pharmacokinetics of PLGA-FK506-NPs were further evaluated by measuring the concentration of FK506 in whole blood, LNs and spleen by using the HPLC/MS system at desired time points. The whole blood FK506 concentration-time curves of free FK506 and PLGA-FK506-NPs are shown in Fig. 5. The results indicated that PLGA-FK506-NPs declined more slowly and had a relatively higher FK506 concentration compared with free FK506. Furthermore, as shown in Table 1, PLGA-FK506-NPs showed a higher AUC (1.69-fold) and MRT (1.29-fold) compared with free FK506, with a significantly slower clearance (1.171 ± 0.138 L/h and 2.032 ± 0.362 L/h, respectively). The higher AUC might be due to the fact that the encapsulated FK506 could be protected from enzymatic degradation 14
in the liver (Li and Huang, 2008). The slower clearance may be attributed to the size of the PLGA-FK506-NPs, which is beneficial to avoid or reduce renal filtration (Duan and Li, 2013). In addition, a significantly longer elimination half-life (t1/2β) was observed for the PLGA-FK506-NP
group.
In
general,
the
pharmacokinetic
behaviors
of
the
PLGA-FK506-NPs were consistent with a pharmacokinetic study of albumin NPs (Gao et al., 2012). The FK506 concentrations in spleen and three different regional LNs were further measured and are shown in Fig. 6. For the FK506 concentration in the spleen, PLGA-FK506-NPs were 3.1-fold higher than free FK506. The spleen is the largest lymphatic organ for the proliferation and activation of T lymphocytes, and it plays an important role in allograft rejection (Chosa et al., 2007). Hence, the targeted delivery of FK506 to the spleen is conducive to generate higher efficacy. A similar situation was found for the FK506 concentration in MLNs, were the PLGA-FK506-NP concentration was 2.9-fold higher in comparison to free FK506, while the FK506 concentrations in both ILNs and ALNs did not show significant differences between free FK506 and PLGA-FK506-NPs. Since it has been reported that the concentration of T lymphocytes in mesenteric lymphatic fluid is 76-fold higher than that in blood (Fanous et al., 2007; Fischer et al., 1996; Yoshida et al., 2016), MLNs would be a suitable target for the delivery of immunosuppressing agents, and higher FK506 concentrations in MLNs should be able to improve overall therapeutic efficacy. There are two main possible targeting mechanisms of PLGA-FK506-NPs. It has been demonstrated that PLGA-FK506-NPs can be taken up by the mononuclear phagocyte system (MPS) (Mistry et al., 2015), and the MPS is mainly distributed in the liver, spleen 15
and lymph nodes, which provides innate conditions for the passive targeted delivery of drugs. Additionally, the small size of PLGA-FK506-NPs allows them to easily pass through the walls of lymphatic capillaries and enter the lymph nodes (Shin et al., 2010). In summary, it can be concluded that PLGA-FK506-NPs are a promising formulation for spleen and LN delivery. 3.5 Toxicity evaluation FK506 toxicity is an important factor causing morbidity and mortality following heart transplantation (Sikma et al., 2015). Herein, the short-term toxicity of PLGA-FK506-NPs was assessed by collecting blood samples and major organs for blood biochemistry and histology analysis. Aspartate aminotransferase (AST), alanine aminotransferase (ALT), blood urea nitrogen (BUN) and serum creatinine (CRE) were chosen as the biochemical indicators to assess the liver and kidney toxicity. As shown in Fig. 7A, a similar level of ALT, AST, BUN and CRE was detected in the PBS, free FK506 and PLGA-FK506-NP groups, suggesting that there was no statistic difference between the free FK506 and PLGA-FK506-NP groups when examining the serology for acute hepatorenal toxicity. Moreover, the histology analysis (shown in Fig. 7B) suggested no toxicity differences among the major organs. These results merely indicate the situation for short-term toxicity, and long-term toxicity analysis requires further intensive studies. Interestingly, Zhao et al. demonstrated that reduced nephrotoxicity has been achieved by reducing the accumulation of FK506 in the kidney (Zhao et al., 2015). The relatively low biodistribution of DiR-labeled PLGA-NPs in the kidney indicate the potential benign toxicity effect of the long-term use of this medication and supports the future use of PLGA-FK506-NPs in transplantation models. 16
3.6 In vivo immunosuppressive effects of PLGA-FK506-NPs To study the therapeutic efficacy of PLGA-FK506-NPs on cardiac allograft acute rejection, heterotopic cardiac transplantation was performed in rat (BN-to-Lewis). The survival curves of cardiac grafts are shown in Fig. 8A. The PLGA-FK506-NP treatment prolonged the survival time more than the free FK506 and PBS treatments. Moreover, treatment with PLGA-FK506-NPs prolonged allograft survival compared with the free FK506 group (mean survival time, 17.1 ± 2.0 versus 13.3 ± 1.7 days; P < 0.01). To investigate the effects of different groups on the development of acute rejection-related pathology, the acute rejection grade in each group was analyzed on POD 7 and is depicted in Fig. 8B and Fig. 8C. As expected, the isograft group, as negative control, exhibited grade 0R rejection, which represents almost no lymphocyte infiltration or myocyte damage. The PBS group exhibited grade 3R acute rejection, which is represented as massive lymphocyte infiltration, myocyte necrosis, scattered hemorrhage, and severe vasculopathy. The rats treated with PLGA-FK506-NPs exhibited only grades 1R or 2R acute rejection, with mild or moderate lymphocyte infiltration in most situations, and this group had more 1R grafts compared with the free FK506 group. T lymphocytes, which play a critical role in acute rejection, are associated with the degree of myocardial injury (Pietra et al., 2000). Thus, the extent of CD3+ T lymphocyte infiltration was further assessed in the myocardium using immunohistochemistry staining. As shown in Fig. 8D, the PLGA-FK506-NP group showed much less CD3+ T lymphocyte infiltration than the free FK506 group. A large number of CD3+ T lymphocytes were infiltrated in the PBS group, whereas there was almost no CD3+ T lymphocyte infiltration in the isograft group. These results demonstrated that 17
