Sialic Acid-Functionalized PEG-PLGA Microspheres Loading

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Sialic Acid-Functionalized PEG-PLGA Microspheres Loading Mitochondrial Targeting Modified Curcumin for Acute Lung Injury Therapy Fei-Yang Jin, Di Liu, Hui Yu, Jing Qi, Yuchan You, Xiaoling Xu, Xuqi Kang, Xiaojuan Wang, Kongjun Lu, Xiao-Ying Ying, Jian You, Yong-Zhong Du, and Jiansong Ji Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/ acs.molpharmaceut.8b00861 • Publication Date (Web): 15 Nov 2018 Downloaded from http://pubs.acs.org on November 16, 2018

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Molecular Pharmaceutics

Sialic Acid-Functionalized PEG-PLGA Microspheres Loading Mitochondrial Targeting Modified Curcumin for Acute Lung Injury Therapy Feiyang Jin1, Di Liu1, Hui Yu1, Jing Qi1, Yuchan You1, Xiaoling Xu1, Xuqi Kang1, Xiaojuan Wang1, Kongjun Lu1, Xiaoying Ying1, Jian You1, Yongzhong Du1,*, Jiansong Ji2,* 1Institute

of Pharmaceutics, College of Pharmaceutical Sciences, Zhejiang University, 866 Yu-Hang-

Tang Road, Hangzhou, 310058, China. E-mail: [email protected] 2Key

Laboratory of Imaging Diagnosis and Minimally Invasive Intervention Research, Lishui Hospital

of Zhejiang University, Lishui 323000, China. E-mail: [email protected]

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: Acute lung injury (ALI) is a serious illness without resultful therapeutic methods commonly. Recent studies indicate the importance of oxidative stress in the occurrence and development of ALI, and mitochondria targeted antioxidant has become a difficult and hot topic in the research of ALI. Therefore, a sialic acid (SA) modified lung-targeted microsphere (MS) for ALI therapy are developed, with triphenylphosphonium cation (TPP) modified curcumin (Cur-TPP) loaded, which could specific target to mitochondria increasing the effect of antioxidant. The results manifest that with the increase of microsphere, lung distribution of microsphere is also increased in murine mice, and after SA modified, microsphere exhibit ideal lung-targeted characteristic in ALI model mice, due to SA efficiently target to E-selectin expressed on inflammatory tissues. Further investigations indicate that SA/Cur-TPP/MS has better antioxidative capacity, decrease intracellular ROS generation, increase mitochondrial membrane potential, contributing to a lower apoptosis rate in HUVECs compared to H2O2 group. In vivo efficacy of SA/Cur-TPP/MS demonstrate that the inflammation has been alleviated markedly and oxidative stress is ameliorated efficiently. Significant histological improvements by SA/Cur-TPP/MS are further proved via HE stains. In conclusion, SA/Cur-TPP/MS might act as a promising drug formulation for ALI therapy. KEYWORDS: acute lung injury, curcumin, mitochondrial targeting, sialic acid, microsphere

1. Introduction As known to all, acute lung injury (ALI) has a close relationship with acute respiratory distress syndrome (ARDS), which does great harm to human body. ALI was determined via serious inflammation with activated neutrophils infiltration into lung tissues and pulmonary edema filled with protein subsequently, the formation of hyaline membrane and interstitial fibrosis, finally with different


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degrees of consequences.1, 2 Despite recent therapeutic progress in pharmacotherapies and supportive care, the mortality and morbidity in ALI/ARDS remain a high level (30%-40%).3, 4 Thus, it is important to find an effective therapeutic approach with less systemic toxicity for ALI cure. Recent research points out that oxidative stress plays a vital role in occurrence and development of ALI, especially the imbalance between the development of reactive oxygen forms and activity of antioxidants has been taken serious increasingly.5 In the early acute lung injury, a great number of neutrophils are activated, releasing a large amount of hydrogen peroxide and oxygen free radicals to eliminate pathogen.6 However, excessive amounts of reactive oxygen species (ROS) can be produced by neutrophils, pulmonary microvascular endothelial cells, alveolar macrophages, and lung parenchyma cells, which are severely traumatized or infected during ALI, resulting acute oxidative stress injury and irreversible damage in normal tissues and cells.7 After the damage of the alveolar barrier, a great number of tissue fluids infiltration aggravate pulmonary edema, and the damaged lung function makes the blood oxygen saturation drop sharply, thus causing the body to be in a serious anoxic state and endanger the safety of life.8 Thus, effective antioxidant and oxygen free radical scavengers are expected to become potential treatment for future ALI therapy.9 It is known that mitochondria are the main organelle attacked by excess ROS in cells. When ALI occurs, the NADPH oxidase in pulmonary vascular endothelial cells is activated to promote the production of ROS in the mitochondria.10 The released ROS can further damage the mitochondrial membrane through the lipid membrane oxidation, which leads to the activation of mitochondrial inherent apoptotic pathway. The apoptotic Bcl-2 family member: BAK is significantly increased, and the apoptotic factor Caspase-3 is also activated, leading to cell apoptosis.11 Thus, there are many basic studies on the treatment of acute lung injury with antioxidant drugs, however, with few clinical



