CXCR4-Receptor-Targeted Liposomes for the Treatment of Peritoneal

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CXCR4-Receptor Targeted Liposomes for the Treatment of Peritoneal Fibrosis Khan Asifullah, Zhanwei Zhou, Weiming He, Kun Gao, Muhammad Waseem Khan, Raza Faisal, Hasnat Muhammad, and Minjie Sun Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.9b00266 • Publication Date (Web): 09 May 2019 Downloaded from http://pubs.acs.org on May 9, 2019

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Molecular Pharmaceutics

CXCR4-Receptor Targeted Liposomes for the Treatment of Peritoneal Fibrosis Khan Asifullah a, Zhanwei Zhou a, Weiming He b, Kun Gao b, Muhammad Waseem Khan c, Raza Faisal a, Hasnat Muhammad a, Minjie Sun a,* aState

key laboratory of Natural Medicine and Department of Pharmaceutics, China

Pharmaceutical University Nanjing, 210009 China bDivision

of Nephrology, Affiliated Hospital of Nanjing University of Chinese Medicine,

Nanjing, 210029 China cSchool

of Pharmacy, Tongji Medical College, Huazhong University of science and technology,

Wuhan Hubei 430030, China

*Corresponding Author:

E-mail address: [email protected] (Minjie Sun)

ACS Paragon Plus Environment

Molecular Pharmaceutics 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

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Abstract:

Peritoneal fibrosis (PF) is a common complication of long term peritoneal dialysis (PD). It is considered the main reason for dialysis inadequacy and PD withdrawal. Transforming growth factor beta (TGF-β) regulates the expression of stromal cellsderived factor -1(SDF-1α) and its receptor C-X-C chemokine receptor type4 (CXCR4) on human peritoneal mesothelial cells (HPMCs), resulting in an increased migratory potential of HPMCs and extracellular matrix (ECM) deposition in scar tissue and eventually fibrosis. Since SDF-1α/CXCR-4 activation has a vital role in the pathogenesis of PF, co-delivery of a CXCR4-receptor targeting agent with an anti-fibrotic agent in a single nanocarrier can be a promising strategy for treating PF. Here for the first time AMD3100

(AMD),

a

CXCR4-receptor

antagonist

was

co-formulated

with

sulfotanshinone IIA sodium (STS IIA) into a liposome (STS-AMD-Lips), to develop a CXCR4-receptor targeting form of combination therapy for PF. CXCR4-targeting increased the ability of liposomes to target fibrotic peritoneal mesothelial cells (PMCs) overexpressing CXCR4 and facilitated the ability of STS IIA treatment at the fibrotic site.


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The liposome had an average diameter of 103 nm with encapsulated efficiencies of above 50%. The in vivo studies confirmed the reversal of peritoneal dialysis solution (PDS)-induced epithelial-mesenchymal transition (EMT) by STS-AMD-Lips in HPMCs. The in vivo studies also revealed the precise biodistribution of the liposomes to Peritoneum. Significant reduction of the morphological lesions and decreased level of ECM proteins were observed in rats treated with STS-AMD-Lips, proving that the liposomal nanocarrier has excellent ability to reverse PF. It has been concluded that the STS-AMD-Lips exhibit specific peritoneal targeting ability and could be used to improve STS-AMD combination delivery for treatment of PF.

Keywords:

Peritoneal dialysis; CXCR4; peritoneal fibrosis; AMD3100; Sulfotanshinone IIA sodium

1. Introduction Peritoneal dialysis (PD) is an effective and well-established renal replacement therapy presently used for patients with end-stage kidney disease. However, exposure to non-



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physiological fluids during long-term PD causes deterioration and alteration to peritoneal membranes and eventually fibrosis. Peritoneal fibrosis (PF) is accompanied by ultrafiltration failure, resulting in termination of PD in patients.1,