PLGA-FK506-NPs successfully alleviated acute rejection and prolonged the graft survival time, which might be attributed to the higher FK506 concentration in the spleen and MLNs compared with free FK506. 3.7 Secretion of cytokines in cardiac allograft In addition to T lymphocytes, cytokines secreted by T lymphocytes play an equivalently important role in the pathogenesis of cardiac acute rejection. IL-2, as an important inflammatory cytokine secreted by activated CD4+ T lymphocytes, is associated with T lymphocyte proliferation and with the differentiation of naïve T lymphocytes into effector T lymphocytes (de Waal Malefyt et al., 1993). IFN-γ, as a pro-inflammatory cytokine secreted mainly by T helper lymphocytes, can enhance cellular immunity mediated by T lymphocytes and aggravate acute rejection (Wang et al., 2003). Therefore, inhibiting the secretion of IL-2 and IFN-γ would significantly reduce the total number of T lymphocytes, the infiltration of effector T lymphocytes, the myocardium damage, and eventually alleviate acute rejection. To verify the effect of PLGA-FK506-NPs on the secretion of cytokines, both IL-2 and IFN-γ in the cardiac allografts were evaluated by immunofluorescence staining. As shown in Fig. 9 and Fig. 10, although both free FK506 and PLGA-FK506-NPs reduced the secretion of IL-2 and IFN-γ significantly compared to the PBS group, the PLGA-FK506-NPs group showed a better result than the free FK506 group. Taken together, these results demonstrate that treatment with PLGA-FK506-NPs effectively inhibits the secretion of these cytokines. 4. Conclusion In this study, PLGA-FK506-NPs were successfully prepared by the O/W emulsion solvent 18
evaporation method. The physicochemical characteristics (i.e., size, zeta potential, entrapment efficiency, and drug loading) and in vivo biodistribution suggested that it is a promising nanoparticulate formulation for passive spleen and LN delivery, and the drug release profiles showed a stable and linear release of FK506. Moreover, PLGA-FK506-NPs showed favorable pharmacokinetics, with increased AUC, MRT, and decreased CL; this treatment also delivered a significantly higher amount of FK506 into spleen and MLNs than that of free FK506 group. Most importantly, the PLGA-FK506-NPs alleviated the acute rejection and prolonged the allograft survival time of rat heterotopic cardiac transplantation, suggesting an improved therapeutic efficacy of FK506. In summary, PLGA-FK506-NPs would be a promising formulation for spleen and lymph node delivery that can be used for the treatment of cardiac allograft acute rejection or other organ transplantations. Authorship Contributions Participated in research design: Cheng Deng, Yihan Chen, Li Zhang, Qing Lv, Yali Yang, Qiaofeng Jin, Mingxing Xie Conducted experiment: Cheng Deng, Yihan Chen, Ya Wu, Huiling Li, Yu Wu, Zhenxing Sun, Bin Wang Performed data analysis: Cheng Deng, Yihan Chen, Li Zhang, Jing Wang, Yuman Li, Qiaofeng Jin, Mingxing Xie Wrote/contributed to the writing of the manuscript: Cheng Deng, Yihan Chen, Li Zhang, Ya Wu, Qiaofeng Jin, Mingxing Xie
19
Conflict of interest The authors report no conflicts of interest in this work. Acknowledgments We are grateful to this work was supported by the National Natural Science Foundation of China [grant numbers 81530056, 81727805,81471678, 81271582, 81671705, 81601540, 81771851, 81701716, 81801716, 81801715] and HUST Interdisciplinary Innovation Team [0118530300]. References Aryal, S., Hu, C.M., Fang, R.H., Dehaini, D., Carpenter, C., Zhang, D.E., Zhang, L., 2013. Erythrocyte membrane-cloaked polymeric nanoparticles for controlled drug loading and release. Nanomedicine (London, England) 8, 1271-1280. Azzi, J., Tang, L., Moore, R., Tong, R., El Haddad, N., Akiyoshi, T., Mfarrej, B., Yang, S., Jurewicz, M., Ichimura, T., Lindeman, N., Cheng, J., Abdi, R., 2010. Polylactide-cyclosporin A nanoparticles for targeted immunosuppression. FASEB journal : official publication of the Federation of American Societies for Experimental Biology 24, 3927-3938. Azzi, J., Yin, Q., Uehara, M., Ohori, S., Tang, L., Cai, K., Ichimura, T., McGrath, M., Maarouf, O., Kefaloyianni, E., Loughhead, S., Petr, J., Sun, Q., Kwon, M., Tullius, S., von Andrian, U.H., Cheng, J., Abdi, R., 2016. Targeted Delivery of Immunomodulators to Lymph Nodes. Cell reports 15, 1202-1213. Bahmani, B., Uehara, M., Jiang, L., Ordikhani, F., Banouni, N., Ichimura, T., Solhjou, Z., Furtmuller, G.J., Brandacher, G., Alvarez, D., von Andrian, U.H., Uchimura, K., Xu, Q., Vohra, I., Yilmam, O.A., Haik, Y., Azzi, J., Kasinath, V., Bromberg, J.S., McGrath, M.M., Abdi, R., 2018a. Targeted delivery of immune therapeutics to lymph nodes prolongs 20
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Fig. 1. Schematic illustration. (A) Preparation of PLGA-FK506-NPs. (B) Dosage regimen and schedule for measuring the survival time and histopathology assay after transplantation. Fig. 2. Characterization of PLGA-FK506-NPs. (A) Scanning electron microscopy (SEM) 26
images. (B) Transmission electron microscopy (TEM) images and (C) size distribution. Fig. 3. In vitro drug release profile of free FK506 and PLGA-FK506-NPs in PBS (pH 7.4). Data are presented as the mean ± SD (n = 3). Fig. 4. Biodistribution of DiR-labeled PLGA-NPs at 24 h after injection. (A) Ex vivo fluorescence image of major organs. (B) Quantitative analysis of nanoparticles in major organs and lymph nodes. Data are presented as the mean ± SD (n = 6). Fig. 5. Rat whole blood concentration-time profiles of FK506. Data are presented as the mean ± SD (n = 6). Fig. 6. Concentration of FK506 in spleen and lymph nodes at 1 h. Data are represented as the mean ± SD (n = 4-6). *** P < 0.001. Fig. 7. In vivo toxicity evaluation of PLGA-FK506-NPs. (A) Serum levels of AST, ALT, BUN, and CRE. (E) H&E staining of major organs. Scale bar = 20 μm. Fig. 8. Effects of PLGA-FK506-NPs on cardiac allografts. (A) Survival curves of the cardiac allografts in rats. (B) Grade of the allograft tissues evaluated on POD 7 (n = 4-5). (C) H&E staining of the tissue sections. Scale bar = 100 μm. (D) Immunohistochemistry staining with CD3+ T lymphocytes. Scale bar = 20 μm. Fig. 9. Immunofluorescence staining of IL-2 secretion in allografts. Scale bar = 50 μm. Fig. 10. Immunofluorescence staining of IFN- γ secretion in allografts. Scale bar = 50 μm.