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applications. The important reason is that most antioxidants do not work in the mitochondria, where produce the most oxidative stress factor and suffer the most severe ROS damage.12 So, how to deliver antioxidants into mitochondria to play its therapeutic effect, has become a difficult and hot topic in the research of ALI. Nowadays, there are many mitochondrial targeting ligands applied in the research, including lipophilic cation and mitochondrial penetrating peptides. The most widely used and studied ligand is triphenylphosphonium cation (TPP) and its derivatives, which has three benzene rings, that can increase the molecular surface area and form delocalized positive charges, which can penetrate the double layer hydrophobic membrane of mitochondria.13 Curcumin (Cur) derived from the rhizome of Curcuma longa is a natural polyphenolic pigment, that has a wide range of pharmacological activities, including antioxidant, anti-inflammatory, antiinfection and anti-tumor capacity.14-16 Curcumin has many functional groups, such as two phthalein backbone and benzene ring, phenolic hydroxyl groups and methoxy and propylene groups enable curcumin to have a strong antioxidant activity.17 Curcumin can also remove superoxide and peroxide, and its scavenging ability is stronger than vitamin E.18 Curcumin has shown significant antiinflammatory effects in ALI phenotypes, which include amelioration of neutrophil recruitment and activation, lung edema, inflammation and cytokine release most likely by affecting NF-κB activation pathway in several ALI models.19 Also, clinical trials have proved that Cur is innoxious to human body when taken at a high dose of 8 g a day for 3 months.20 However, due to poor water solubility and fast degradation resulting low bioavailability, the clinical application of Cur is limited.21 Therefore, it is necessary to develop a drug delivery system of Cur formulation for better treatment efficacy. We intend to use TPP to modify the Cur via chemical bonding to synthesize Cur prodrug (Cur-TPP), which has


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good mitochondrial targeting ability and better ability to scavenge ROS to reduce oxidative stress damage compared to previous curcumin delivery system. And then, considering its low bioavailability, Poly (lactic-co-glycolic acid) (PLGA) polymer modified with Polyethylene glycol (PEG) is chosen to fabricate a pulmonary targeting microspheres, and simultaneously Cur-TPP is entrapped. The major challenge to synthesis a pulmonary targeting microspheres is to control the size range (0.5 – 5 μM) for particles to be trapped by pulmonary capillaries.22 PLGA, the US Food and Drug Administration (FDA) approved biodegradable polymer for in vivo use, is now applied in several parenteral microspheres in the market. Because of PLGA possess a good biocompatibility and sustained slow drug release, it is proper to be applied in an injectable microsphere system, which shows good characteristics to deliver Cur in vivo for a long-term use. To further establish a drug delivery system effectively targeted to inflammatory pulmonary tissues, a small natural molecular: sialic acid (SA), was applied to modify the microspheres on its surface. It has been well proved that SA as the termini of mammalian cell surface glycolipids locating in the outmost layer of cell membranes that could specific bind to E-selectin.

23

Previous researches

indicated that SA was an effective ligand for active targeting activity for tumor metastases,24 rheumatoid arthritis25 and acute kidney injury26 by modulating cell to cell interactions among endothelial cells, leukocytes and tumor cells. In the development of ALI, vascular endothelial cells (VECs) could highly and stably express integrin, E-selectin and other adhesive molecules due to inflammatory responses. SA-modified polymer microspheres due to E-selectin over expressed on inflammatory VECs could be an accurate cure to deliver drugs at ALI sites. In this study, we report a SA-induced Cur-TPP loaded multifunctional drug delivery system (Abstract Graphic), which could bind with E-selectin expressed on inflammatory pulmonary



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endothelium and improve accumulation of microspheres into ALI tissues, with sustained releasing Cur-TPP targeting to mitochondria to exert potential treatment efficacy. Cur was modified with TPP via chemical bonding, and SA-PEG-PLGA conjugate was synthesized by esterification reaction, and then SA modified microspheres loaded with Cur-TPP were fabricated via emulsion solvent evaporation method. The characteristics of microspheres including drug loading, entrapment efficiency, potential, size and in vitro drug release profile and cytotoxicity, cellular uptake and in vivo distribution were examined in detail. The therapeutic potential of microspheres was evaluated in LPSactivated ALI murine model, and the pharmacodynamics was determined.

2. Experimental section 2.1 Material and animals: Curcumin was purchased from Sigma Aladdin (Shanghai, China). Sialic acid was obtained from Sigma Aladdin (Shanghai, China). HOOC-PEG-COOH (MW = 2.0 kDa) was obtained

from

Dai

Gang

Biotechnology

(Jinan,

China),

USA.

MPEG-PLGA

(MW=2000,15000,PLGA:75/25) and OH-PLGA-COOR (MW=15000, LA/GA = 50 ∶ 50) were purchased from Dai Gang Biotechnology (Jinan, China). 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) and ICG were purchased from Sigma Aladdin (Shanghai, China). Primary antibodies, including Anti-Bcl-2, Anti-Bax, Anti-CD62E, Anti-caspase-3 antibodies were obtained from Abcam (UK). The ELISA kits of IL-6 and TNF-α were purchased from Boster Biotechnology (Wuhan, China). Anti-RAGE, Anti-CD68, Anti-SP-D antibodies were purchased from Proteintech (Wuhan, China). ICR male mice aged 3-4 weeks had 20-30g weights were obtained from Zhejiang Medical Animal Centre (Hangzhou, China). 2.2 Cell culture: The human umbilical vein endothelial cells (HUVECs) were obtained from Chinese academy of sciences cell bank (Shanghai, China). HUVECs were maintained in DMEM high glucose


