Folate Receptor-Mediated Renal-Targeting Nanoplatform for the

Subscriber access provided by UNIV AUTONOMA DE COAHUILA UADEC

Controlled Release and Delivery Systems

Folate Receptor-mediated Renal-targeting Nanoplatform for the Specific Delivery of Triptolide to Treat Renal Ischemia/Reperfusion Injury Caili Huang, Tao Zeng, Jianwen Li, Lishan Tan, Xiulong Deng, Yanchao Pan, Qi Chen, Aiqing Li, and Jianqiang Hu ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.9b00119 • Publication Date (Web): 13 May 2019 Downloaded from http://pubs.acs.org on May 18, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 46 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Biomaterials Science & Engineering

Folate

Receptor-mediated

Renal-targeting

Nanoplatform for the Specific Delivery of Triptolide to Treat Renal Ischemia/Reperfusion Injury Caili Huang,‡a Tao Zeng,‡b Jiawen Li,‡b Lishan Tan,b Xiulong Deng,a Yanchao Pan,a Qi Chen,a Aiqing Li,*b and Jianqiang Hu*a a

Nanobiological medicine Center, Key Laboratory of Fuel Cell Technology of Guangdong

Province, Department of Chemistry, School of Chemistry and Chemical Engineering, South China University of Technology, Guangzhou, 510640, China. b

State Key Laboratory of Organ Failure Research, Nanfang Hospital, Southern Medical

University, Guangzhou, 510515, China. ‡

Co-first author.

*Corresponding author: Jianqiang Hu, PhD (Email: [email protected]) Aiqing Li, PhD ([email protected]) KEYWORDS Triplitode, folate, nanoparticles, kidney targeting, renal ischemia/reperfusion injury.

ACS Paragon Plus Environment

1

ACS Biomaterials Science & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 46

ABSTRACT Triptolide (TP) has been widely used in clinical medicine, however it is in a dilemma due to its toxicity and non-specificity. Here, we reported a biocompability and high-efficiency renaltargeting nanoplatform for renal ischemia/reperfusion injury (IRI) therapy, in which the toxic drug of TP was encapsulated into folate (FA)-modified Pluronic F127/P123 nanoparticles (FPNPs). The TP-loaded FPNPs (TP-FPNPs) had good stability and could effectively reduce the cytotoxicity of TP. Compared with the Pluronic nanoparticles (PNPs) group, cellular uptake ability of FPNPs significantly improved because of folate receptor-mediated endocytosis effect. Ex vivo organ imaging and pharmacokinetic results indicated that FPNPs possessed high kidney selectivity and long retention time. The therapeutic effect of TP-FPNPs on renal IRI was more superior to that of free TP, such as lower acute tubular injury index (2.9-fold), renal function indexes of serum creatinine (4.3-fold), urea nitrogen (2.0-fold) and western blotting (2.4-fold). Systemic toxicity assay suggested that TP-FPNPs had much lower nephrotoxicity, hepatotoxicity and genital system toxicity than free TP. Thus, renal-targeting FPNPs will be a potential delivery platform of hydrophobic drugs for treatment of renal diseases.

INTRODUCTION Acute kidney injury (AKI) has attracted extensive attention due to its high morbidity and mortality and easily progressing into chronic kidney disease.1,2 Renal ischemia/reperfusion injury (IRI), as the main cause of AKI,3 usually occurs in kidney transplantation, surgery of cardiopulmonary resuscitation and aortic bypass, trauma, hemorrhage, hypotension and burns.4 At present, pathophysiology of IRI in kidney is still very complex, which involves some pathological pathways such as neutrophils activating, reactive oxygen species releasing and other inflammatory mediators.5 Previous studies have demonstrated that some agents, such as doxycycline,6 leptin,7


2

Page 3 of 46 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


levosimendan,8 iloprost9 and ascorbic acid,10 have beneficial effects on renal IRI. However, despite these drugs can alleviate renal IRI to a certain extent, few therapeutic options can realize effective treatment of renal IRI to date.11 Therefore, to reduce the incidence and death of renal IRI, it is crucial to find an effective therapeutic drug. Recently, several active compounds from traditional Chinese herbs have displayed positive effects on renal IRI.12-14 As an important bioactive compound, triptolide (TP) has multiple pharmacological activities, e.g., immune-modulation, anti-inflammatory, anticancer activities, anti-cystogenesis and anti-proliferative, which is widely applied in treating autoimmune and inflammatory diseases, tumors and organ and tissue transplantation induced diseases.15,16 Furthermore, TP can also effectively treat nephritis and renal IRI.17 Although TP has many advantages in treatment of clinical diseases, its low solubility, poor selectivity, fast clearance and severe toxicity still limit its wide applications.18,19 To improve biocompability, water solubility and kidney-targeting of TP, many strategies have developed. Zhang’s group has constructed different targeting ligands of TP, such as 14-succinyl triptolidelysozyme,20 triptolide-glucosamine conjugate21 and triptolide aminoglycoside.17 However, TP modified with small molecular aptamers is still easily cleared and has certain toxicity due to the exposed TP. It is well known that folate (FA) receptor (FR) has high expression in tumor.22-24 However, for normal tissues and organs, FR usually expresses in choroid plexus, placenta, kidney and intestine membranes.25 Moreover, several FRs (e.g., FRα, FRβ and FRγ) can specifically express in renal proximal tubule epithelial cells (PTECs) as well as high affinity between FA and FR, thus resulting in that FA modified vehicles can deliver drugs to the kidney by FR-mediated endocytosis.26,27 Shillingford and his co-workers have demonstrated that FA-rapamycin conjugate could achieve kidney-targeting delivery by FR–mediated endocytosis.28 Taylor’s group also created a 4-hydroxy-


3


Page 4 of 46

Tempo-FA to target PTECs for preventing renal IRI.29 Although drug-FA conjugates can achieve kidney targeting, they still have some deficiencies such as short retention time in blood circulation and inefficient drug release. Nevertheless, drug nanocarriers (e.g., lipids or polymers) have obvious superiority in prolonging drug retention time and improving drug release efficiency in vivo.30-34 Pluronic copolymers, as biocompatible and easily modified nanocarriers, can form a core–shell structure to incorporate lipophilic drugs.35,36 Pluronics that consists of hydrophilic polyethylene oxide (PEO) and hydrophobic polypropylene oxide (PPO) blocks has been extensively studied as carriers of hydrophobic drugs.37-39 A disulfide core-crosslinked Pluronic F127 nanoparticles loading PTX was developed to realize redox stimulus-responsive of intracellular drug release.40 A pH-dependent noncovalent incorporation and release of DOX in Pluronic P85-poly (acrylic acid) block copolymer was reported by Tian et al, in which DOX was loaded via electrostatic interactions.41 Moreover, the ultrasound-enhanced intracellular uptake of DOX administered with the Pluronic P105 micelles was demonstrated in vitro.42 Furthermore, Kanoujia et al. constructed gatifloxacin Pluronic F127 micelles with a high drug loading efficiency and sustained drug release process, in which drug released mainly through dissolution and diffusion.43 Therefore, the results indicated that Pluronics showed great advantages in drug loading and controlled drug release as drug carriers. Combined advantages of FA and Pluronic copolymers, we constructed a biocompability and highefficiency renal-targeting nanoplatform for renal IRI therapy, in which the toxic drug of TP was encapsulated into FA-modified Pluronic F127/P123 nanoparticles (FPNPs) (Scheme 1). FPNPs was prepared by a thin-film hydrated self-assembly strategy, in which FPNPs could realize renal targeting by FR-mediated endocytosis. After injected into the renal IRI model mice by tail vein, TP-FPNPs could reach the kidney and phagocytized by renal tubular cells. In tubular cells, TP


4

Page 5 of 46 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


would be slowly released from TP-FPNPs by a diffuse-controlled process at the beginning stage and subsequently the TP-FPNPs was degraded, thus realizing renal IRI treatment. The FPNPs with low toxicity, long retention time, kidney-targeting, high cellular uptake efficiency and superior curative effect will promise the great potential to be a delivery platform of hydrophobic drugs for treatment of renal diseases.