27
Table 1. Pharmacokinetic parameters of FK506 after tail vein injection of free FK506 and PLGA-FK506-NPs in rats. Parameter
FK506
PLGA-FK506-NPs
t1/2α (h)
2.117 ± 0.667
4.548 ± 1.617*
t1/2β (h)
14.95 ± 2.243
47.007 ± 10.398**
AUC0-t (μg. h/L)
488.868 ± 72.647
830.604 ± 102.219**
MRT0-t (h)
9.708 ± 1.256
12.586 ± 1.662**
CL (L/h/kg)
2.032 ± 0.362
1.171 ± 0.138**
t1/2α, distribution half-life; t1/2β, elimination half-life; AUC0-t, area under plasma concentration versus time curve; MRT, mean residence time; CL, total clearance; * P < 0.05; ** P < 0.01
28
S0378-5173(19)30996-2 https://doi.org/10.1016/j.ijpharm.2019.118951 IJP 118951
To appear in:
International Journal of Pharmaceutics
Received Date: Revised Date: Accepted Date:
3 August 2019 3 December 2019 11 December 2019
Please cite this article as: C. Deng, Y. Chen, L. Zhang, Y. Wu, H. Li, Y. Wu, B. Wang, Z. Sun, Y. Li, Q. Lv, Y. Yang, J. Wang, Q. Jin, M. Xie, Delivery of FK506-loaded PLGA nanoparticles prolongs cardiac allograft survival, International Journal of Pharmaceutics (2019), doi: https://doi.org/10.1016/j.ijpharm.2019.118951
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© 2019 Published by Elsevier B.V.
Delivery of FK506-loaded PLGA nanoparticles prolongs cardiac allograft survival
Cheng Denga,b,1, Yihan Chena,b,1, Li Zhanga,b, Ya Wua,b, Huiling Lia,b, Yu Wua,b, Bin Wanga,b, Zhenxing Suna,b, Yuman Lia,b, Qing Lva,b, Yali Yanga,b, Jing Wanga,b, Qiaofeng Jina,b,*,[email protected], Mingxing Xiea,b,*,[email protected]
aDepartment
of Ultrasound, Union Hospital, Tongji Medical College, Huazhong University
of Science and Technology, Wuhan 430022, China bHubei
Province Key Laboratory of Molecular Imaging
*Corresponding
author.at: Department of Ultrasound, Union Hospital, Tongji Medical College,
Huazhong University of Science and Technology, 1277 Jiefang Avenue, Wuhan 430022, China 1 Equal
contributiors
Graphical abstract
Abstract In this study, FK506-loaded poly(lactide-co-glycolide) nanoparticles (PLGA-FK506-NPs) 1
were
developed
using
an
O/W
emulsion
solvent
evaporation
method.
The
PLGA-FK506-NPs were observed to be monodispersed and spherical by transmission and scanning electron microscopy. The mean size and zeta potential measured by dynamic light scattering were 110 ± 1.3 nm and -20.56 ± 3.65 mV, respectively. The FK506 entrapment and loading efficiency were 94.46 ± 1.88% and 5.38 ± 0.24%, respectively. Moreover, a pharmacokinetics study revealed that the PLGA-FK506-NPs behaved significantly different than free FK506 by exhibiting a higher area under curve (1.69-fold), higher mean residence time (1.29-fold), slower clearance and longer elimination half-life. Notably, the concentrations of FK506 in the spleen and mesenteric lymph nodes of the PLGA-FK506-NP group were 3.1-fold and 2.9-fold higher than those of the free FK506 group. Furthermore, the immunosuppressive efficacy was evaluated in a rat heterotopic heart transplantation model, and the results showed that PLGA-FK506-NP treatment could successfully alleviate acute rejection and prolong allograft survival compared with the free FK506 treatment (mean survival time, 17.1 ± 2.0 versus 13.3 ± 1.7 days). In conclusion, PLGA-FK506-NPs are a promising formulation for spleen and lymph node delivery and have potential use in the treatment of cardiac allograft acute rejection.
Keywords: FK506; PLGA; Nanoparticle; Heart transplantation; Acute rejection; Lymph node
2
1. Introduction Heart transplantation has become a standard treatment for thousands of patients with irreversible end-stage heart failure (Olymbios et al., 2018; Stehlik et al., 2018). However, cardiac allograft acute rejection remains one of the most important risk factors causing cardiac allograft loss within the first few months after heart transplantation (Stehlik et al., 2010). Fortunately, the incidence of acute rejection has been greatly reduced since the application of efficient immunosuppressive agents, such as FK506, which can inhibit T lymphocyte proliferation and activation efficiently through binding to FK506-binding protein 12 (FKBP12) to block the activation of calcineurin within the T lymphocytes (Schreiber and Crabtree, 1992). Although FK506 has shown a high efficiency, the long-term systemic administration of FK506 inevitably induces side-effects, including nephrotoxicity, neurotoxicity, hypertension and diabetogenic effects. In addition, complications such as opportunistic infections and malignancies often occur after nonspecific entire host immune system suppression following daily FK506 uptake (Lund et al., 2017; Stehlik et al., 2012). Furthermore, current FK506 formulations have various disadvantages including poor bioavailability, high pharmacokinetic variability and a narrow therapeutic window (5-15 ng/mL) (Sikma et al., 2015), which have greatly affected its therapeutic efficacy. Several recent studies have demonstrated that therapeutic efficacy is relevant to the FK506 concentration within the lymphocytes (Bahmany et al., 2019; Capron et al., 2016). Notably, a previous study reported that the effective concentration of FK506 in lymphocytes is only up to 0.26 ng/mL, which is 30 times lower than that in whole blood (Blanchet et al., 2006). Therefore, the targeted delivery of FK506 to the T lymphocytes would have immense 3
potential to promote therapeutic efficacy because T lymphocytes play a dominant role during acute rejection. It is well known that the proliferation and activation of T lymphocytes occurs primarily in the spleen and lymph nodes (LNs) (Lakkis et al., 2000). The targeted delivery of FK506 to the spleen and LNs would be an effective strategy to inhibit T lymphocyte activation.