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medium supplemented with 10 % fetal bovine serum (Gibco) and 1 % penicillin and streptomycin and were incubated at 37 °C in a humidified atmosphere of 5 % CO2. 2.3 Synthesis and characterization of Microsphere: Microspheres were fabricated via a modified method of the oil-in-water(O/W) emulsion solvent evaporation. Firstly, PEG2K-PLGA15K (60mg) were dissolved in dichloromethane (1.5ml) and ultrasonicated until polymer were completely dissolved to form the oil (O) phase. The oil phase was then emulsified in 1% (w/v) aqueous PVA solution (15ml) applying a probe-type ultrasonicator at different ultrasonic power and duration as shown in Table 1 to obtain MS-1, MS-2 and MS-3. Then the emulsion was agitated on a magnetic stirrer at room temperature until solvent removal. Microspheres were then recovered by centrifuging at 16000 × g for 5 minutes. The microspheres were then washed three times with deionized water and lyophilized. The microsphere size and its distributions were measured by DLS after feasible dilution. 2.4 Cell uptake of various size of microspheres: To evaluate cellular uptake of MS-1, MS-2 and MS3, drug curcumin(Cur) was entrapped into microspheres using previous method. HUVECs were seeded in a 12-well plate at a density of 1 × 105 cells/well and incubated overnight. Then the cells were incubated with Cur-loaded MS-1, MS-2, MS-3(1μM) respectively for 2, 4, 8 and 12 hours in 37℃. Afterwards, the medium was removed and the cells were resuspended in PBS and fluorescence intensity was measured by flow cytometry. 2.5 Biodistribution of various size of microsphere-ICG in mice: Tetrabutylammonium iodide and ICG were weighed according to 1:2 molecular molar ratio and dissolved in DMSO stirring overnight. The ICG-tetrabutylammonium iodide complexes was then formed and entrapped into microspheres. The ICR mice were intravenously injected with ICG-tetrabutylammonium iodide complex-loaded MS1, MS-2 and MS-3 at a dose of 1.0 mg/kg. After 6 hours, mice were sacrificed and organs were harvest.



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The Maestro in vivo imaging system was used to measure the fluorescence signal in organs. 2.6 Synthesis and characterization of Cur-TPP: Synthesis of Curcumin-(4-Carboxybutyl) Triphenylphosphonium (Cur-TPP) conjugate was accomplished as described (Figure S1). Curcumin (Cur) (1.995g, 0.005mol), (4-carboxybutyl) triphenyl bromide (TPP) (2.412g, 0.005mol) were placed in a dry round bottom flask with a magnetic stirring bar, adding 30mL anhydrous dimethyl sulfoxide(DMSO). After dissolved, dichlorosulfoxide (SOCl2) (0.4ml, 0.0075mol) was brought in. The reaction mixture was stirred at 60℃ for 24 hours with nitrogen protection. Then reaction solution was then dialyzed using deionized water for 2 days. Finally, the precipitation product was lyophilized and Cur-TPP was got. The structure of Cur-TPP was confirmed by 1H NMR. Samples were dissolved at 15 mg·mL-1 in dimethylsulfoxide-d6 for measurement. 2.7 Synthesis and characterization of SA-PEG-PLGA conjugates: SA-PEG-PLGA conjugates were compounded according to the method reported previously 24 and obtained via esterification reaction in two steps’ reaction: (1) esterification reaction between hydroxyl groups of PLGA and carboxyl groups of PEG; (2) esterification reaction between hydroxyl groups of SA and carboxyl groups of PEG-PLGA. Briefly, PEG (200 mg, 0.1 mmol), DCC (123.6 mg, 0.6 mmol) and DMAP (7.32 mg, 0.06 mmol) were dissolved DMSO. The solution was stirred for 1 h under nitrogen protection at 60 ℃ to activate the carboxyl groups. After that, DMSO solution containing PLGA (400 mg, 0.1 mmol) was added. The mixture solution was kept stirring for a day. Then, SA (30.9 mg, 0.1 mmol) was added and the reaction was carried out for another 24 hours. The resultant solution was further dialyzed with distilled water (DI) water for 48 h to remove hydrophilic impurities. Then, the polymer was obtained after centrifuge at 5000 rpm for 15 minutes to remove water-insoluble byproducts and lyophilized for further use.