MATERIALS AND METHODS Materials and Instruments Pluronic F127 (PF127, EO100PO69EO100, Mn~12600 g/mol), Pluronic P123 (P123, EO20PO70EO20, ~5800 g/mol), trypsin-EDTA, 4’,6-diamidino-2-phenylindole (DAPI) were purchased from Sigma-Aldrich Co. (St. Louis, MO). Folate (FA), dicyclohexylcarbodiimide (DCC), 4dimethylaminopyridine (DMAP), triptolide (TP), anhydrous dimethyl sulfoxide (DMSO) were purchased from Aladdin Reagent Database Inc. (Shanghai, China). Cyanine7 NHS ester (Cy7) was purchased from Lumiprobe (America). Purified deionized water was prepared by a Milli-Q water (18.2 MΩ·cm). Other reagents and solvents were analytical grade and used as received without further purification. Ultraviolet−visible (UV-vis) absorption spectrum was recorded using a Hitachi U-3010 UV−vis spectrophotometer. 1H nuclear magnetic resonance (1H NMR) spectrum was measured by an Avance IIITM 1H NMR (600 MHz, Bruker, Switzerland) and DMSO-d6 used as solvent. The luminescence spectrum was collected by a Hitachi F-4500 fluorescence spectrophotometer (Japan). Hydrodynamic size and zeta potential were measured by Dynamic Light Scattering (DLS) using a Zetasizer Nano ZS (Malvern Instruments, Worcestershire, UK). Size and morphology of NPs stained with 1% phosphotungstic acid were characterized by a 200 kV JEOL 2010-F


5


Page 6 of 46

transmission electron microscope (TEM, Japan) and a ZEISS Merlin scanning electron microscope (SEM, Germany). The concentration of TP was determined by high performance liquid chromatography-mass spectrometric (HPLC-MS) using AcquityTM Waters LC and Waters TQ-S MS systems. Cell viability was measured at 450 nm using a microplate reader (Multiskan GO, Thermo Scientific). The fluorescence of cell and tissue was observed using an epifluorescence microscope (Axio Imager Z2, Zeiss, Oberkochen, Germany). Cellular uptake analysis was performed using a flow cytometer (FACS Canto II, BD Biosciences, USA). Ex vivo fluorescence imaging was captured using a Kodak in-Vivo Imaging System (Bruker, USA), which the imaging parameters included excitation (710/10 nm), emission (790/20 nm) and exposure time (30 s). Western blot images were detected by an Odyssey detector (LI-COR, NE, USA). Cell Lines and Animals Normal rat kidney proximal tubular epithelial (NRK-52E) cells, normal rat renal fibroblasts (NRK49F) cells and human cervical cancer (SiHa) cells were obtained from American Type Culture Collection (ATCC, VA, USA). Cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco, high glucose) containing 100 IU/mL penicillin, 10% fetal bovine serum solutions (FBS) and 100 mg/mL streptomycin (37 °C, 5% CO2 and 95% relative humidity). When ~80% confluence was reached, cells were switched to other plates for subsequent experiments. Male C57 BL/6 mice (3−4 weeks, 20-25 g) were supplied by the Health Science Center, Southern Medical University (Guangzhou, China), and all involving mice were approved by the National Institute of Biological Science and Animal Care Research Advisory Committee of Southern Medical University, all animal experiments were carried out according to guidelines of their Animal Research Ethics Board. All mice were maintained under standard housing conditions for 24 h before used.


6

Page 7 of 46 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Synthesis of FA-PF127 Conjugate FA-PF127 was prepared by a modified procedure.35 Typically, PF127 (10 g) was dissolved in anhydrous DMSO (20 mL) under vigorous stirring. FA (0.53 g), DCC (0.25 g) and DMAP (0.15 g) were then dissolved in DMSO (10 mL). Subsequently, the two solutions were mixed and stirred slowly for 24 h in dark at room temperature. The reaction solution was purified by dialysis (MW1000) against deionized water for 3 days, followed by centrifugation to remove 1,3dicyclohexylurea. Finally, the purified solution was frozen and lyophilized. The as-prepared FAPF127 conjugate was characterized by UV-vis and 1H NMR spectra. The purity of FA-PF127 was determined by adding 0.015 mmol dibromomethane served as internal standard solution in FAPF127 (350 mg). Preparation of TP-FPNPs TP-FPNPs was prepared by a modified thin-film hydrated method. TP, FA-PF127 and P123 were dissolved in methanol (6 mL), followed by rotating evaporation at 50 °C to remove solvent. The thin film obtained was then hydrated with deionized water (5 mL). Next, the hydrated solution was homogenized (17000 rpm, IKA, Germany) for 2 min and subsequently stirred for 6 h. Finally, the TP-FPNPs were obtained by centrifugation and filtration (10000 rpm, 10 min) using ultrafiltration centrifugal tube (30 kDa). To achieve Cy7-FPNPs, Cy7 (500 μg) was mixed with TP, FA-PF127 and P123 and was subsequently performed by the same preparation procedure. The Cy7 would entangle with hydrophobic group of FPNPs due to its hydrophobicity. The entrapment efficiency (EE %) and drug-loading (DL %) efficiency of TP could be calculated through the following equations:44

EE% =

weight of drug in NPs × 100% weight of feeding drug

(1)


7


DL% =

weight of drug in NPs × 100% weight of feeding polymer and drug

Page 8 of 46

(2)

Stability and in vitro Release Study To evaluate physical stability of TP-FPNPs. The TP-FPNPs were incubated with phosphate buffer solution (PBS, 10 mM, pH 7.4) and 10 % FBS by shaking (37 °C, 1000 rpm). Finally, the size of TP-FPNPs at different time intervals (0, 1, 3, 6, 12, 24 and 48 h) was measured by DLS. In vitro release behavior of TP from TP-FPNPs was monitored by a dialysis method. Briefly, TPFPNPs (2 mL) were placed in a dialysis bag (30 kDa). Subsequently, the dialysis bag was immerged in 10 mM PBS (pH 7.4, 50 mL) containing 10% 1, 2-propanediol and gently stirred at 100 rpm (37.0 ± 0.5 °C). Then, 1 mL dialyzed solution was collected at 0, 1, 3, 6, 12, 24 and 48 h. Finally, the extracted solution was filtered and injected into HPLC-MS to determine the TP concentration. In vitro release of free TP was measured via the same way. Cytotoxicity Assay in Vitro NRK-52E cells were trypsinzed using trypsin-EDTA (0.05% at 37 °C) and grown in 96-well plates at a density of 1×105 cells/well. Then, the cells were cultured with DMEM medium containing 10% FBS for 24 h, and the medium was subsequently replaced with DMEM basic medium for starvation treatment for 12 h. After 12 h, the media in holes were sucked away and replaced with DMEM (200 μL) media containing TP or TP-FPNPs with different concentrations of TP (0-120 ng/mL, each concentration has five pores). Finally, the cell viability was evaluated by a cell counting kit CCK-8 and microplate reader. Cellular Uptake Assay