Recently,
numerous
approaches
have
been
developed
to
deliver
immunosuppressive agents specifically to the lymphatic system (Azzi et al., 2016; Bahmani et al., 2018a; Bahmani et al., 2018b). Formulation modification is one of the most accessible approaches to promote biodistribution and to eventually achieve lymphatic system delivery. Nanoparticle (NP)-based drug delivery systems can deliver drugs to a targeted site, and they release their payload in a sustained manner (Bahmani et al., 2018a). Among various NPs, poly(lactide-co-glycolide) (PLGA) has been widely studied for its biocompatibility, biodegradability, non-toxicity and non-immunogenicity and has been designed to load various immunoregulation agents for the treatment of acute rejection in liver, corneal, and islet transplantation (Azzi et al., 2016; Miyamoto et al., 2004; Pham et al., 2018; Shirali et al., 2011; Tang et al., 2012; Wang et al., 2004; Wu et al., 2019; Xu et al., 2014). Particularly, FK506-loaded PLGA NPs have been reported to have an improved lymphatic-targeting capability in normal rats (Shin et al., 2010). However, their therapeutic efficacy remains to be verified in the transplantation models. In this study, a rat heterotopic heart transplantation model was established to verify the therapeutic efficacy of PLGA NPs loaded with FK506 (PLGA-FK506-NPs), which were prepared by the O/W emulsion solvent evaporation method. We characterized not only their physicochemical properties and the in vitro release patterns, but also the toxicity, 4
biodistribution and pharmacokinetics of PLGA-FK506-NPs. Finally, the therapeutic efficacy of PLGA-FK506-NPs for cardiac allograft was evaluated by measuring the survival rate of allografts and scored by the graft histologic section according to the criteria of the 2005 International Society for Heart and Lung Transplantation (ISHLT). PLGA-FK506-NPs were demonstrated to efficiently deliver FK506 to the spleen and LNs to promote the therapeutic efficacy. 2. Materials and methods 2.1 Materials and animals FK506 was purchased from MCE (New Jersey, USA). PLGA (lactide: glycolide ratio 50:50, molecular weight = 30,000-60,000) was obtained from Sigma-Aldrich (St. Louis, MO, USA).
Pluronic®
F-68
was
purchased
from
Solarbio
(Beijing,
China).
1,1 ′
-dioctadecyl-3,3,3′,3′-tetramethylindotricarbocyanine iodide (DiR) was purchased from AAT Bioquest (California, USA). All the other chemicals used in this study were of analytical grade. Male Brown Norway (BN, 200-250 g) and Lewis (200-250 g) rats were purchased from Vital River Laboratory (Beijing, China). All the animals were fasted for 12 h prior to the experiments. Animal experiments were approved by the Animal Care and Use Committee of Huazhong University of Science and Technology and performed in accordance with the experimental animal care guidelines of the Animal Experimentation Ethics Committee of Huazhong University of Science and Technology. 2.2 Preparation of PLGA-FK506-NPs 5
PLGA-FK506-NPs were prepared by the O/W emulsion solvent evaporation method (Fig. 1A) as previously reported with some modifications (Mistry et al., 2015). Briefly, FK506 (1 mg) and PLGA (16 mg) were dissolved in a mixture of ethyl acetate (0.25 mL) and dichloromethane (0.25 mL). This mixture was added to 2 mL of F-68 solution (1.25 % w/v) and was sonicated for 5 min with a probe sonicator (LC1000N, Ultrasonic processor, China) to generate an oil-in-water emulsion. The resultant emulsion was diluted to 10 mL with F-68 solution (0.5% w/v). The solvents were then removed using a rotary vacuum evaporator for approximately 15 min at 35℃. The resultant dispersion was centrifuged for 15 min at 4000 rpm using an Amicon Ultra-4 centrifugal filter (MWCO: 10 k, Millipore, USA), as reported in a previous study (Aryal et al., 2013; Azzi et al., 2010). After the aqueous dispersion was filtered, free FK506 was separated from the nanoparticles, and the residue was collected and lyophilized using trehalose (10% w/v) as cryoprotectant. DiR-labeled PLGA-NPs were prepared in the same method by adding 50 µL of DiR (1 mg/mL) to the initial organic mixture. 2.3 Characterization of PLGA-FK506-NPs 2.3.1 Size distribution, zeta potential and polydispersity The size distribution, zeta potential and polydispersity index (PDI) of the PLGA-FK506-NPs and DiR-labeled PLGA-NPs were determined by dynamic light scattering (DLS) using a zeta potential analyzer (ZetaPALS, Brookhaven Instruments, USA), with a scattering angle of 90° at 25℃. All measurements were repeated in triplicate.