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2.8 Sialic acid modified microsphere fabrication and drug loading: SA-PEG-PLGA (6mg) and PEG-PLGA (60mg) and moderate Cur-TPP were dissolved in a 4:1 mixture of Dichloromethane (1.2mL) and ethyl acetate (0.4mL) and ultrasonicated until both polymer and drug were completely dissolved. The 25% ethyl acetate was indispensable to achieve Cur-TPP solubility. Then oil phase was injected into 1% PVA solution (15mL) stirring to form emulsion and then using a probe-type ultrasonicator at 150 W for 1 minutes (active every 4 s for a 3 s duration) to obtain a uniform emulsion. Then the emulsion was agitated on a magnetic stirrer at room temperature until solvent removal. Microspheres were then recovered by centrifuging at 16000 × g for 5 minutes. Finally, microspheres were washed three times with DI water and lyophilized. 2.9 Characterization of sialic acid modified microsphere: The microsphere zeta potential, size and size distribution were measured by DLS. The morphology of microspheres were observed by TEM and SEM. Drug loading and entrapment efficiency in microspheres were measured by completely dissolving microspheres in DMSO, centrifuging at 5,000×g for 10 min, and analyzing Cur-TPP content in the supernatant by ultraviolet spectrophotometer. Drug loading (DL) and Encapsulation efficiency (EE) were calculated by the following formulas: DL (%) = (mass of Cur-TPP entrapped in microspheres/mass of microspheres) ×100 %, EE (%) = (mass of Cur-TPP entrapped in microspheres/mass of Cur-TPP added) ×100 %. 2.10 In vitro drug release: The release profiles of Cur-TPP-loaded SA/PEG-PLGA/MS and PEGPLGA/MS were studied in PBS (pH 7.4) solution containing Tween-80 (0.5%, v/v). Briefly, same amount of microspheres were well-suspended in 40mL release medium and were shaken in an incubator at 100 rpm under 37°C. At different times, 1ml release medium was removed and the same amount of PBS was added. The specific amount of released drug was measured via HPLC from the



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standard Cur-TPP curve. 2.11 Biodistribution of ICG-labelled microsphere in ALI mice: The ALI murine model was established via intratracheal instillation of LPS (4 mg/Kg) which was dissolved in saline.27 Briefly, mice were randomly distributed into four groups: the control (healthy mice) administrated with sialic acid modified microsphere (SA/PEG-PLGA/MS); the control administrated with PEG-PLGA/MS; the ALI model administrated with SA/PEG-PLGA/MS; the ALI administrated with PEG-PLGA/MS. ICG-tetrabutylammonium iodide complexes was still used as the fluorescent probe entrapped into microspheres. Then mice were intravenously injected with ICGloaded microspheres at the dose of 1mg/kg after LPS intratracheal instillation of LPS for 4 h. After 6 hours, the mice were euthanized followed by collecting organs. Fluorescence signal in representative organs was observed by using Maestro in vivo imaging system. And then lungs in ALI model were quickly moved to liquid nitrogen for immunohistochemical stain. E-selection receptors were stained with TRITC conjugated E-selection antibody and DAPI was used to stain cell nucleus. And the relative traits between E-selection receptors and microspheres were observed by confocal microscope. The assessment of drug concentration in lungs at different time points. The ALI murine model was established, and mice were intravenously injected with SA/Cur-TPP/MS. After 2, 4, 8, 12, 24 h injection, mice were sacrificed and lungs were collected. Lungs were than ground in PBS buffer on ice, and the supernate were extracted drug with acetonitrile. Then drug was assessed by HPLC assay. The distribution of SA/Cur-TPP/MS in different types of cells in vivo. The ALI murine model was established, and mice were intravenously injected with SA/Cur-TPP/MS. After 6h, mice were sacrificed and lungs were collected for immunochemistry staining. Among lung tissue sections, endothelial cells were stained with TRITC conjugated E-selectin antibody. Macrophages were stained


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with anti-CD68 antibody. Type 2 epithelial cells were stained with TRITC conjugated anti-SP-D antibody. Type 1 epithelial cells were stained with TRITC conjugated anti-RAGE antibody. DAPI was used to stain cell nucleus. And the relative traits between cells and microspheres were observed by confocal microscope. 2.12 Cell viability assay: The cytotoxicity of blank SA/PEG-PLGA/MS and PEG-PLGA/MS, free Cur-TPP and Cur-TPP-loaded sialic acid microspheres (Cur-TPP/MS) on HUVECs was determined by MTT assay. Cells were incubated with complete medium containing SA/PEG-PLGA/MS and PEGPLGA/MS at a series concentration from 3 μg/mL to 200 μg/mL. In the same way, free Cur-TPP and Cur-TPP/MS was also added into the culture medium at different concentration from 3 μM to 40 μM. The cells were cultured for another 24 hours. And then, 20μL of MTT (C18H16BrN5S) was added a for another 4 h at 37 °C. Later 100 μL DMSO was used to replace the medium. Finally, absorbance of the solution in each well was measured at 570 nm by Bio-Rad 680 microplate reader. Cell viability (%) =(Asample-Ablank) / (Acontrol-Ablank) ×100% 2.13 Targeted cellular uptake of SA/PEG-PLGA/MS: HUVECs were seeded in a 12-well plate for 24 h, and then exposed to LPS (400 ng/ml). After 4 hours, LPS-activated HUVECs and non-activated HUVECs were respectively incubated with Cur-TPP-loaded SA/PEG-PLGA/MS for 6 h in 37 ℃, CurTPP-loaded PEG-PLGA/MS as control group. The fluorescent intensity was observed by confocal microscope. HUVECs were pretreated with various concentration of SA solution for 1 hour and then incubated with SA/PEG-PLGA/MS for another 6 h. The fluorescence intensity was measured via flow cytometry. 2.14 Mitochondrial targeting study: Free Cur and Cur-TPP was added to HUVEC culture medium at a dose of 5 μM for 6 hours. Later, the medium was removed and mitochondria were stained by