2, 3

Moreover, PF also

leads to progression of some most serious complications. Encapsulating peritoneal sclerosis (EPS) is the most serious and pondered to be one of the reasons for PD avoidance in patients with end-stage renal disease (ESRD).4, 5

Histologically PF is described by the myofibroblast proliferation with increased production and accumulation of the extracellular matrix (ECM) in the peritoneal mesothelium upon undertaking PD.6, 7 Mechanism of the pathogenesis of PF has been extensively established through ample research studies. Epithelial-to-mesenchymal transition (EMT) of human peritoneal mesothelial cells (HPMCs) is believed to be the main mechanism involved in the origination and advancement of PF.8, 9, 10, 11 Long term exposure to PD solution induces oxidative stress in HPMCs and initiates Mitogenactivated protein kinase p38 (p38MAPK) pathway which is involved in activation of TGFβ.12,

13, 14

TGF-β regulates the expression of SDF-1α and its receptor CXCR4 on


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HPMCs, resulting in an increased migratory potential of HPMCs and extracellular matrix (ECM) deposition in scar tissue and fibrosis.15, 16

The discovery of PF mechanism and characterization of fibrosis-promoting molecules or pathways in PMCs have provided new insights into treating PF. The current therapeutic strategies mainly evolved around these mechanisms and include; Strategies for inhibiting angiogenesis, Strategies for reducing inflammation, Strategies for inhibiting collagen synthesis, Strategies for reducing oxidative stress.17 Angiogenesis occurs during the advancement of various pathologic disorders, including fibrosis. It is established that the inhibition of angiogenesis is a suitable strategy to prevent PF. Hiroaki et al

18

reported the blockade of vascular endothelial growth factor (VEGF) by the neutralizing

antibody significantly attenuated the formation of new blood vessels (neoangiogenesis) and thickening of the submesothelial compact zone. Inflammation is another most important factor contributing to PF. Targeting macrophages infiltration is a promising approach to inhibit inflammation and PF. Kushiyama T et al

19

employed liposomal clodronate in a rat model to

deplete macrophages and reverse PF. Collagen synthesis leads to ECM deposition and plays a vital role in the progression of PF. Heat shock protein 47 (HSP47) is a collagen-specific chaperone, co-expressed in α–SMA positive myofibroblast and collagen III and is essential for the biosynthesis and secretion of collagen. Targeting HSP47 is also a potential therapeutic candidate in PF.20, 21 Increase in oxidative stress has been proposed to perform an important part in PF. Yao Zhou et al

22

demonstrated that STS IIA ameliorate PDS induced HPMC injury via

suppression of ASK1-P38-mediated oxidative stress. These approaches have been reported to be



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promising, however, they might have limitations and there is an urgent need for the development of novel approaches to enhance the prognosis of all PD patients.23,

24

CXCR-4-receptor overexpressed on HPMCs 15, 25 could be an excellent novel target and it is possible to combine a CXCR4-receptor antagonist with another antifibrotic agent to provide both targeting and combine anti-fibrotic effect for enhanced therapy.

Here we hypothesized that AMD3100-equipped liposomes loaded with STS (STS-AMDLips) can be utilized to target CXCR4-overexpressing HPMCs and treat PF. AMD3100, a bicyclam derivative selectively antagonizes the CXCR4.26 The AMD chemotactic response was caused by the CXCR4/SDF-1 axis in various cells.27, 26 It is considered an excellent choice for CXCR4 specific therapies.28AMD3100 attenuates TGF-β1 stimulated proliferation and migration of cells.29TanshinoneIIa (Danshen) is the most abundant and therapeutically active natural constituent obtained from the root of Salvia miltiorrhiza. TanshinoneIIa is widely used in many cardiovascular diseases.30 Sulfotanshinone IIA Sodium (STS IIA) is a new hydrophilic derivative of tanshinone IIA. Its therapeutic use is well established in various cardiovascular and cerebrovascular