6
2.3.2 Morphological characterization The morphology of the above PLGA-FK506-NPs was evaluated by transmission electron microscopy (TEM, Hitachi HT7700, Japan) and scanning electron microscopy (SEM, Hitachi SU8010, Japan). For TEM, a drop of the PLGA-FK506-NP dispersion was placed on a copper grid coated with carbon film and dried in air. The sample was negatively stained with 1% phosphotungstic acid and dried overnight at room temperature. For SEM, the freeze-dried PLGA-FK506-NPs were placed on tinfoil and coated with platinum via ion sputter (EM ACE 200, Leica, Germany) for 5 min. 2.3.3 FK506 entrapment and loading efficiency The entrapment efficiency (EE) and drug-loading efficiency (LE) were measured by high-performance liquid chromatography (HPLC) with a C18 column (4.6 mm × 250 mm, 5 µm) at 40 ℃. The mobile phase was a mixture of acetonitrile/0.1% phosphoric acid in water (70/30, v/v), and the flow rate was 1 mL/min. The FK506 was detected at a wavelength of 210 nm (Tung Thanh et al., 2018). EE and LE were calculated as follows: entrapment efficiency (%) = (weight of FK506 in nanoparticles/weight of FK506 added) × 100 and drug-loading efficiency (%) = (weight of FK506 in nanoparticles/weight of nanoparticles) × 100. 2.3.4 In-vitro drug release The in-vitro release tests were conducted using a dialysis method and were performed under the sink condition, as previously reported (Mistry et al., 2015; Xu et al., 2014). Briefly, 7
1 mL of PLGA-FK506-NP dispersion (equal to 1.0 mg of FK506) was added to a dialysis bag (MWCO: 8–1.4 kDa), and 1 mL of FK506 in ethanol (1 mg/mL) was used as control. The dialysis bag was submerged in a 50-mL tube that contained 40 mL of phosphate-buffered saline (PBS, pH = 7.4) and 0.5% (v/v) Tween-80 and was shaken at 100 rpm at 37 ℃. At predetermined time intervals (4, 8, 16, 24, 48, 72, 96, 120, 144, and 168 h), 1 mL of the release medium was withdrawn and fresh release medium was added. The released drug was determined by the HPLC method described above. 2.4 Biodistribution Lewis rats were used to study the biodistribution. The rats (n = 6) were intravenously administered 5 mg of DiR-labeled PLGA-NPs via the tail vein. Trafficking of the DiR-labeled PLGA-NPs was evaluated using a small animal imaging system (In-Vivo FX PRO, Bruker, USA) equipped with a 750 nm excitation filter and a 790 nm emission filter. The hearts, livers, spleens, lung, kidneys, mesenteric lymph nodes (MLNs), inguinal lymph nodes (ILNs) and axillary lymph nodes (ALNs) were harvested and analyzed for biodistribution 24 h post injection. 2.5 Pharmacokinetics study 2.5.1 In vivo experiments To
compare
the
pharmacokinetic
characteristics
between
free
FK506
and
PLGA-FK506-NPs, Lewis rats (n = 6) were divided into two groups and were administrated either free FK506 solution or PLGA-FK506-NP dispersion through the tail vein at a single 1 mg/kg dose of FK506. At predetermined time intervals (2, 4, 8, 12, 24, and 48 h), whole 8
blood (approximately 200 µL) was collected from the jugular vein into a tube with EDTA, as previously described (Wei et al., 2014). To determine the FK506 concentration in spleen and LNs, the rats (n = 4-6) were divided into two groups and administrated each formulation as mentioned above. The spleen, MLNs, ILNs and ALNs were then isolated and weighed 1 h after administration. All the samples were stored at -80 ℃ until assay. 2.5.2 Blood and tissue sample analysis The concentration of FK506 in whole blood and tissues was measured by an HPLC-mass spectroscopy (HPLC-MS) system (UltiMate 3000 RS and TSQ Quantum, Thermo Fisher Scientific Inc., USA) using a previously reported method with some modifications (Shin et al., 2010). Briefly, 100 µL of whole-blood sample and 300 µL of acetonitrile were mixed and vortexed for 5 min, followed by centrifugation at 15000 rpm for 10 min. Next, 10 µL of cyclosporin A (2 µg/mL) was added to the supernatant as an internal standard. The solution was measured on an HPLC-MS system equipped with a C18 column (2.1 mm × 100 mm, 1.9 µm) at 35℃. Methanol and 0.1% aqueous formic acid were used as the mobile phases. The flow rate was 0.4 mL/min, and the injection volume was 5 µL. To evaluate the concentration of FK506 in spleen and LNs, the tissue was homogenized for 2-3 min in 1.0 mL of dichloromethane. After centrifugation for 10 min at 14000 rpm, 100 µL of the organic solvent was transferred to a tube and dried under nitrogen. The residue was then dissolved in 150 µL of methanol and 10 µL of cyclosporin A (2 µg/mL), vortexed for 1 min, and injected onto the HPLC-MS system for analysis. Pharmacokinetic analysis was performed using the DAS2.0 program. 9
2.6 Toxicity assay The in vivo toxicity of PLGA-FK506-NPs was examined by measuring blood biochemistry indicators of liver and kidney function. A total of 18 Lewis rats (n = 6 per group) were intravenously injected with either 400 µL of PBS, or PBS containing PLGA-FK506, or free FK506. All rats were euthanized 24 h after injection, and their blood samples, hearts, livers, spleens, lungs, and kidneys were collected. The organs were fixed in formalin, embedded in paraffin and sectioned at 4 µm. The sections were stained with hematoxylin and eosin (H&E) and observed with an optical microscope (IX73, Olympus, Japan).
2.7 Preparation of BN-to-Lewis rat heterotopic cardiac transplantation model Heterotopic heart transplantation was performed using a microsurgical technique, as previously described (Liu et al., 2018). The rats were anesthetized with 3.5% isoflurane and maintained at 2%. The donor heart was harvested following the ligation of superior and inferior vena cava, and the dissection of the ascending aorta and pulmonary artery. The harvested donor heart was immediately immersed in cold saline solution until transplantation. Following the abdominal incision of the recipient rat, the abdominal aorta and the vena cava were isolated and clamped. The ascending aorta and pulmonary artery of the donor heart were sutured to the abdominal aorta and the vena cava of the recipient rat, respectively. After the transplanted heart began to beat again, the abdomen was closed. BN hearts were transplanted into Lewis recipients as allografts, and Lewis hearts were transplanted into Lewis recipients as isografts. The allograft rats were then randomized into three groups (4-6 10