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MitoTracker Deep Red FM dye. And then, cells were washed with PBS 3 times, and stained DAPI. The fluorescence in cells was observed by a fluorescence microscope. Similarly, Cur-loaded MS and Cur-TPP-loaded MS were also incubated with HUVECs, and then stain the cells using the same method. Finally, cells were observed by LSCM. The fluorescence Professional was used software Image J to analyze quantitatively. 2.15 Microspheres protect HUVECs from H2O2: The protection effect of microspheres was determined by MTT assays under oxidative stress created by H2O2. HUVECs were cultured into a 96well plate and then treated for 4 h with blank microspheres (MS), free Cur, Cur-TPP, Cur-loaded microspheres (Cur/MS) and Cur-TPP-loaded microspheres (Cur-TPP/MS) in culture medium at various content from 3 μM to 20 μM, and then 250 μM was added to the media prior to create an oxidative stress. After 24 h, cells were analyzed by MTT. Meanwhile, Cells were then treated for 4 h with MS, free Cur, Cur-TPP, Cur/MS and Cur-TPP/MS in fresh DMEM medium at a Cur/Cur-TPP concentration of 10 μM, and then varied doses of H2O2 were added into culture medium. Finally, cell viability was evaluated via MTT assay. 2.16 HUVECs intracellular ROS measurement in vitro: HUVECs were cultured in a 6-well plate,

and then cells were treated with free Cur, Cur-TPP, Cur/MS and Cur-TPP/MS in culture medium at a Cur/Cur-TPP concentration of 5 μM for 12 h, and after that H2O2 was added at concentration of 400 μM. After 2 h, the medium was removed, and cells were washed with PBS 3 times, and then incubated with10 mM DCFH-DA dye for 30 min. ROS-induced, intracellular fluorescence was viewed via fluorescence microscope and measured via flow cytometry after cells were harvest in PBS. 2.17 SOD, MDA, NO detection: The levels of SOD, MDA, NO were assessed via SOD assay kit, MDA assay kit, and NO assay kit (Beyotime Biotech, China) respectively. HUVECs were treated with


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Cur, Cur-TPP, Cur/MS, Cur-TPP/MS at concentration of 5 μM for 4 h, and then 250 μM H2O2 was added to medium. After 24 h, cells were lysed and collected by centrifuging at 13,000 rpm for 10 min. The supernatant was used for the measurement. 2.18 Mitochondrial Membrane Potential (Δψm) Measurement: The mitochondrial membrane potential was evaluated by JC-1 kit assay (Beyotime Biotech, China). HUVEC cells were handled as depicted in SOD detection. Then cells were arranged as the product instruction. At last, cells were observed by fluorescence microscope and analyzed using flow cytometer. 2.19 Cell apoptosis: The apoptosis effect of microspheres was measured via Annexin V-FITC/PI apoptosis detection kit (Beyotime Biotech, China). HUVEC cells were cultured into 6-well plates overnight. And then cells were handled as depicted in SOD detection. Later cells were arranged as the product instruction. At last, the cells were measured via flow cytometer. 2.20 Acute lung injury treatment: ALI model was established as depicted before. And then Mice were randomly divided into 5 groups: (1) control + saline group (n = 6); (2) LPS + saline group (n = 6); (3) LPS + Cur/MS (n = 6); (4) LPS + Cur-TPP/MS (n = 6); (5) LPS +SA/Cur-TPP/MS (n = 6). The treatment groups were intravenously injected with Cur/Cur-TPP at the dose of 2.4 mg/kg after LPS challenge for 4 hours. The animals were executed after drug administration for 24 and 48 hours. Afterwards, bronchoalveolar lavage fluid (BALF) and lungs were gathered for pharmacology researches in vivo. Wet/dry ratio of lung measurement: The collected lungs were cleaned and weighed as wet weight. Then, the lungs were heated at 70°C for 48 h until constant weight to measure the dry weight. The wetto-dry weight ratio was calculated by dividing the wet weight by the dry weight (Wet/dry = wet weight / dry weight).



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Measurement of pro-inflammatory cytokines and oxidative stress in lungs: The lungs collected were cleaned and lysed by centrifuging at 13,000 rpm for 15 min. The supernatant was applied for the detection. The levels of TNF-α and IL-6 as pro-inflammatory were measured by ELISA kits as the guidelines of product introductions. The levels of SOD and MDA were measured using commercial kits as the guidelines of product introductions. The protein concentrations were measured by a bicinchoninic acid assay. All samples were measured triplicate. BALF collection and lung inflammation evaluation: Mouse BALF was collected as the protocal reported in advance.28 With consistent practice, ice PBS was introduced and the collection of BALF could reach to 80%. The recovered BALF was centrifuged at 1300 rpm for 10 min, and the supernatant was collected followed by storage at -80 ℃. The remainder of cell suspensions was employed to measure neutrophils level in BALF as previous assay reported.29 The ratio of neutrophils versus total cells in BALF was measured by flow cytometer and the data were evaluated by flowjo software. Histological analysis: Lungs were fixed in 4.5% buffered formalin and embedded in paraffin. Five micrometer-thick sections were stained using hematoxylin and eosin (H&E) for morphology analysis of lungs with light microscopy. 2.21 Statistics: Statistical significance between two groups was determined using Student’s t-tests. p Value < 0.05 was considered significant.