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pathologies.31, 32, 33, 34, 35 Latest studies revealed that STS IIA exerts a protective effect in HPMCs undergoing PD.36,

37

Liposomes (a class based on phospholipids) have

attracted much more attention than the other systems due to various meritorious features in delivering the various drugs to the target site.38 Therefore, a CXCR4targeted liposome (STS-AMD-Lips) was formulated to target peritoneal fibrosis via a multifunctional nanocarrier co-delivering AMD3100 and STS IIA. AMD3100, incorporated into the liposomes, aided dual functions; it worked as a targeting moiety and also inhibited the advancement of fibrosis.39, 40 STS IIA protect HPMCs against toxicity of glucose-based PD solution through inhibition of p38 MAPK activated oxidative stress22 as shown in the scheme. 1. We studied the Cell viability and cellular uptake of STS-AMD-Lips in the HPMC. We also examined the CXCR4 antagonism and peritoneal targeting by STSAMD-Lips both in vitro and in vivo. Lastly, we verified the anti-fibrotic effect of STSAMD-Lips in PDS induced PF model /SD rats.



Scheme 1. Schematic illustration of the design and mechanism of action of STS-AMDLips in the treatment of peritoneal fibrosis (PF), peritoneal administration of STS-AMDLips achieves superior antifibrotic effect due to combining CXCR4 antagonism and p38MAPK inhibition. AMD3100 improves the STS-AMD-Lips ability to target HPMCs overexpressing CXCR4 and also assist STS IIA treatment at the fibrotic site.

2. Materials and methods

2.1 Materials


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Soybean phosphatidylcholine (PC) was purchased from Toshisun Biology& Technology Co., Ltd. (Shanghai, China) and Shanghai A.V.T. Pharmaceutical Co. Ltd. (Shanghai, China). Sulfotanshinone IIA sodium (STS IIA) was from Shanghai Pharma (Shanghai China). AMD3100 was from Biochempartner, Inc. (Shanghai, China). PDS was from Baxter (Baxter, China), IR780 was from Sigma Aldrich (Saint Louis, Missouri, United States). Methylglyoxal solution (MGO) obtained from Sigma-Aldrich. Coumarin6 (C6) was from TCI (Japan). Fetal bovine serum (FBS) from Hyclone (GE healthcare life sciences) and Human SDF-1α from PeproTech, Inc. (USA), RPMI-1640 medium were from KeyGen biotech (Nanjing, China).DMEM medium from Gibco (Thermo Fisher, Scientific). Cell counting Kit-8 reagent (CCK-8) was from Dojindo Inc.(Shanghai China) Antibodies against phosphor-p38 mitogen-activated protein kinase (MAPK) were from CST Reagents Company Limited (Shanghai, China) while antibodies against α-SMA and CXCR4 were from Abcam (Cambridge, MA).

2.2. Cell culture and animals

HPMCs (HMrSv5) were cultured in RPMI-1640 supplemented with 10% FBS, 100 U/mL



penicillin G, 100 mg/mL streptomycin, and 0.25 mg/mL amphotericin B at 37 °C in 5% CO2.41 U2OS cells cultured in DMEM supplemented with 10% FBS, 2×10-3M LGlutamine, 1% Pen-Strep and 0.5 mg/mL G418. U2OS express functional EGFPCXCR4 fusion proteins.

Female BALB/c mice (20-25 g) and SD rats weighing 180-200 g were supplied by the Qinglong mountain animal farm, Nanjing. All animal studies were performed according to the official protocols assessed and approved by the ethical committee of China Pharmaceutical University.