rats per group). PBS, PLGA-FK506-NPs or free FK506 solution was injected intravenously via the tail vein at a dose of 1 mg/kg on postoperative day (POD) 1 to 5 (Fig. 1B). The isograft rats were used as the negative control. The survival of grafts was determined by daily palpation. The endpoint was defined as complete cessation of heart beats during a follow-up time of 28 days, and the grade of graft rejection was determined by histological examination. 2.8 Histological analysis Allografts were harvested on POD 7 or at the time of complete cessation of heart beats. The tissues were fixed in formalin, embedded in paraffin and sectioned at 4 µm. The sections were stained with hematoxylin and eosin to assess the grade of acute rejection according to the criteria of ISHLT (Stewart et al., 2005). T lymphocyte infiltration of the allografts was evaluated by immunohistochemical staining with anti-CD3 antibody (ab5690, Abcam, Cambridge,
UK). The
secretion
of
inflammatory
cytokines
was
evaluated
by
immunofluorescence with anti-IFN-γ (ab216642, Abcam) and anti-IL-2 antibody (ZI091710A, R&D). 2.9 Statistical analysis Statistical analysis was performed by Student’s t-test (α = 0.05) or one-way analysis of variance (ANOVA) using SPSS version 19.0 (IBM, Armonk, NY, USA). All data are presented as the mean value ± standard deviation (SD). P-values less than 0.05 are considered to be statistically significant. 3.Results and discussion 11
3.1 Characterization of PLGA-FK506-NPs and DiR-labeled PLGA-NPs The PLGA-FK506-NPs were prepared by the O/W emulsion solvent evaporation method with FK506 encapsulated into the core of the NPs. The morphology of the PLGA-FK506-NPs examined by SEM and TEM is shown in Fig. 2A and 2B, respectively. The PLGA-FK506-NPs were monodispersed and spherical in shape under SEM and TEM, with a narrow size distribution (PDI = 0.14 ± 0.01). These findings conformed well with the mean size of the NPs (110 ± 1.3 nm) measured by DLS, as shown in Fig. 2C. In addition, the zeta potential of the NPs, which predicts their stability, was -20.56 ± 3.65 mV. The mean diameter and zeta potential of the DiR-labeled PLGA-NPs were 117.6 ± 1.3 nm and -26.83 ± 2.56 mV, respectively. According to HPLC analysis, the FK506 entrapment and loading efficiencies of the PLGA-FK506-NPs were 94.46 ± 1.88% and 5.38 ± 0.24%, respectively, suggesting that most of the added FK506 was effectively encapsulated into the PLGA NPs. These results demonstrated that the PLGA-FK506-NPs were successfully fabricated for use in subsequent studies. It has been reported that NPs will passively be delivered to the LNs in a size-dependent manner and that NPs with a size of 20 to 200 nm are easily taken up by dendritic cells or macrophages and migrate to the LNs (Manolova et al., 2008). Furthermore, a negative zeta potential of NPs can not only influence the stability of the NPs but also contributes to the enhanced mononuclear phagocyte system (MPS) uptake (Reis et al., 2006), which may trigger NP uptake by macrophages, followed by drainage into the regional LNs and the spleen (Rao et al., 2010). Therefore, the prepared PLGA-FK506-NPs may have the advantage of delivering FK506 into the lymphatic system and improving the concentration of FK506 in this system to inhibit the activation and proliferation of T lymphocytes. 12
3.2 In vitro release The in vitro release of FK506 from PLGA-FK506-NPs was investigated by a dialysis method at preset times. As shown in Fig. 3, free FK506 solution exhibited a rapid release behavior; more than 90% of FK506 was released within 16 h, and almost 100% was released within 24 h. In contrast, the PLGA-FK506-NPs exhibited a nearly linear FK506 release profile; approximately 22.68 ± 2.65% of the FK506 was released in the initial 2 days at a relatively fast speed, and approximately 50.99 ± 1.87% was released within the following 5 days in a sustained manner. This drug release profile showed no distinct burst release behavior but rather a relatively smooth process, suggesting that FK506 was mainly released by diffusing from the NPs, while little was released from the surface of the NPs. The release process was sustained for more than 7 days, and approximately half of the residual drug might become further released with the degradation of the PLGA matrix. Generally, the FK506 release could be explained by a triphasic release pattern (Busatto et al., 2018; Hu et al., 2011b). These release profiles indicate that PLGA-FK506-NPs are promising carriers for controlling the release of FK506. 3.3 Biodistribution To study biodistribution, the major organs (hearts, livers, spleens, lung, kidneys) and lymph nodes (MLNs, ILNs, ALNs) were harvested 24 h post injection of the DiR-labeled PLGA-NPs and detected by a small animal imaging system. As shown in Fig. 4A, the NPs were mainly accumulated in liver and spleen, which was consistent with a previous report (Hu et al., 2011a). Except for liver and spleen, the fluorescence signal in LNs, especially in MLNs, was higher than that in other organs. Furthermore, the quantitative results shown in 13
Fig. 4B further confirmed that the fluorescence intensities of the spleen and MLNs were approximately 10.3 times and 2.6 times higher than that of the kidney. This phenomenon may have been caused by mononuclear phagocytic uptake and regional LN drainage. It is well known that the spleen and LNs, as the secondary lymphatic organs, are critical sites for the immune response and play an important role in regulating the immune response and allograft rejection (Burrell et al., 2011; Lakkis et al., 2000). Thus, if the DiR-labeled PLGA-NPs are substituted with PLGA-FK506-NPs, higher accumulation of FK506 in lymphatic organs and stronger immunosuppression can be expected. In a previous study, Azzi et al. demonstrated that longer graft survival can be achieved by active targeting of the LNs through modifying FK506-loaded microparticles so that they will bind high endothelial venules in LNs (Azzi et al., 2016). 3.4 Pharmacokinetic analysis After demonstrating that DiR-labeled PLGA-NPs can be passively targeted to the spleen and LNs, the pharmacokinetics of PLGA-FK506-NPs were further evaluated by measuring the concentration of FK506 in whole blood, LNs and spleen by using the HPLC/MS system at desired time points. The whole blood FK506 concentration-time curves of free FK506 and PLGA-FK506-NPs are shown in Fig. 5. The results indicated that PLGA-FK506-NPs declined more slowly and had a relatively higher FK506 concentration compared with free FK506. Furthermore, as shown in Table 1, PLGA-FK506-NPs showed a higher AUC (1.69-fold) and MRT (1.29-fold) compared with free FK506, with a significantly slower clearance (1.171 ± 0.138 L/h and 2.032 ± 0.362 L/h, respectively). The higher AUC might be due to the fact that the encapsulated FK506 could be protected from enzymatic degradation 14
in the liver (Li and Huang, 2008). The slower clearance may be attributed to the size of the PLGA-FK506-NPs, which is beneficial to avoid or reduce renal filtration (Duan and Li, 2013). In addition, a significantly longer elimination half-life (t1/2β) was observed for the PLGA-FK506-NP
group.