8

Page 9 of 46 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


NRK-52E cells were planted into coverslip at density of 1×105 cells/well and cultured with DMEM medium containing 10% FBS for 24 h, and then exposed to DMEM media containing Cy7-PNPs or Cy7-FPNPs and incubated for another 2 h. For free-FA competition studies, cells were cultured in presence of 100 μM FA-contained DMEM for 30 min and then exposed to Cy7-FPNPs. After 2 h of cell uptake, the cells were washed with PBS for 3 times and stained with DAPI for 10 min in dark at 37 °C. Finally, these cells were observed by confocal laser scanning microscopy. Moreover, flow cytometry was also used to measure fluorescence intensity of Cy7 at excitation and emission wavelengths of 750 and 773 nm, respectively. Each group was performed three times. Ex Vivo Fluorescence Imaging and Biodistribution of FPNPs C57 BL/6 mice were injected with Cy7-FPNPs (0.5 mg/kg TP) via tail vein. After injection, the mice were sacrificed, and their organs (heart, liver, spleen, lung, kidney, intestine, thymus, muscle and brain) were excised and collected at 3, 12, 24 and 48 h post-injection, followed by washing with physiological saline 3 times and immediately imaged. The corresponding fluorescence intensity of Cy7 distribution in each organ was counted using the Kodak in-Vivo Imaging System software. At each time point, three parallel mice were used to conduct the imaging. Pharmacokinetics and Body Distribution of TP Pharmacokinetic profiles and body distribution of free TP and TP-FPNPs (0.5 mg/kg TP) were studied in C57 BL/6 mice via tail vein injection. Three mice were applied in each group. At predetermined time points (1, 6, 12, 24 and 48 h) after injected, blood samples were collected in heparinized tubes and heart, liver, spleen, lung, kidney, intestine and brain were isolated and stored at -80 °C before used. Separation of plasma was conducted by centrifugation (3000 rpm, 5 min). The organs were washed with cold saline and weighed and then homogenized with two-fold volume of 0.9% saline (g/mL). Next, 120 μL of carbamazepine internal standard solution (10


9


Page 10 of 46

ng/mL in methanol) was added into plasma or tissue homogenate (1 mL), and extracted with 5 mL acetic ether for vortex-mixing 5 min, followed by centrifugation (12000 rpm, 10 min). Finally, the upper organic phase was transferred and evaporated, dissolved with 1% formic acid aqueous solution. An aliquot (5 μL) was analyzed by HPLC-MS to quantify concentration of TP.21,45 Pharmacodynamics Study on Renal IRI Model Renal IRI model in C57 BL/6 mice was established according to previous studies.20,21 Mice were randomly divided into four groups: (i) sham operation group, mice were received identical surgical procedure besides renal IRI (n = 5); (ii) IRI + saline group, mice were injected with normal saline 3 days before renal ischemia (n = 5); (iii) IRI + free TP group: mice were injected TP (0.01 mg/kg/day) 3 days before renal ischemia (n = 5); (iv) IRI + TP-FPNPs group: mice were injected TP-FPNPs (equivalent to 0.01 mg/kg/day of TP) 3 days before renal ischemia (n = 5). After injection, mice were anesthetized using pentobarbital sodium for 35 min and maintained under anesthesia for the duration of ischemia. Mice were subjected to midline laparotomy, and then micro-aneurysm clamps was used to occlude renal arteries and veins of mice to bilateral renal ischemia for 30 min. After observed for 5 min and no abnormity found, the mice were sutured and returned to their cages where they were allowed to recover from anesthesia. All animals were evaluated for renal function and histological morphology, and kidney tissues were lysed with protein cracking liquid lysis buffer for 30 min on ice to determine protein levels of p-ERK. In Vivo Toxicological Evaluation In vivo toxicities were studied in male C57 BL/6 mice. First, mice were randomly divided into four groups: Free TP (0.7 mg/kg), FPNPs (65 mg/kg), TP-FPNPs (equal to 0.7 mg/kg of TP) and saline (5 mL/kg). These mice were injected with free TP, FPNPs, TP-FPNPs and saline 3 times per week for 3 weeks. Then, the mice were sacrificed at 21th day and their kidney, liver and testis were


10

Page 11 of 46 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


collected, sectioned and stained with hematoxylin and eosin (H&E) staining to investigate their histological morphology. Renal toxicity was monitored by index of serum creatinine (SCR) and blood urea nitrogen (BUN), while hepatic toxicity was surveyed by quantitative analysis of alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Statistical Analysis Comparisons between the groups were determined by one-way ANOVA, followed by Student’s ttest (IBM SPSS software, version 23.0). Differences were considered to be statistically significant at p < 0.05. All experiment data were obtained from three or five replicates (as detailed throughout the paper) for each data point, all data are expressed as the mean value ± standard deviation (SD). For microscopic images, the experiments were performed in several replicates, and representative images were given.

RESULTS AND DISCUSSION Preparation and Characterization of TP-FPNPs To achieve kidney targeting of Pluronic F127/P123 NPs, PF127 was first modified with FA. Synthetic route of FA-PF127 and 1H NMR characterizations of FA, PF127 and FA-PF127 were illustrated in Figure S1. 1H NMR spectrum peaks at 1.03 and 3.30-3.50 ppm could be clearly discerned, which were characteristic of methyl and methylene groups of PF127, respectively.36 The 1H NMR peaks at 8.67, 7.42 and 6.60 ppm corresponded pteridine proton and aromatic protons of FA, respectively.46 Besides, the peak at 11.49 ppm deriving from terminal carboxyl proton in FA disappeared in 1H NMR spectrum of FA-PF127, indicating that FA was successfully modified onto PF127. By integrated and compared peak areas between the peak at 5.39 ppm of internal standard solution and the peak at 7.42 ppm of aromatic protons in FA, the purity of FA-PF127 was


11


Page 12 of 46

estimated to be about 82.5%. Moreover, two peaks at 280 and 350 nm that perhaps came from aromatic ring of FA47 could be observed in the UV spectrum of FA-PF127 (Figure S2), which further demonstrated successful modification of FA on PF127. Using FA-PF127 as an assembly component, a renal-targeting nanoplatform was constructed by a thin-film hydrated self-assembly strategy. Previous study had indicated that the aggregation behavior of binary Pluronics was PPO dependent and Pluronics with similar PPO moieties exhibited cooperative aggregation.48 The similar-length PPO chains of PF127 and P123 easily intertwined each other by hydrogen bonds and hydrophobic interactions to form a hydrophobic core. While the PEO chains would form hydrophilic shells around the core, the terminal-modified FA in FA-PF127 of which would expose on the outside of the FPNPs. The sizes and polydispersity index (PDI) of FPNPs closely associated with emulsification method, FA-PF127 and P123 amounts and hydration temperature (Table S1).49 By adjusting the ratio of P123 to FA-PF127 at 2:1, 1:1, 1:2 and 1:3, the different FPNPs were formed. When the ratio of P123 to FA-PF127 was 1:2, the FPNPs prepared had the best homogeneity, which was perhaps because the appropriate ratio of P123 to FA-PF127 was propitious to form uniform circular aggregates.39 Besides, the most uniform FPNPs was formed by the hydrated method at 50 °C, indicating that moderate temperature was favorable to fabricate homogeneous NPs. To sum up, the optimum conditions for uniform FPNPs were homogenization 2 min, 50 mg P123, 100 mg FA-PF127 and hydration at 50 °C. Under the above-mentioned optimal conditions, TP served as a self-assembly unit was easily encapsulated into FPNPs due to its hydrophobic property. For example, when 2 mg TP was added, its EE % and DL % were 75.0 ± 2.0% and 1.11 ± 0.03% respectively. Moreover, its zeta potential was -11.9 ± 1.2 mV. Figure 1A,B showed SEM and TEM images of TP-FPNPs prepared by the present method. The spherical TP-FPNPs had well-dispersed and the average size of about 90 nm,


12

Page 13 of 46 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


which was similar to its hydrodynamic size (Figure 1C). Compared with FPNPs (~70 nm), the TP-FPNPs had larger size, which demonstrated that TP was successfully packaged in FPNPs (Figure S3). Stability and In Vitro Release Characterizations of TP-FPNPs The good stability of nanomaterials was important for its biological applications.50 To investigate stability of TP-FPNPs, 10 mM PBS (pH 7.4) and 10% FBS were used (Figure 1D). No significant change in the particle size could be observed within 48 h in the two media, which displayed that TP-FPNPs was not degraded in PBS and FBS and thus had good stability in blood circulation. It was worth noting that the particle size of TP-FPNPs in FBS slightly increased in comparison with that in PBS, which was perhaps due to the adsorbed cationic proteins on the surface of the negatively charged TP-FPNPs. Moreover, TP-FPNPs that adsorbed hydrophobic protein corona had stable interface and thus prevented TP-FPNPs aggregations. Therefore, TP-FPNPs prepared by the present method had good stability and was hopeful to realize high-efficiency delivery of TP in vivo. In vitro cumulative release profiles of TP-FPNPs and free TP were illustrated in Figure 1E. Most of TP (99.2%) would release within 12 h, which suggested that free TP could rapidly release within short time. Comparatively, only ~50% of TP released from TP-FPNPs within 12 h, which was much slower than free TP. The small-quantity release of TP in TP-FPNPs was mainly because of drug diffusion and erosion and swelling of nanocarriers.51 Moreover, the TP released only 68.3% after 48 h, which could be attributed to effective encapsulation in FPNPs. This indicated that TPFPNPs presented a sustained release process and thus prolonged retention time of drug, which would be a good foundation for in vivo applications of TP-FPNPs.