3. Results 3.1 Synthesis and characterization of microsphere The microspheres were synthesized via emulsion solvent evaporation method, and the change of ultrasonic power and time is the key to control the size of microspheres. As depicted in Table 1, MS1 was fabricated under 180 W of ultrasonic power for 3 min, forming size = 250 ± 9.16 nm, PDI =


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0.137±0.013. Also, MS-2 was fabricated under 180 W for 2 min, gaining size = 423±5.57 nm, PDI = 0.288±0.065. MS-3 was fabricated under 150 W for 1min, forming size = 851±31.80 and PDI = 0.316±0.072. The size distributions were detected by dynamic light scattering (Figure 1A). Table 1. Physicochemical characterization of microspheres Sample

Ultrasonic Power

Time

Size/nm

PDI

MS-1

180W

3min(4s on, 3s off)

250±9.16

0.137±0.013

MS-2

180W

2min(4s on, 3s off)

423±5.57

0.288±0.065

MS-3

150W

1min(4s on, 3s off)

851±31.80

0.316±0.072

The ultrasonic power indicated the working power of probe-type ultrasonicator, and Time represented the total time with 4 seconds’ ultrasonic working and 3 seconds’ interval.

3.2 Cell uptake of various size of microsphere MS-1, MS-2, MS-3 loaded curcumin as fluorescent probes were incubated with HUVECs for predetermined time, and then fluorescence in cells were detected by flow cytometry, reflecting the cellular uptake levels. At 2 and 4 h, fluorescence intensity of MS-1 is much greater than MS-3 (p < 0.01), as shown in Figure 1B. However, with the incubated time extending, the gap of cell uptake between MS-1, MS-2, MS-3 was narrowed. And there was no significant difference between various size of microspheres.

3.3 Biodistribution of various size of microsphere-ICG in mice The passive targeting efficiency of microspheres was evaluated in male mice. As shown in Figure 1C, the fluorescent intensity in lungs between various size of microspheres were tremendously



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different. Apparently, the fluorescent signals of lung in ICG-loaded MS-3 treated group was much stronger than those of ICG-loaded MS-1 and MS-2 treated group, indicating MS-3 had the best passive pulmonary targeting efficiency. The quantitative values of lung fluorescence signals in mice injected with MS-1, MS-2 and MS-3 were analyzed and presented in Figure 1D. MS-3 was chosen for the continue study.

Figure 1. Synthesis and characterization of Microsphere. (A) Hydrodynamic size distribution of MS1, MS-2, MS-3. (B) Assessment on cellular uptake of MS-1, MS-2 and MS-3 in vitro and the fluorescence intensity observed by flow cytometry. (C) Bio-distribution of MS-1, MS-2, MS-3 were


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viewed by in vivo imaging system after intravenous injection for 6 h. (D) The quantitative analysis of fluorescence intensity in heart, lung, liver, spleen and kidney of Figure 1C. (n = 3)

3.4 Synthesis and characterization of Cur-TPP The synthesis route of Cur-TPP was illustrated as Figure S1(a). Firstly, SOCl2 was used as the catalyst, and the carboxylic acid on triphenyl bromide (4-carboxybutyl) was first formed to acyl chloride, and then the nucleophilic addition reaction of the phenolic hydroxyl group in curcumin produced the ester. To improve the reaction activity, the whole process needed to be carried out under the condition of no water, so fresh distilled DMSO was taken as the reaction solvent, and nitrogen protection was carried out. Excessive SOCl2 would be decomposed into SO2 and HCl and removed, in the process of dialysis. The structural analysis of Cur-TPP was confirmed by 1H NMR spectra (Figure S1(b)). The peaks at about 3.98 ppm belonging to Cur (-OCH3) and peaks at about 1.713 and 1.576 ppm belonging to TPP (-CH2-CH2-) were both observed in the spectrum of Cur-TPP, and at the same time, the proton peak of the carboxyl group (-COOH, 12.1ppm) at the end of TPP could not be observed, which indicated that TPP was successfully grafted to Cur.

3.5 Synthesis and characterization of SA-PEG-PLGA conjugates The synthesis route of SA-PEG-PLGA was depicted as Figure S2(a). Firstly, use DCC as dehydrating agent, DMAP as catalyst, PLGA free hydroxyl and free carboxyl on one end of the PEG as reactive groups to produce carboxylic ester dehydration reaction. After 24h reaction, SA was added to make the terminal hydroxyl on the SA to be esterified and dehydrated with the carboxyl on the other side of PEG. The by-product was removed by dialysis. As shown in Figure S2(b), the structure of SA-



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PEG-PLGA was confirmed by 1H NMR spectra. The peaks at about 3.50 ppm belonging to PEG (CH2-CH2-) and peaks at about 1.49 ppm belonging to PLGA (-CH3) and peaks at about 8.25 ppm belonging to SA (-COOH) were both observed in the spectrum of SA-PEG-PLGA, which demonstrated SA-PEG-PLGA conjugate was successfully synthesized.