In
general,
the
pharmacokinetic
behaviors
of
the
PLGA-FK506-NPs were consistent with a pharmacokinetic study of albumin NPs (Gao et al., 2012). The FK506 concentrations in spleen and three different regional LNs were further measured and are shown in Fig. 6. For the FK506 concentration in the spleen, PLGA-FK506-NPs were 3.1-fold higher than free FK506. The spleen is the largest lymphatic organ for the proliferation and activation of T lymphocytes, and it plays an important role in allograft rejection (Chosa et al., 2007). Hence, the targeted delivery of FK506 to the spleen is conducive to generate higher efficacy. A similar situation was found for the FK506 concentration in MLNs, were the PLGA-FK506-NP concentration was 2.9-fold higher in comparison to free FK506, while the FK506 concentrations in both ILNs and ALNs did not show significant differences between free FK506 and PLGA-FK506-NPs. Since it has been reported that the concentration of T lymphocytes in mesenteric lymphatic fluid is 76-fold higher than that in blood (Fanous et al., 2007; Fischer et al., 1996; Yoshida et al., 2016), MLNs would be a suitable target for the delivery of immunosuppressing agents, and higher FK506 concentrations in MLNs should be able to improve overall therapeutic efficacy. There are two main possible targeting mechanisms of PLGA-FK506-NPs. It has been demonstrated that PLGA-FK506-NPs can be taken up by the mononuclear phagocyte system (MPS) (Mistry et al., 2015), and the MPS is mainly distributed in the liver, spleen 15
and lymph nodes, which provides innate conditions for the passive targeted delivery of drugs. Additionally, the small size of PLGA-FK506-NPs allows them to easily pass through the walls of lymphatic capillaries and enter the lymph nodes (Shin et al., 2010). In summary, it can be concluded that PLGA-FK506-NPs are a promising formulation for spleen and LN delivery. 3.5 Toxicity evaluation FK506 toxicity is an important factor causing morbidity and mortality following heart transplantation (Sikma et al., 2015). Herein, the short-term toxicity of PLGA-FK506-NPs was assessed by collecting blood samples and major organs for blood biochemistry and histology analysis. Aspartate aminotransferase (AST), alanine aminotransferase (ALT), blood urea nitrogen (BUN) and serum creatinine (CRE) were chosen as the biochemical indicators to assess the liver and kidney toxicity. As shown in Fig. 7A, a similar level of ALT, AST, BUN and CRE was detected in the PBS, free FK506 and PLGA-FK506-NP groups, suggesting that there was no statistic difference between the free FK506 and PLGA-FK506-NP groups when examining the serology for acute hepatorenal toxicity. Moreover, the histology analysis (shown in Fig. 7B) suggested no toxicity differences among the major organs. These results merely indicate the situation for short-term toxicity, and long-term toxicity analysis requires further intensive studies. Interestingly, Zhao et al. demonstrated that reduced nephrotoxicity has been achieved by reducing the accumulation of FK506 in the kidney (Zhao et al., 2015). The relatively low biodistribution of DiR-labeled PLGA-NPs in the kidney indicate the potential benign toxicity effect of the long-term use of this medication and supports the future use of PLGA-FK506-NPs in transplantation models. 16
3.6 In vivo immunosuppressive effects of PLGA-FK506-NPs To study the therapeutic efficacy of PLGA-FK506-NPs on cardiac allograft acute rejection, heterotopic cardiac transplantation was performed in rat (BN-to-Lewis). The survival curves of cardiac grafts are shown in Fig. 8A. The PLGA-FK506-NP treatment prolonged the survival time more than the free FK506 and PBS treatments. Moreover, treatment with PLGA-FK506-NPs prolonged allograft survival compared with the free FK506 group (mean survival time, 17.1 ± 2.0 versus 13.3 ± 1.7 days; P < 0.01). To investigate the effects of different groups on the development of acute rejection-related pathology, the acute rejection grade in each group was analyzed on POD 7 and is depicted in Fig. 8B and Fig. 8C. As expected, the isograft group, as negative control, exhibited grade 0R rejection, which represents almost no lymphocyte infiltration or myocyte damage. The PBS group exhibited grade 3R acute rejection, which is represented as massive lymphocyte infiltration, myocyte necrosis, scattered hemorrhage, and severe vasculopathy. The rats treated with PLGA-FK506-NPs exhibited only grades 1R or 2R acute rejection, with mild or moderate lymphocyte infiltration in most situations, and this group had more 1R grafts compared with the free FK506 group. T lymphocytes, which play a critical role in acute rejection, are associated with the degree of myocardial injury (Pietra et al., 2000). Thus, the extent of CD3+ T lymphocyte infiltration was further assessed in the myocardium using immunohistochemistry staining. As shown in Fig. 8D, the PLGA-FK506-NP group showed much less CD3+ T lymphocyte infiltration than the free FK506 group. A large number of CD3+ T lymphocytes were infiltrated in the PBS group, whereas there was almost no CD3+ T lymphocyte infiltration in the isograft group. These results demonstrated that 17
PLGA-FK506-NPs successfully alleviated acute rejection and prolonged the graft survival time, which might be attributed to the higher FK506 concentration in the spleen and MLNs compared with free FK506. 3.7 Secretion of cytokines in cardiac allograft In addition to T lymphocytes, cytokines secreted by T lymphocytes play an equivalently important role in the pathogenesis of cardiac acute rejection. IL-2, as an important inflammatory cytokine secreted by activated CD4+ T lymphocytes, is associated with T lymphocyte proliferation and with the differentiation of naïve T lymphocytes into effector T lymphocytes (de Waal Malefyt et al., 1993). IFN-γ, as a pro-inflammatory cytokine secreted mainly by T helper lymphocytes, can enhance cellular immunity mediated by T lymphocytes and aggravate acute rejection (Wang et al., 2003). Therefore, inhibiting the secretion of IL-2 and IFN-γ would significantly reduce the total number of T lymphocytes, the infiltration of effector T lymphocytes, the myocardium damage, and eventually alleviate acute rejection. To verify the effect of PLGA-FK506-NPs on the secretion of cytokines, both IL-2 and IFN-γ in the cardiac allografts were evaluated by immunofluorescence staining. As shown in Fig. 9 and Fig. 10, although both free FK506 and PLGA-FK506-NPs reduced the secretion of IL-2 and IFN-γ significantly compared to the PBS group, the PLGA-FK506-NPs group showed a better result than the free FK506 group. Taken together, these results demonstrate that treatment with PLGA-FK506-NPs effectively inhibits the secretion of these cytokines. 4. Conclusion In this study, PLGA-FK506-NPs were successfully prepared by the O/W emulsion solvent 18