2.3. Preparation and characterization of STS-AMD-Lips

Solvent evaporation and hydration method were used to prepare the liposome.42 In brief, for Drug loaded liposome 75 mg phospholipid, 30 mg cholesterol was well dissolved in a 40 mL solvents mixture of ethanol and dichloromethane (4:1, v/v). AMD was added to this mixture of solvent. The vacuum was applied for 1h at 32 ℃ to evaporate the organic solvents. The dried film obtained after solvent evaporation was hydrated with 5 mL distilled water containing STS IIA at 34 ℃ for about 30 minutes. The


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resulting solution was subsequently emulsified by ultra-sonication (800 s at 300 W/cm2). After preparation, the un-entrapped STS IIA was separated by a cellulose nitrate membrane filter (0.22 µm). The un-encapsulated AMD3100 was eliminated by ultrafiltration tubes (Millipore, USA) applying centrifugation at 4 ℃ at 12,000 rpm for 30 minutes. Zeta potential, Particle size, and a polydispersity index of the STS-AMD-Lips were evaluated by zetaPlus (Brookhaven instruments corporation, USA). The morphology and size distribution of STS-AMD-Lips was revealed by transmission electron microscopy with H-600; TEM (Hitachi, Japan). The amount of AMD3100 and STS IIA in drug-loaded liposomes (STS-AMD-Lips) were examined using a highperformance liquid chromatography system (HPLC, Waters Alliance Corporation, USA). For the detection of STS IIA and AMD3100, the absorption wavelengths were set at 270 nm and 215 nm. The eluent comprised of a mixture of methanol, acetonitrile, and triethylamine (37:2:61, v/v/v) for STS IIA and for AMD3100 mixture of tetra butyl ammonium hydrogen sulfate (10 mM, pH3.3) and acetonitrile (58:42, v/v). The flow set to 1 mL/min. The separation was achieved using a diamond ODS C18 column (150 mm × 4.6 mm ×5 µm).43,

44, 45

The drug encapsulation efficiency (EE %) of STS IIA and



AMD3100 was analyzed according to equation (Eq). (1) :

EE (%) =We/Wt×100

Where We was the analyzed concentration of entrapped STS IIA and AMD3100 in STSAMD-lips; Wt was the total analyzed concentration of STS IIA and AMD3100.

2.4. In vitro drug release The in vitro drug release of STS IIA and AMD3100 from the free drug and liposomes was done by taking known quantity (1 mL) of the drug-loaded liposome (STS-AMD-Lips) in dialysis bag. The dialysis bag was then incubated into a beaker containing 20 mL of the release medium, phosphate-buffered saline (PBS) pH 7.4 at 37 ℃ under mild agitation in a water bath, hence complying with the sink conditions for experiments. At a predetermined time (0 h, 0.3 h, 2 h, 4 h, 12 h, 24 h, 48 h, and 92 h) samples (500 µL) was taken and analyzed for STS IIA and AMD3100 by HPLC. The percentage of the released drug was determined via the following Eq (2):

Release (%) = Drugreleased / DrugLoaded


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2.5. In vitro stability study of liposomes

For the stability study, STS-AMD-Lips were incubated in 10 mM PBS having 140 mM NaCl (pH 7.4) at 37 °C under moderate stirring. At given time intervals (0 h, 12 h, 24 h, 48 h, and 72 h) 2 mL solution was withdrawn and polydispersity index and particle size were measured by DLS.

2.6. Assessment of Cell Viability HPMCs were inoculated into 96-well plates in RPMI1640 media having 10% FBS and kept at 37 °C in a humidified incubator of 95% air and 5% CO2. The cells were allowed to grow until confluence. After getting proper confluence standard medium containing 10% FBS were removed and cells were exposed to different concentrations of reagents and drug formulations (Free dug mix, STS-AMD-Lips) for indicated time points. 10 µL of CCK-8 solution was added into to each well and further incubated for 1 h in an incubator. The optical density was assessed with a spectrophotometer (Synergy2 multifunctional microplate reader) at a wavelength of 450 nm. Each measurement was performed in triplicate. Cell viability was demonstrated as a percentage of the control



group and calculated according to the Eq. (3):

Cell viability (%) = (ODsample / ODcontrol) × 100

Where ODsample was the optical density of the cells groups exposed to different reagents and formulations; ODcontrol indicated the optical density of untreated cells groups.