13


Page 14 of 46

Cytotoxicity Assay and Cellular Uptake Mechanism Study To assess the cytotoxicity of TP-FPNPs, NRK-52E cells were incubated with TP and TP-FPNPs (Figure 2A). After incubated with TP, the viability of NRK-52E cells gradually decreased with increasing TP concentrations. When TP concentration was 30 ng/mL, its cell viability was about 60%. Further, its cell viability would be down up to only 40% when the TP concentration reached 120 ng/mL, showing severe cytotoxicity. Compared with free TP, cell viability of TP-FPNPs were more than 80% within the given concentration ranges, indicating that TP-FPNPs had good biocompatibility. The slight decrease of cell viability of TP-FPNPs was perhaps due to tracequantity release of TP in TP-FPNPs. Therefore, TP encapsulated with FPNPs could greatly lower its cytotoxicity. To track intracellular uptake efficiency, PNPs, FPNPs and FA-FPNPs were labelled with Cy7 and incubated with NRK-52E cells, respectively. There appeared excitation and emission fluorescence peaks at around 750 and 773 nm of Cy7-FPNPs, indicating successful encapsulation of Cy7 (Figure S4).52 Flow cytometric analysis of Cy7-PNPs, Cy7-FPNPs and FA + Cy7-FPNPs were performed in NRK-52E cells to understand its uptake mechanism (Figure 2B). In addition, folate receptor overexpressed SiHa cells and folate receptor deficient NRK-49F cells were also used. After incubation for 2 h at 37 °C, uptake of Cy7-FPNPs by SiHa and NRK-52E cells was much higher than that of Cy7-PNPs (Figure S5). However, compared with Cy7-FPNPs, uptake of the competitive inhibition group which was pre-treated with FA for 30 min was much lower than Cy7FPNPs. The excellent cellular uptake efficiency of Cy7-FPNPs was perhaps due to the existence of FR on cell surface and thus facilitated the interaction between Cy7-FPNPs and cellular membranes. Besides, for the folate receptor deficient cells of NRK-49F cells, the fluorescence intensities among Cy7-PNPs, Cy7-FPNPs and FA + Cy7-FPNPs were similar, which proved FR-


14

Page 15 of 46 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


mediated endocytosis pathway. Figure 2C-E shows fluorescent images of Cy7-FPNPs. The red fluorescence of Cy7 did not overlap with the green fluorescence of the nucleus, indicating that Cy7-FPNPs mainly existed in cytoplasm. This indicated that Cy7-FPNPs could be effective uptake by cells. Further, to illustrate the endo/lysosome escape mechanism of FPNPs, Zeta potential of FPNPs at different pH values were measured (Figure S6). When the pH values decreased from 7.4 to 5.0, the Zeta potential of FPNPs positively shifted from -11.9 ± 1.2 to -0.5 ± 0.1 mV. This was perhaps due to the protonation of amines/imines protons in FA under acidic microenvironment, thus neutralizing the negative potential of FPNPs. Therefore, when FPNPs entered the endosome (approximately pH 5) through endocytosis, the “proton sponge” effect would occur, leading to the rupture of endo/lysosomal membrane53,54. Finally, the FPNPs easily released and entered into the cytosol. In Vivo Fluorescence Imaging and Pharmacokinetic Characterization To explore renal targeting of FPNPs, X-ray photoelectron spectra (XPS) of N 1s peaks of PNPs and FPNPs were recorded (Figure S6). N 1s peak at ca. 398.4 eV deriving from FA55 was found in FPNPs, demonstrating that FA exposed on surface of FPNPs. Furthermore, to evaluate kidneytargeting ability of FPNPs, tissue distribution of Cy7-FPNPs at different time points was investigated by organ fluorescence imaging (Figure 3A-D). The fluorescence intensity of kidneys was much stronger than those of other organs at all time points and reached its maximum at 12 h post-injection, indicating the selective accumulation of Cy7-FPNPs in kidneys. Moreover, there still existed a strong fluorescence in kidney until 48 h post-injection, which demonstrated the long kidney retention time of Cy7-FPNPs. The specific renal-targeting ability of Cy7-FPNPs could be attributed to FR-mediated endocytosis of renal tubular cells.25 In addition, liver had a relatively weak fluorescence intensity in the short time although NPs was usually excreted through liver,56


15


Page 16 of 46

which also implied that Cy7-FPNPs could easily escape from reticuloendothelial system. The corresponding fluorescence intensity of Cy7 distribution in each organ examined from fluorescence imaging was illustrated in Figure 3E, which indicated specific kidney targeting and satisfactory renal retention capability of Cy7-FPNPs. To further prove the selectivity of FPNPs to kidney, free TP and TP-FPNPs were injected into mice intravenously and their tissue distribution was analyzed (Figure 4). As shown in Figure 4A, the concentrations of TP were nearly similar among different organs, indicating that TP had not specific targeting toward kidney. Different from TP, TP-FPNPs began to preferentially accumulate in the kidneys after 6 h (Figure 4B). And the TP-FPNPs concentration in kidneys reached 1994.4 ng/g at 12 h, which was 3.2-fold higher than that of TP (Figure 4C). The concentration of TPFPNPs always remained higher in the kidneys than those in other organs over the examined 48 h observation period. Furthermore, TP-FPNPs always possessed a high plasma concentration and the plasma concentrations of TP-FPNPs were higher than that of TP at all observed time points (Figure 4D). And until 48 h post-injection, the plasma concentration of TP-FPNPs was still 302.6 ng/g, which demonstrated its long retention time. Consequently, FPNPs changed the distribution of TP and significantly increased the concentration of TP in kidneys and blood, suggesting that TP-FPNPs had renal targeting ability and long renal retention time. Therapeutic Efficacy of TP-FPNPs against Renal IRI To investigate the treatment effect of TP-FPNPs against renal IRI, renal IRI model mice were constructed with male C57 BL/6 mice. Figure 5A displays histological sections of kidneys from different operated mice by H&E staining. Renal morphology of the saline-treated IRI group displayed severe renal damages, shown as tubular lumen dilated, renal tubular epithelial cells degenerated, leukocytes infiltrated and protein casts formed (Figure S7). Compared with the