3.6 Sialic acid modified microsphere fabrication and characterization As previous mentioned, MS-3 were both biocompatible and had high accumulation in lungs, so MS-3 was modified with sialic acid and then it was applied to entrap Cur-TPP to fabricate lungtargeting drug delivery system. To entrap Cur-TPP into microsphere and modify microsphere with SA, we dissolved PEG-PLGA, SA-PEG-PLGA and Cur-TPP as synthesized previously in a 4:1 mixture of dichloromethane and ethyl acetate to get a clear solute. Afterwards, according to the method to fabricate MS-3, Cur-TPP-loaded SA modified microspheres were synthesized (SA/PEG-PLGA/MS). As shown in Table 2, with the increase of drug/polymer from 5% to 15%, the drug loading (DL%) upregulated from 3.24±0.01 to 8.14±0.04, and entrapment efficiency (EE%) decreased from 68±0.88 to 63.5±0.24 without significant change to size, PDI and zeta potential, so 15% of drug/polymer was chosen to optimize forms. The zeta potential of SA/PEG-PLGA/MS and PEG-PLGA/MS were 24.2±2.1 mV and -21.6±0.6 mV (Figure 2A and 2B). Moreover, the scanning electron microscope (SEM) images (Figure 2C and 2D) and transmission electron microscopy (TEM) images (Figure 2A and 2B) were taken to indicate the round and same morphology of microspheres with the result of dynamic light scattering (DLS).

Table 2. Physicochemical characterization of sialic acid modified microspheres


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Formulation SA/PEGPLGA/MS

PEGPLGA/MS

Drug/polymer

Size/nm

PDI

DL%

EE%

Zeta potential

5%

834±31.80

0.291±0.052

3.24±0.01

68±0.88

-22.9±2.0

10%

868±27.35

0.288±0.045

6.03±0.02

66.3±0.56

-23.5±1.3

15%

852±30.70

0.334±0.074

8.14±0.04

63.5±0.24

-24.2±2.1

5%

863±28.43

0.213±0.048

3.38±0.02

70.9±0.34

-22.9±1.5

10%

821±35.37

0.273±0.065

5.78±0.04

63.6±0.28

-20.5±0.5

15%

882±31.23

0.235±0.031

8.07±0.04

61.9±0.33

-21.6±0.6

Drug/polymer represented the mass ratio of Cur-TPP to polymer. The PDI, DL and EE represented the polydispersity index, drug drug loading content and encapsulation efficiency, respectively. The data represent the mean ± SD (n = 3).

3.7 In vitro drug release As displayed in Figure 2E, the release of Cur-TPP from the SA/PEG-PLGA/MS showed a proper sustained release profile, an about 20 % burst release at 3 hours was revealed and then the release continued to 72 h with a final release of 63 %. And the same release manner was observed in Cur-TPP loaded PEG-PLGA/MS.

3.8 Biodistribution of ICG-labelled microsphere in ALI mice To assess the lung-targeting efficacy of SA modified microspheres in vivo, ALI murine models or healthy were injected intravenously with ICG-loaded PEG-PLGA/MS or SA/PEG-PLGA/MS. After administration for 6 h, mice were executed, and their organs were collected. Compared to healthy mice, much higher lung fluorescence signal was shown in ALI mice administrated with SA/PEGPLGA/MS (Figure 2F). Besides, the lung fluorescence intensity in ALI mice treated by SA/PEGPLGA/MS was superior than that treated with PEG-PLGA/MS. Meanwhile in Figure 2H, E-selectin was highly expressed on pulmonary endothelium, and SA/PEG-PLGA/MS shows a remarkably higher



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accumulation than PEG-PLGA/MS in lung tissues. The concentration-time curve of SA/PEGPLGA/MS in lungs was shown in Figure S5, which demonstrate the specific accumulation effect in lungs. Meanwhile, the distribution of SA/Cur-TPP/MS in different types of cells in lung tissues was observed in Figure S6, which prove it could be uptake by those cells and exert its treatment efficacy.

Figure 2. Sialic acid modified microsphere fabrication and characterization. Zeta potential of sialic acid modified PEG-PLGA microspheres (A), PEG-PLGA microspheres (B), obtained by DLS, and the inserted images were obtained by TEM (scale bar = 1 μM). The morphology of SA/PEG-PLGA/MS (C) and PEG-PLGA/MS (D) were observed by SEM (scale bar = 1 μM). (E) In vitro release manner of Cur-TPP from SA/PEG-PLGA/MS and PEG-PLGA/MS. (F) Bio-distribution of SA/PEG-


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PLGA/MS in vivo. The fluorescence images of excised organs from mice treated with LPS or not. (G) The quantitative analysis of fluorescence intensity in heart, lung, liver, spleen and kidney of Figure 2F. (n = 3) (H) Imaging of E-selectin receptors stained with a fluorescent E-selectin antibody with Cur-TPP loaded microspheres, and nuclei were stained with DAPI. (scale bar = 50 μm)

3.9 Cytotoxicity study of microspheres The cell viability of blank SA/PEG-PLGA/MS and PEG-PLGA/MS were evaluated in HUVECs by MTT assay with a wide range of concentrations. In Supporting Figure S1, the blank microspheres displayed minimum cytotoxicity with over 90% HUVECs survive at the highest concentration of 200 µg·mL-1, that demonstrated that the synthesized polymer microspheres had low toxicity and good biocompatibility. A dose-dependent cell viability was revealed in Supporting Figure S2, which demonstrated that Cur-TPP-loaded microspheres could up-regulate cell viability compared to free CurTPP at same concentration.