2.7. CXCR4 redistribution assay 46

CXCR4 redistribution assay was utilized to determine the antagonizing ability of STSAMD-Lip against CXCR4. Briefly, U2OS cells bearing (enhanced green fluorescent protein) EGFP-CXCR4 receptors were cultured in 96 wells black plates (each well carrying 8000 cells) with optical bottoms at least 24 hours before the experiment. The cells were initially washed with 100 µL of assay buffer (DMEM medium supplemented with 1% FBS, 2 mM L-glutamine, 1% Penicillin-Streptomycin and 10 mM HEPES) and then incubated with STS-Lips and STS-AMD-Lips in assay buffer containing 0.25% DMSO at 37 °C for 30 minutes. Afterward, SDF-1α at a concentration of 10 nM was added to each well. The negative control was assigned to cells treated with SDF-1α alone. After 1 hour of incubation, fixation of the cell was done with 4% formaldehyde at


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room temperature for 20 minutes. The cells were then washed with PBS 4 times. Hoechst (1 mM) in PBS containing 0.5% Triton X-100 was added to stain the nuclei. EVOS FL microscope was used to take Images at 20 X magnification.

2.8. In vitro cellular uptake

The cellular uptake STS-AMD-Lips was examined by employing confocal laser scanning microscope (CLSM) and flow cytometry together, to confirm the CXCR4 receptor targeting efficiency and reflect the intracellular mechanism of the liposomes. We used coumarin6 (C6) as the tracer molecule. C6 solution, C6-Lips, and C6-AMD-Lips was dispersed in FBS free RPMI1640 media and was added to PDS treated HPMCs. The cells were seeded in 35 mm dishes and cultured at 37 ℃. After incubation for 4 h, the old medium was removed and washed twice with ice-cold buffer. Then 4, 6 diamidino-2phenylindole (DAPI) was used for 15 minutes to stain the cells nuclei. Subsequently, the cells were examined by a confocal laser scanning microscope (CLSM, Zeiss).

Flow cytometry analysis carried out described briefly as; HPMCs were seeded in 24 well plates (104 cells/well) and incubated in RPMI1640 medium for 24 h. To assess time-



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dependent cellular uptake mechanism, culture medium was exchanged and cells were treated with C6-AMD-Lips having C6 at concentrations of 0.5 mg/mL and incubated at 37 ℃ for 1, 2 and 4 h. Cells were washed with cold PBS thrice, collected by Trypsinization and the fluorescence intensity was determined by a flow cytometer.

To examine the CXCR4 mediated uptake of STS-AMD-Lips HPMCs were incubated with free C6, C6-Lips, C6-AMD-Lips and C6-AMD-Lips (prior treated with AMD) for 4 h. The C6 concentration in all formulations is 0.25, 0.5 or 0.1 µg/mL. After incubation at 37 ℃, cells were washed with the cold PBS thrice and collected carefully by Trypsinization. Subsequently, fluorescence intensity was determined by a flow cytometer.

2.9. Western blot analysis

Western blotting procedures used were described in.22 Briefly, protein lysates extracted from

cells

were

resolved

by

10%

or

7.5%

SDS-polyacrylamide

gels

and

electrophoretically transferred to polyvinylidene difluoride (PVD) membrane using protein electrophoresis and blotting apparatus (Bio-Red, China). Following blocking in 5% fat-free dry milk in PBS for 2 h at room temperature, the membrane was probed with


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primary antibodies overnight at 4 ℃. The membrane was washed and incubated with the secondary antibody (anti-rabbit IgG horseradish peroxidase-conjugated antibody) for 1 h at room temperature. ChemiDoc imaging system (Bio-Red) was used to anticipate the blotting bands. β-Tubulin or β-Actin were used as internal controls.