16

Page 17 of 46 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


saline-treated IRI group, the renal damages of the IRI mice received by free TP were relieved to a certain degree. Interestingly, only mild swelling or degeneration of tubular lumen and tubular cells were observed in the IRI group received by TP-FPNPs. After TP-FPNPs treatment, acute tubular injury index significantly decreased up to 0.77 ± 0.07, which was about 3-fold lower than those (2.53 ± 0.09 and 2.24 ± 0.21) for saline and free TP groups (Figure 5B). Moreover, the SCR level (29.0 ± 1.0) after TP-FPNPs treatment was about 4.3-fold lower than that (125.3 ± 30.7) after TP treatment (Figure 5C). The BUN level (10.1 ± 2.2) after TP-FPNPs treatment was about 2.0-fold lower than that (20.2 ± 0.5) after TP treatment (Figure 5D). These results revealed that TP-FPNPs had a superior curative effect in renal IRI therapy. This could be attributed to the specific kidneytargeting ability and long kidney residence time of TP-FPNPs in the renal IRI model mice. Western blot analysis was employed to measure protein expression of acute tubular injury-related signaling pathways, in which the expression level of downstream proteins of p-ERK was studied (Figure 6A). The expression level of p-ERK was 2.6-fold lower after TP-FPNPs treatment than the saline group (Figure 6B), suggesting that TP-FPNPs could greatly alleviate acute tubular injury. This was probably due to ERK/MAP kinase signaling pathways blocked by TP-FPNPs in the renal IRI model.57 However, the expression of p-ERK was not significantly suppressed in the free TP group (0.01 mg/kg for 3 days), which was mainly due to the easy clearance and non-kidney targeting of TP. In addition, the expression level of p-ERK treated with TP-FPNPs (equal to 0.01 mg/kg TP for 3 days) was comparable or even lower than that of 10-fold TP concentration (0.1 mg/kg),20 which suggested that TP-FPNPs with a low TP concentration could inhibit acute tubular injury and possessed a strong inhibition effect on renal IRI. In Vivo Organ Toxicity Study


17


Page 18 of 46

Previous studies have demonstrated that as the dose increased (≥ 0.2 mg/kg), TP showed potential toxicity on liver, kidney and reproductive systems.18 So we studied the systemic toxicity of TPFPNPs to assess its utility as a renal targeting nanoplatform. Liver sections staining showed that the liver appeared smooth and normal in color for the saline, FPNPs and TP-FPNPs treated groups (Figure 7A), suggesting that the saline, FPNPs and TP-FPNPs had no toxicity on liver. However, the liver of mice treated with TP appeared inflammatory infiltration, vacuolation, swelling and necrosis. Furthermore, the indexes of liver function were also evaluated (Figure 7C,D). The ALT and AST in the serum significantly elevated (2.2 and 4.1-fold) after treated with TP (0.7 mg/kg) 3 times a week for 21 days, indicating its serious hepatic toxicity. Hepatotoxicity induced by TP might be due to the accumulation of TP in the liver. Nevertheless, after treated with FPNPs and TP-FPNPs, the ALT and AST levels nearly did not rise compared with saline group, indicating that FPNPs and TP-FPNPs nearly had not toxicity on liver. To further evaluate systemic toxicity of TP-FPNPs, mice were treated with saline, TP, FPNPs and TP-FPNPs, then their testis tissues were sectioned and stained. The interstitial tissues of the testes had full complement of leydig cells and were normal in the saline and FPNPs treated groups (Figure 7B). However, severe testicular degeneration (e.g., disruption or disappearance in the interstitial tissue) and focal areas of necrosis of leydig cells in the interstitial tissue appeared in TP treated group, which demonstrated its severe testicular toxicity. While in TP-FPNPs group, only slight degree of testicular degeneration could be observed, indicating that TP-FPNPs had relatively good biocompability and greatly relieved TP toxicity on testis due to FPNPs capping. Furthermore, the kidney function indexes of SCR and BUN were investigated to evaluate nephrotoxicity of TPFPNPs (Figure 7E,F). The serum SCR and BUN in mice treated with TP significantly elevated (2.2 and 2.8-fold) than the saline group, indicating a severe nephrotoxicity of TP. However, the


18

Page 19 of 46 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


SCR and BUN levels in the serum treated with FPNPs or TP-FPNPs at concentrations up to 65 mg/kg/day (equal to 0.7 mg/kg/day of TP) for 21 days were no significant change and nearly as the same as the saline group, which indicated that FPNPs and TP-FPNPs were nontoxic to kidney. These results indicated that TP-FPNPs possessed excellent biocompability and dramatically lowered TP toxicity on kidney, liver and reproductive systems.

CONCLUSIONS In summary, we have successfully fabricated biocompability and high-efficiency renal-targeting nanoplatform of TP-FPNPs for effective renal IRI therapy in vivo, in which the TP with severe toxicity was capped in biocompable FPNPs. The TP-FPNPs possessed excellent biocompability (e.g., lower nephrotoxicity, hepatotoxicity and genital system toxicity) and dramatically lowered TP toxicity due to the TP encapsulated by FPNPs. Our results revealed that the TP-FPNPs possessed long retention time, high cellular uptake efficiency and thus good renal targeting. In vivo renal IRI therapy, TP-FPNPs was much more efficient than free TP, such as lower acute tubular injury index (2.9-fold), renal function indexes of serum creatinine (4.3-fold), urea nitrogen (2.0fold) and western blotting (2.4-fold). With these attractive characteristics, we believe that the renal-targeting FPNPs will be a promising drug delivery platform of hydrophobic drugs for treatment of renal diseases.

SUPPORTING INFORMATION Figure S1 provides synthesis and 1H NMR characterization of FA-PF127. Figure S2 provides UV spectra of FA, PF127 and FA-PF127. Figure S3 provides hydrodynamic sizes of FPNPs and TPFPNPs measured by DLS. Figure S4 shows excitation and emission fluorescence spectra of Cy7-


19


Page 20 of 46

FPNPs. Figure S5 provides flow cytometric analysis images of NRK-49F, SiHa and NRK-52E cells incubated with PBS, Cy7-PNPs, Cy7-FPNPs and Cy7-FPNPs + FA. Figure S6 shows Zeta potentials of FPNPs under different pH conditions. Figure S7 provides XPS spectra of N 1s peaks from PNPs and FPNPs. Figure S8 provides representative light photomicrographs of H&E-stained kidney tissue. Table S1 shows hydrodynamic size and PDI characteristic of FPNPs prepared by the different methods. AUTHOR INFORMATION Corresponding Author *Correspondence should be addressed to Jianqiang Hu ([email protected]). Author Contributions Caili Huang, Tao Zeng and Jiawen Li designed and performed experiments. Jianqiang Hu, Aiqing Li, Lishan Tan, Xiulong Deng, Yanchao Pan and Qi Chen supervised the project. Caili Huang, Tao Zeng and Jiawen Li analyzed data; Caili Huang, Tao Zeng and Jianqiang Hu wrote the manuscript. All authors edited and approved the final manuscript. ‡These authors contributed equally. Notes There are no conflicts to declare. ACKNOWLEDGMENT This work was supported by grants from the National Nature and Science Foundation of China (Nos. 81770727, 21673081, 51273070, 81270825), GDUPS (2017), Key project of Guangdong Natural Science Foundation (2018B0303110002), Key Project of Guangzhou Science Technology