evaporation method. The physicochemical characteristics (i.e., size, zeta potential, entrapment efficiency, and drug loading) and in vivo biodistribution suggested that it is a promising nanoparticulate formulation for passive spleen and LN delivery, and the drug release profiles showed a stable and linear release of FK506. Moreover, PLGA-FK506-NPs showed favorable pharmacokinetics, with increased AUC, MRT, and decreased CL; this treatment also delivered a significantly higher amount of FK506 into spleen and MLNs than that of free FK506 group. Most importantly, the PLGA-FK506-NPs alleviated the acute rejection and prolonged the allograft survival time of rat heterotopic cardiac transplantation, suggesting an improved therapeutic efficacy of FK506. In summary, PLGA-FK506-NPs would be a promising formulation for spleen and lymph node delivery that can be used for the treatment of cardiac allograft acute rejection or other organ transplantations. Authorship Contributions Participated in research design: Cheng Deng, Yihan Chen, Li Zhang, Qing Lv, Yali Yang, Qiaofeng Jin, Mingxing Xie Conducted experiment: Cheng Deng, Yihan Chen, Ya Wu, Huiling Li, Yu Wu, Zhenxing Sun, Bin Wang Performed data analysis: Cheng Deng, Yihan Chen, Li Zhang, Jing Wang, Yuman Li, Qiaofeng Jin, Mingxing Xie Wrote/contributed to the writing of the manuscript: Cheng Deng, Yihan Chen, Li Zhang, Ya Wu, Qiaofeng Jin, Mingxing Xie
19
Conflict of interest The authors report no conflicts of interest in this work. Acknowledgments We are grateful to this work was supported by the National Natural Science Foundation of China [grant numbers 81530056, 81727805,81471678, 81271582, 81671705, 81601540, 81771851, 81701716, 81801716, 81801715] and HUST Interdisciplinary Innovation Team [0118530300]. References Aryal, S., Hu, C.M., Fang, R.H., Dehaini, D., Carpenter, C., Zhang, D.E., Zhang, L., 2013. Erythrocyte membrane-cloaked polymeric nanoparticles for controlled drug loading and release. Nanomedicine (London, England) 8, 1271-1280. Azzi, J., Tang, L., Moore, R., Tong, R., El Haddad, N., Akiyoshi, T., Mfarrej, B., Yang, S., Jurewicz, M., Ichimura, T., Lindeman, N., Cheng, J., Abdi, R., 2010. Polylactide-cyclosporin A nanoparticles for targeted immunosuppression. FASEB journal : official publication of the Federation of American Societies for Experimental Biology 24, 3927-3938. Azzi, J., Yin, Q., Uehara, M., Ohori, S., Tang, L., Cai, K., Ichimura, T., McGrath, M., Maarouf, O., Kefaloyianni, E., Loughhead, S., Petr, J., Sun, Q., Kwon, M., Tullius, S., von Andrian, U.H., Cheng, J., Abdi, R., 2016. Targeted Delivery of Immunomodulators to Lymph Nodes. Cell reports 15, 1202-1213. Bahmani, B., Uehara, M., Jiang, L., Ordikhani, F., Banouni, N., Ichimura, T., Solhjou, Z., Furtmuller, G.J., Brandacher, G., Alvarez, D., von Andrian, U.H., Uchimura, K., Xu, Q., Vohra, I., Yilmam, O.A., Haik, Y., Azzi, J., Kasinath, V., Bromberg, J.S., McGrath, M.M., Abdi, R., 2018a. Targeted delivery of immune therapeutics to lymph nodes prolongs 20
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Fig. 1. Schematic illustration. (A) Preparation of PLGA-FK506-NPs. (B) Dosage regimen and schedule for measuring the survival time and histopathology assay after transplantation. Fig. 2. Characterization of PLGA-FK506-NPs. (A) Scanning electron microscopy (SEM) 26
images. (B) Transmission electron microscopy (TEM) images and (C) size distribution. Fig. 3. In vitro drug release profile of free FK506 and PLGA-FK506-NPs in PBS (pH 7.4). Data are presented as the mean ± SD (n = 3). Fig. 4. Biodistribution of DiR-labeled PLGA-NPs at 24 h after injection. (A) Ex vivo fluorescence image of major organs. (B) Quantitative analysis of nanoparticles in major organs and lymph nodes. Data are presented as the mean ± SD (n = 6). Fig. 5. Rat whole blood concentration-time profiles of FK506. Data are presented as the mean ± SD (n = 6). Fig. 6. Concentration of FK506 in spleen and lymph nodes at 1 h. Data are represented as the mean ± SD (n = 4-6). *** P < 0.001. Fig. 7. In vivo toxicity evaluation of PLGA-FK506-NPs. (A) Serum levels of AST, ALT, BUN, and CRE. (E) H&E staining of major organs. Scale bar = 20 μm. Fig. 8. Effects of PLGA-FK506-NPs on cardiac allografts. (A) Survival curves of the cardiac allografts in rats. (B) Grade of the allograft tissues evaluated on POD 7 (n = 4-5). (C) H&E staining of the tissue sections. Scale bar = 100 μm. (D) Immunohistochemistry staining with CD3+ T lymphocytes. Scale bar = 20 μm. Fig. 9. Immunofluorescence staining of IL-2 secretion in allografts. Scale bar = 50 μm. Fig. 10. Immunofluorescence staining of IFN- γ secretion in allografts. Scale bar = 50 μm.
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Table 1. Pharmacokinetic parameters of FK506 after tail vein injection of free FK506 and PLGA-FK506-NPs in rats. Parameter
FK506
PLGA-FK506-NPs
t1/2α (h)
2.117 ± 0.667
4.548 ± 1.617*
t1/2β (h)
14.95 ± 2.243
47.007 ± 10.398**
AUC0-t (μg. h/L)
488.868 ± 72.647
830.604 ± 102.219**
MRT0-t (h)
9.708 ± 1.256
12.586 ± 1.662**
CL (L/h/kg)
2.032 ± 0.362
1.171 ± 0.138**
t1/2α, distribution half-life; t1/2β, elimination half-life; AUC0-t, area under plasma concentration versus time curve; MRT, mean residence time; CL, total clearance; * P < 0.05; ** P < 0.01
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