2.10. Pharmacokinetics study

SD rats weighing 180-200 g were divided into three different groups i.e. free STS IIA, free AMD3100, and STS-AMD-Lips. Each group assigned 4 rats (n=4). Rats were treated by intravenous (i.v) injection at a dose of 10 mg/kg equivalent. At predetermined time intervals (0.17, 0.5, 1, 2, 4, 8, 12 and 24 h) blood sample was taken in heparin tubes and centrifuged for 10 minutes at 5000 rpm. The plasma supernatant was isolated carefully and analyzed with HPLC.43,

45

PK-Solver was used to calculate the

pharmacokinetic parameters.

2.11. In vivo imaging and biodistribution analysis

PDS induced peritoneal fibrosis BALB/C mice were used to investigate the biodistribution of formulations. The mice used in this study were divided into two groups



(n=3). IR70-Lips and IR70-AMD-Lips formulations were administered to mice via tail vein injection. At time points of 2 h, 4 h, 8 h, and 24 h, images were taken using the in

vivo imaging system. After 12 hours post-treatment, the mice were sacrificed; organs were separated and washed with PBS to verify the biodistribution analysis. Accumulation and retention of dye were determined and evaluated by taking images using Carestream Molecular Imaging Software V5.3.5. The IR780 dye was fixed at excitation and emission wavelength of 720 nm and 790 nm respectively.

2.12. Histology and immunochemistry

To evaluate the STS-AMD-Lips effect on cell density and thickening in PDS induced peritoneal fibrosis, we carried out Hematoxylin-eosin staining (H&E) and Masson’s trichrome staining. This study was performed in SD rats having 180-200 g weight. The rats were assigned randomly into four different treatment groups (n=5) i.e. (1) Control group, that received 20 mL saline via i.v injection (2) PDS group, intraperitoneal injection (IP) of 20 mL 40 mM MGO +PDS (3) Free drug mix, i.v injection of free drug STS and AMD at STS IIA dose of 10 mg/kg (4) i.v injection of STS-AMD-Lips at STS IIA


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dose of 10 mg/kg. These solutions were administered for 3 weeks. On day 21, rats were sacrificed and peritoneum tissue of the upper right wall of the abdominal wall was collected.

For histological analysis peritoneum tissue was fixed in 4% formaldehyde solution and embedded in paraffin. Sections were made at 4 µm thick, deparaffined and stained using HE and Masson’s reagent according to the manufacturer’s manual. Sections were then observed under light microscopy, photographed and quantified in 10 selected fields (200X). For immunostaining analysis, Paraffin section was first boiled in 10 nM Nacitrate solution (pH 6.0) for 10 minutes to retrieve antigens and then stained with an antibody against TGF- β1, α-SMA, collagen I and collagen III for light microscope analysis. The number of positive cells for α-SMA and TGF-β1 were counted in the submesothelial dense region in 10 fields at 200X magnification. The area having collagen I and collagen III were evaluated at 200X magnification in the predetermined fields of the dense region taken by a digital camera and evaluated by using ImageJ in 10 fields.



2.13. Biological safety evaluation of STS-AMD-Lips

The male SD rats were used in this study. Rats were allocated into 4 different groups (n=5) and different formulations including saline, PDS+MGO (IP), Free drug mix, and STS-AMD-Lips were injected by i.v tail injections at a concentration of 10 mg/kg every day for 3 weeks and body weight changes were monitored after every 3-days.

2.14. Statistical analysis

All experiments were done in triplicate. Data were analyzed using Prism 6 (GraphPad Software, San Diego, CA). Data were expressed as the mean ± standard deviation (SD). Statistical analysis was performed using Student’s t-tests or analysis of variance (ANOVA). Comparisons between two groups were made using student’s t-test and ANOVA followed by Tukey’s test for analysis of more than two groups. Statistical significance was set at *P

CXCR4-Receptor-Targeted Liposomes for the Treatment of Peritoneal

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