20

Page 21 of 46 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


and Innovation Commission (201804020054) and Science and Technology Planning Project of Guangdong Province (2017A010103041) and China Postdoctoral Science Foundation (Nos. 2017M620369, 2018M643088). REFERENCES 1. Sharp, C. N.; Siskind, L. J. Developing better mouse models to study cisplatin-induced kidney injury. Am. J. Physiol-Renal 2017, 313, F835-F841, doi:10.1152/ajprenal.00285.2017. 2. Kang, R.; Rovin, B. Advances and challenges on new therapies and clinical targets of ccute kidney injury. Toxicol. Pathol. 2018, 10, 1-5, doi:10.1177/0192623318800688. 3. Chertow, G. M.; Burdick, E.; Honour, M.; Bonventre, J. V.; Bates, D. W. Acute kidney injury, mortality, length of stay, and costs in hospitalized patients. J. Am. Soc. Nephrol. 2005, 16, 33653370, doi:10.1681/ASN.2004090740. 4. Kumar, S.; Allen, D. A.; Kieswich, J. E.; Patel, N. S.; Harwood, S.; Mazzon, E.; Cuzzocrea, S.; Raftery, M. J.; Thiemermann, C.; Yaqoob, M. M. Dexamethasone ameliorates renal ischemiareperfusion injury. J. Am. Soc. Nephrol. 2009, 20, 2412-2425, doi:10.1681/ASN.2008080868. 5. Malek, M.; Nematbakhsh, M. Renal ischemia/reperfusion injury; from pathophysiology to treatment. J. Renal Inj. Prev. 2015, 4, 20-27, doi:10.12861/jrip.2015.06. 6. Ihtiyar, E.; Yasar, N. F.; Erkasap, N.; Koken, T.; Tosun, M.; Oner, S.; Erkasap, S. Effects of doxycycline on renal ischemia reperfusion injury induced by abdominal compartment syndrome. J. Surg. Res. 2011, 167, 113-120, doi:10.1016/j.jss.2009.09.048. 7. Robertson, L. T.; Trevino-Villarreal, J. H.; Mejia, P.; Grondin, Y.; Harputlugil, E.; Hine, C.; Vargas, D.; Zheng, H.; Ozaki, C. K.; Kristal, B. S.; Simpson, S. J.; Mitchell, J. R. Protein and calorie restriction contribute additively to protection from renal ischemia reperfusion injury partly via leptin reduction in male mice. J. Nutr. 2015, 145, 1717-1727, doi:10.3945/jn.114.199380.


21


Page 22 of 46

8. Grossini, E.; Molinari, C.; Pollesello, P.; Bellomo, G.; Valente, G.; Mary, D.; Vacca, G.; Caimmi, P. Levosimendan protection against kidney ischemia/reperfusion injuries in anesthetized pigs. J. Pharmacol. Exp. Ther. 2012, 342, 376-388, doi:10.1124/jpet.112.193961. 9. Erer, D.; Ozer, A.; Demirtas, H.; Gonul, II; Kara, H.; Arpaci, H.; Comu, F. M.; Oktar, G. L.; Arslan, M.; Kucuk, A. Effects of alprostadil and iloprost on renal, lung, and skeletal muscle injury following hindlimb ischemia-reperfusion injury in rats. Drug Des. Devel. Ther. 2016, 10, 26512658, doi:10.2147/DDDT.S110529. 10. Korkmaz, A.; Kolankaya, D. The protective effects of ascorbic acid against renal ischemiareperfusion injury in male rats. Renal Failure 2009, 31, 36-43, doi:10.1080/08860220802546271. 11. Wu, M. Y.; Yiang, G. T.; Liao, W. T.; Tsai, A. P.; Cheng, Y. L.; Cheng, P. W.; Li, C. Y.; Li, C. J. Current mechanistic concepts in ischemia and reperfusion injury. Cell. Physiol. Biochem. 2018, 46, 1650-1667, doi:10.1159/000489241. 12. Zhong, Y.; Deng, Y.; Chen, Y.; Chuang, P. Y.; J., C. H. Therapeutic use of traditional Chinese herbal medications for chronic kidney diseases. Kidney Int. 2013, 84, 1108-1118, doi:10.1038/ki.2013.276. 13. Qiao, H.; Sun, M.; Su, Z.; Xie, Y.; Chen, M.; Zong, L.; Gao, Y.; Li, H.; Qi, J.; Zhao, Q.; Gu, X.; Ping, Q. Kidney-specific drug delivery system for renal fibrosis based on coordination-driven assembly

of

catechol-derived

chitosan.

Biomaterials

2014,

35,

7157-7171,

doi:10.1016/j.biomaterials.2014.04.106. 14. Li, J.; Zhang, C.; He, W.; Qiao, H.; Chen, J.; Wang, K.; Oupicky, D.; Sun, M. Coordinationdriven assembly of catechol-modified chitosan for the kidney-specific delivery of salvianolic acid B to treat renal fibrosis. Biomater. Sci. 2017, 6, 179-188, doi:10.1039/c7bm00811b.


22

Page 23 of 46 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


15. Zhou, Z. L.; Yang, Y. X.; Ding, J.; Li, Y. C.; Miao, Z. H. Triptolide: structural modifications, structure-activity relationships, bioactivities, clinical development and mechanisms. Nat. Prod. Rep. 2012, 29, 457-475, doi:10.1039/c2np00088a. 16. Liu, Q. Triptolide and its expanding multiple pharmacological functions. Int. Immunopharmacol. 2011, 11, 377-383, doi:10.1016/j.intimp.2011.01.012. 17. Qi, B.; Wang, X.; Zhou, Y.; Han, Q.; He, L.; Gong, T.; Sun, X.; Fu, Y.; Zhang, Z. A renaltargeted triptolide aminoglycoside (TPAG) conjugate for lowering systemic toxicities of triptolide. Fitoterapia 2015, 103, 242-251, doi:10.1016/j.fitote.2015.04.008. 18. Xue, X.; Gong, L.; Qi, X.; Wu, Y.; Xing, G.; Yao, J.; Luan, Y.; Xiao, Y.; Li, Y.; Wu, X.; Chen, M.; Gu, J.; Ren, J. Knockout of hepatic P450 reductase aggravates triptolide-induced toxicity. Toxicol. Lett. 2011, 205, 47-54, doi:10.1016/j.toxlet.2011.05.003. 19. Xi, C.; Peng, S.; Wu, Z.; Zhou, Q.; Zhou, J. Toxicity of triptolide and the molecular mechanisms

involved.

Biomed.

Pharmacother.

2017,

90,

531-541,

doi:10.1016/j.biopha.2017.04.003. 20. Zhang, Z.; Zheng, Q.; Han, J.; Gao, G.; Liu, J.; Gong, T.; Gu, Z.; Huang, Y.; Sun, X.; He, Q. The targeting of 14-succinate triptolide-lysozyme conjugate to proximal renal tubular epithelial cells. Biomaterials 2009, 30, 1372-1381, doi:10.1016/j.biomaterials.2008.11.035. 21. Zhou, P.; Sun, X.; Gong, T.; Zhang, Z.; Zhang, L. Conjugating glucosamine to triptolide to enhance its protective effect against renal ischemia-reperfusion injury and reduce its toxicity. J. Drug Target. 2014, 22, 200-210, doi:10.3109/1061186x.2013.856011. 22. Abouelmagd, S. A.; Meng, F.; Kim, B. K.; Hyun, H.; Yeo, Y. Tannic acid-mediated surface functionalization of polymeric nanoparticles. ACS Biomater. Sci. Eng. 2016, 2, 2294-2303, doi:10.1021/acsbiomaterials.6b00497.


23


Page 24 of 46

23. Liu, S.; Gao, Y.; A, S.; Zhou, D.; Greiser, U.; Guo, T.; Guo, R.; Wang, W. Biodegradable Highly Branched Poly(β-Amino Ester)s for Targeted Cancer Cell Gene Transfection. ACS Biomater. Sci. Eng. 2016, 3, 1283-1286, doi:10.1021/acsbiomaterials.6b00503. 24. Zhang, Z.; Wan, J.; Sun, L.; Li, Y.; Guo, J.; Wang, C. Zinc finger-inspired nanohydrogels with glutathione/pH triggered degradation based on coordination substitution for highly efficient delivery

of

anti-cancer

drugs.

J.

Control.

Release

2016,

225,

96-108,

doi:10.1016/j.jconrel.2016.01.035. 25. Chen, C.; Ke, J.; Zhou, X. E.; Yi, W.; Brunzelle, J. S.; Li, J.; Yong, E. L.; Xu, H. E.; Melcher, K. Structural basis for molecular recognition of folic acid by folate receptors. Nature 2013, 500, 486-489, doi:10.1038/nature12327. 26. Dolman, M. E.; Harmsen, S.; Storm, G.; Hennink, W. E.; Kok, R. J. Drug targeting to the kidney: Advances in the active targeting of therapeutics to proximal tubular cells. Adv. Drug Deliver. Rev. 2010, 62, 1344-1357, doi:10.1016/j.addr.2010.07.011. 27. Birn, H.; Spiegelstein, O.; Christensen, E. I.; Finnell, R. H. Renal tubular reabsorption of folate mediated by folate binding protein 1. J. Am. Soc. Nephrol. 2005, 16, 608-615, doi:10.1681/ASN.2004080711. 28. Shillingford, J. M.; Leamon, C. P.; Vlahov, I. R.; Weimbs, T. Folate-conjugated rapamycin slows progression of polycystic kidney disease. J. Am. Soc. Nephrol. 2012, 23, 1674-1681, doi:10.1681/ASN.2012040367. 29. Knight, S. F.; Kundu, K.; Joseph, G.; Dikalov, S.; Weiss, D.; Murthy, N.; Taylor, W. R. Folate receptor-targeted antioxidant therapy ameliorates renal ischemia-reperfusion injury. J. Am. Soc. Nephrol. 2012, 23, 793-800, doi:10.1681/ASN.2011070711.


24

Page 25 of 46 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


30. Mura, S.; Nicolas, J.; Couvreur, P. Stimuli-responsive nanocarriers for drug delivery. Nature Mater. 2013, 12, 991-1003, doi:10.1038/nmat3776. 31. Cao, W.; Li, Y.; Yi, Y.; Ji, S.; Zeng, L.; Sun, Z.; Xu, H. Coordination-responsive seleniumcontaining polymer micelles for controlled drug release. Chemical Science 2012, 3, 3403, doi:10.1039/c2sc21315j. 32. Chen, C. Y.; Kim, T. H.; Wu, W. C.; Huang, C. M.; Wei, H.; Mount, C. W.; Tian, Y.; Jang, S. H.; Pun, S. H.; Jen, A. K. pH-dependent, thermosensitive polymeric nanocarriers for drug delivery to solid tumors. Biomaterials 2013, 34, 4501-4509, doi:10.1016/j.biomaterials.2013.02.049. 33. Ma, G.; Liu, J.; He, J.; Zhang, M.; Ni, P. Dual-Responsive Polyphosphoester-Doxorubicin Prodrug Containing a Diselenide Bond: Synthesis, Characterization, and Drug Delivery. ACS Biomater. Sci. Eng. 2018, 4, 2443-2452, doi:10.1021/acsbiomaterials.8b00429. 34. Liu, K.-F.; Liu, Y.-X.; Li, C.-X.; Wang, L.-Y.; Liu, J.; Lei, J.-D. Self-Assembled pH and Redox Dual Responsive Carboxymethylcellulose-Based Polymeric Nanoparticles for Efficient Anticancer

Drug

Codelivery.

ACS

Biomater.

Sci.

Eng.

2018,

4,

4200-4207,

doi:10.1021/acsbiomaterials.8b00920. 35. D. S. Ling; H. P. Xia; W. Park; M. J. Hackett; C. Y. Song; Na, K. pH-sensitive nanoformulated triptolide as a targeted therapeutic strategy for hepatocellular carcinoma. ACS nano 2014, 8, 80278039, doi:10.1021/nn502074x. 36. Lin, J.; Chen, J.; Huang, S.; Ko, J.; Wang, Y.; Chen, T.; Wang, L. Folic acid-Pluronic F127 magnetic nanoparticle clusters for combined targeting, diagnosis, and therapy applications. Biomaterials 2009, 30, 5114-5124, doi:10.1016/j.biomaterials.2009.06.004.


25


Page 26 of 46

37. Batrakova, E. V.; Kabanov, A. V. Pluronic block copolymers: evolution of drug delivery concept from inert nanocarriers to biological response modifiers. J. Control. Release. 2008, 130, 98-106, doi:10.1016/j.jconrel.2008.04.013. 38. Lee, E. S.; Oh, Y. T.; Youn, Y. S.; Nam, M.; Park, B.; Yun, J.; Kim, J. H.; Song, H. T.; Oh, K. T. Binary mixing of micelles using Pluronics for a nano-sized drug delivery system. Colloid. Surface. B. 2011, 82, 190-195, doi:10.1016/j.colsurfb.2010.08.033. 39. Wei, Z.; Hao, J.; Yuan, S.; Li, Y.; Juan, W.; Sha, X.; Fang, X. Paclitaxel-loaded Pluronic P123/F127 mixed polymeric micelles: formulation, optimization and in vitro characterization. Int. J. Pharmaceut. 2009, 376, 176-185, doi:10.1016/j.ijpharm.2009.04.030. 40. Abdullah Al, N.; Lee, H.; Lee, Y. S.; Lee, K. D.; Park, S. Y. Development of disulfide corecrosslinked pluronic nanoparticles as an effective anticancer-drug-delivery system. Macromol. Biosci. 2011, 11, 1264-1271, doi:10.1002/mabi.201100083. 41. Tian, Y.; Bromberg, L.; Lin, S. N.; Hatton, T. A.; Tam, K. C. Complexation and release of doxorubicin from its complexes with pluronic P85-b-poly(acrylic acid) block copolymers. J. Control. Release 2007, 121, 137-145, doi:10.1016/j.jconrel.2007.05.010. 42. Husseini, G. A.; Rosa, M. A., Diaz De La; Tasneem, G.; Yi, Z.; Christensen, D. A.; Pitt, W. G. Release of doxorubicin from unstabilized and stabilized micelles under the action of ultrasound. J. Nanosci. Nanotechnol. 2007, 7, 1028-1033, doi:10.1166/jnn.2007.218. 43. Jovita, K.; Priya Singh, K.; Saraf, S. A. Evaluation of gatifloxacin pluronic micelles and development of its formulation for ocular delivery. Drug Deliv. and Transl. Res. 2014, 4, 334-343, doi:10.1007/s13346-014-0194-y.


26

Page 27 of 46 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


44. Zhao, L.; Du, J.; Duan, Y.; Zang, Y.; Zhang, H.; Yang, C.; Cao, F.; Zhai, G. Curcumin loaded mixed micelles composed of Pluronic P123 and F68: preparation, optimization and in vitro characterization. Colloid. Surface. B. 2012, 97, 101-108, doi:10.1016/j.colsurfb.2012.04.017. 45. Meng, F.; Wang, J.; Ping, Q.; Yeo, Y. Quantitative Assessment of Nanoparticle Biodistribution by

Fluorescence

Imaging,

Revisited.

ACS

Nano

2018,

12,

6458–6468,

doi:10.1021/acsnano.8b02881. 46. Zhang, W.; Shi, Y.; Chen, Y.; Ye, J.; Sha, X.; Fang, X. Multifunctional Pluronic P123/F127 mixed polymeric micelles loaded with paclitaxel for the treatment of multidrug resistant tumors. Biomaterials 2011, 32, 2894-2906, doi:10.1016/j.biomaterials.2010.12.039. 47. Hao, J.; Tong, T.; Jin, K.; Zhuang, Q.; Han, T.; Bi, Y.; Wang, J.; Wang, X. Folic acidfunctionalized drug delivery platform of resveratrol based on Pluronic 127/D-alpha-tocopheryl polyethylene glycol 1000 succinate mixed micelles. Int. J. Nanomed. 2017, 12, 2279-2292, doi:10.2147/IJN.S130094. 48. Chaibundit, C.; Ricardo, G. M. P. S.; Costa, V. d. M. L. L.; Yeates, S. G.; Booth, C. Micellization and Gelation of Mixed Copolymers P123 and F127 in Aqueous Solution. Langmuir 2007, 23, 9229-9236, doi:10.1021/la701157j. 49. Kowalczuk, A.; Trzcinska, R.; Trzebicka, B.; Müller, A. H. E.; Dworak, A.; Tsvetanov, C. B. Loading of polymer nanocarriers: Factors, mechanisms and applications. Prog. Polym. Sci. 2014, 39, 43-86, doi:10.1016/j.progpolymsci.2013.10.004. 50. Yang, X.; Grailer, J. J.; J., R. I.; Javadi, A.; Hurley, S. A.; Matson, V. Z.; Steeber, D. A.; Gong, S. Multifunctional stable and pH responsive polymer vesicles formed by heterofunctional triblock copolymer for targeted anticancer drug delivery and ultrasensitive MR imaging. ACS nano 2010, 4, 6805-6817, doi:10.1021/nn101670k.


27


Page 28 of 46

51. Kim, K. S.; Park, S. J. Effect of porous silica on sustained release behaviors of pH sensitive Pluronic F127/poly(acrylic acid) hydrogels containing tulobuterol. Colloid. Surface. B. 2010, 80, 240-246, doi:10.1016/j.colsurfb.2010.06.017. 52. Lin, X.; Zhu, H.; Luo, Z.; Hong, Y.; Zhang, H.; Liu, X.; Ding, H.; Tian, H.; Yang, Z. Nearinfrared fluorescence imaging of non-Hodgkin's Lymphoma CD20 expression using Cy7conjugated obinutuzumab. Mol. Imaging. Biol. 2014, 16, 877-887, doi:10.1007/s11307-014-07423). 53. Pack, D. W.; Hoffman, A. S.; Pun, S.; Stayton, P. S. Design and development of polymers for gene delivery. Nat. Review. Drug Discov. 2005, 4, 581-593, doi:10.1038/nrd1775. 54. Yang, S. R.; Lee, H. J.; Kim, J. D. Histidine-conjugated poly(amino acid) derivatives for the novel endosomolytic delivery carrier of doxorubicin. J. Control. Release 2006, 114, 60-68, doi:10.1016/j.jconrel.2006.05.016. 55. Varshosaz, J.; Hassanzadeh, F.; Aliabadi, H. S.; Banitalebi, M.; Rostami, M.; Nayebsadrian, M. Novel worm-like amphiphilic micelles of folate-targeted cyclodextrin/retinoic acid for delivery of doxorubicin in KG-1 cells. Colloid. Polym. Sci. 2014, 292, 2647-2662, doi:10.1007/s00396014-3307-8. 56. Lai, X.; Tan, L.; Deng, X.; Liu, J.; Li, A.; Liu, J.; Hu, J. Coordinatively self-assembled luminescent gold nanoparticles: Fluorescence turn-on system for high-efficiency passive tumor imaging. ACS Appl. Mater. Inter. 2017, 9, 5118-5127, doi:10.1021/acsami.6b14681. 57. Geng, X.; Zhang, M.; Lai, X.; Tan, L.; Liu, J.; Yu, M.; Deng, X.; Hu, J.; Li, A. Small-sized cationic miRi-PCNPs selectively target the kidneys for high-efficiency antifibrosis treatment. Adv. Healthc. Mater. 2018, 7, 1800558, doi:10.1002/adhm.201800558.


28

Page 29 of 46 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Scheme 1.

Scheme 1. Schematic diagram of TP-FPNPs preparation and renal-targeting drug delivery.


29


Page 30 of 46

Figure 1.

Figure 1. Physicochemical characteristic of TP-FPNPs. (A) SEM and (B) TEM images of TPFPNPs; (C) Size distribution of TP-FPNPs determined by DLS; (D) Stability of the TP-FPNPs in 10 mM PBS (pH 7.4) and 10% FBS solutions obtained from DLS; (E) Release profiles of TP from free TP and TP-FPNPs in PBS (pH 7.4). Data were expressed as mean ± SD (n = 3).


30

Page 31 of 46 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 2.

Figure 2. Cytotoxicity and cellular uptake. (A) Cytotoxicity of TP and TP-FPNPs in NRK-52E cells after incubated with various concentrations of TP for 24 h. (B) Quantitative fluorescence analysis by flow cytometry of NRK-49F, SiHa and NRK-52E cells incubated with PBS, Cy7-PNPs, Cy7-FPNPs and FA + Cy7-FPNPs. Fluorescent images of (C) Cy7, (D) DAPI staining nucleus and (E) merged of Cy7 and DAPI. Cells were incubated with Cy7-FPNPs for 2 h. Images were taken at 400× magnification. Data were expressed as mean ± SD (n=3).


31


Page 32 of 46

Figure 3.

Figure 3. Ex vivo fluorescence images and biodistribution of Cy7 in organs (1-9: heart, liver, spleen, lung, kidneys, intestine, thymus, muscle and brain) of mice after injection of Cy7-FPNPs at (A) 3, (B) 12, (C) 24 and (D) 48 h; (E) The corresponding fluorescence intensity of Cy7 distribution in each organs examined from (A-D). Data were expressed as mean ± SD (n = 3).


32

Page 33 of 46 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 4.

Figure 4. Pharmacokinetic evaluation of TP-FPNPs. Biodistribution of (A) TP and (B) TP-FPNPs in mice at different time points after intravenous injection. (C) Biodistribution of TP and TPFPNPs in mice at 12 h post-injection. (D) Concentration-time curves of TP and TP-FPNPs in plasma after intravenous injection. Data were expressed as mean ± SD (n=3).


33


Page 34 of 46

Figure 5.

Figure 5. Pathological staining analysis and kidney function index. (A) Histological sections of the kidneys from sham-operated mice, IRI mice and IRI mice treated with TP or TP-FPNPs by H&E staining. (B) Acute tubular injury scored from H&E staining images. (C) SCR and (D) BUN of sham-operated mice, IRI mice and IRI mice treated with TP (0.01 mg/kg) or TP-FPNPs 3 days (where TP concentration was about 0.01 mg/kg). All images were taken at 400× magnification. Data were expressed as mean ± SD (n=5). Statistical significance: *p < 0.05 versus the sham group; #p < 0.05 versus the IRI + TP group.


34

Page 35 of 46 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 6.

Figure 6. Protein expression levels of mice treated with different groups: sham-operated mice, IRI mice and IRI mice treated with TP (0.1 mg/kg for 3 days), TP (0.01 mg/kg for 3 days) or TPFPNPs (equal to 0.01 mg/kg TP for 3 days). (A) Expression levels of p-ERK and T-ERK and (B) Statistic analyses of protein levels of p-ERK, which was normalized by the protein level of TERK. Data were expressed as mean ± SD (n = 5). *p < 0.05 versus the saline group. #p < 0.05 versus the IRI + 0.01 mg/kg TP group.


35


Page 36 of 46

Figure 7.

Figure 7. System toxicity study. (A,B) Representative histomorphologies of livers and testis from mice treated with saline, TP, FPNPs and TP-FPNPs, in which these mice were killed after 3 weeks of treatment. Variations of liver and kidney function indexes of (C) ALT, (D) AST, (E) SCR and (F) BUN after 3 weeks of treatment. Data were expressed as mean ± SD (n=5).


36

Page 37 of 46 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


For Table of Contents Use Only Folate Receptor-mediated Renal-targeting Nanoplatform for the Specific Delivery of Triptolide to Treat Renal Ischemia/Reperfusion Injury Caili Huang,‡a Tao Zeng,‡b Jiawen Li,‡b Lishan Tan,b Xiulong Deng,a Yanchao Pan,a Qi Chen,a Aiqing Li,*b and Jianqiang Hu*a


37


Scheme 1 177x150mm (300 x 300 DPI)


Page 38 of 46

Page 39 of 46 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 1.



Figure 2 177x119mm (300 x 300 DPI)


Page 40 of 46

Page 41 of 46 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 3.



Figure 4.


Page 42 of 46

Page 43 of 46 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 5.



Figure 6. 150x150mm (300 x 300 DPI)


Page 44 of 46

Page 45 of 46 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 7. 145x150mm (300 x 300 DPI)



Table of Contents 69x35mm (600 x 600 DPI)


Page 46 of 46

Folate Receptor-Mediated Renal-Targeting Nanoplatform for the

Recommend Documents