3.10 Targeted cellular uptake of SA/PEG-PLGA/MS To intimate Inflammatory endothelial cells in vivo which could over-express E-selectin on its surface, lipopolysaccharide (LPS) was applied to simulate HUVECs in vitro. Upon previous studies, E-selectin could be expressed maximally when HUVECs incubated with LPS for 4 h.30 To inquiry the targeting ability of SA modified microspheres, cell uptake experiments for SA-PEG-PLGA and PEGPLGA microspheres on LPS simulated HUVECs at various times (2, 4, 8 and 12 h) were completed and tested by flow cytometry. And the results were shown in Figure 3B, indicating SA-PEG-PLGA and PEG-PLGA microspheres were internalized by HUVECs in a time-dependent way. Meanwhile,



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cells incubated with SA-PEG-PLGA microspheres obtained higher fluorescence intensity than those incubated with microspheres without SA modified with different time points (4, 8 and 12 h), which had significant differences (p < 0.05). The mechanism of cellular uptake which might be related with the anchoring of SA on E-selectin over-expressed on inflammatory cells remained unclear. Thus, SA modified microspheres were incubated with LPS stimulated cells for 6 h, and then E-selectin on cell membranes were stained with a fluorescent E-selectin antibody (red). The result shows a positive correlation with the cellar internalization of the SA modified microspheres and the expression of E-selectin, and PEG-PLGA microspheres compared to SA-PEG-PLGA microspheres showing a much lower level of cellular uptake. Also, cells without LPS-activated did not show high expression of E-selectin and had much lower microspheres internalization (Figure 3A). To further confirm the mechanism, competitive cellular uptake experiment was taken. LPS-activated HUVECs were blocked with different concentration of free SA previously, and the results (Figure 3C) detected by flow cytometry show a gradual decrease on cell internalization of SA-PEG-PLGA microspheres with the improvement of SA concentration. All these results demonstrated the cellular uptake mechanism that microspheres largely internalization via SA specific binding to E-selectin expressed on inflammatory cells.


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Figure 3. Targeted cellular uptake study of SA/PEG-PLGA/MS. (A) LPS-activated HUVECs and normal ones were treated with Cur-TPP loaded SA-PEG-PLGA and PEG-PLGA microspheres for 6 hours, respectively. Scale bar=50 μm. (B) LPS-activated HUVECs were treated with PEG-PLGA and SA-PEG-PLGA microspheres for 2, 4, 8 and 12 h, and the fluorescent intensity was detected via flow cytometry (* p < 0.05, n = 3). (C) Cells were blocked with various content SA in advance, and then those were incubated with SA-PEG-PLGA/MS respectively, using flow cytometry to get results.

3.11 Mitochondrial targeting study in vitro After the HUVECs had been incubated with free Cur, Cur-TPP respectively for predetermined time (2 h, 4 h and 6 h), the cells were washed and co-localization of drug was examined by fluorescence microscope (Figure 4A). Since the Cur-TPP was designed to target mitochondria, mitochondrial localization was determined by fluorescence dual-staining with a mitochondrial molecular probe, Mito-Tracker Red FM. Compared with untargeted free Cur-treated cells, the cells treated with Cur-



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TPP showed obvious higher level of internalization by cells at 2 h, 4 h and 6 h, and the semiquantitative results in Figure 4C also showed same tendency. As shown in Figure 4B, cells incubated with Cur-TPP for 6 h was observed by confocal microscope, showing obvious co-localization between the mitochondrial probe Mito-Tracker Red FM and Cur-TPP. Also, Cur-loaded microspheres (Cur/MS) and Cur-TPP loaded microspheres (Cur-TPP/MS) were treated with cells for 2 h, 4 h, 8h respectively and the microspheres had the same characteristics except the different drug-loading. Compared with untargeted Cur/MS-treated cells, the cells treated with Cur-TPP/MS showed remarkable colocalization between the mitochondrial probe and drug (Figure 4D). The yellow area of the overlapping image represented a co-localization of Cur-TPP/MS and Mito-Tracker Red FM. These results demonstrated that Cur-TPP/MS exhibited better selectivity for mitochondria in HUVEC cells.

Figure 4. Mitochondrial targeting study in vitro. (A) Imaging of cells were treated with Cur, Cur-TPP at at a dose of 5 μM for 2, 4 and 6h with mitochondria stained by MitoTracker Deep Red FM (Red). scale bar = 50 μm. (B) Imaging of cells treated with Cur-TPP for 6 h with mitochondria stained. scale


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bar = 10 μm. (C) The quantitative analysis of fluorescent intensity of Cur-TPP in cells from Figure 4A. scale bar = 40 μm. (D) Imaging of cells were incubated with Cur/MS and Cur-TPP/MS at a dose of 5 μM for 2, 4, 8 h. Also, mitochondria were stained via MitoTracker Deep Red FM (Red), and nuclei was stained by DAPI (Blue).

3.12 In vitro antioxidant activity of Cur-TPP loaded microspheres The above results indicated that SA-modified Cur-TPP-loaded microspheres (Cur-TPP/MS) could enter HUVECs through SA-derived targeting activity with Cur-TPP releasing, which could selectively accumulate in mitochondria to improve its therapeutic effects. To further confirm the mechanism by which Cur-TPP, Cur-TPP/MS developed much more antioxidant activity than free Cur and Cur/MS to cells, the following experiments were carried out. 3.12.1 Microspheres protect HUVECs from H2O2 Next, the ability of MS, free Cur, Cur-TPP, Cur/MS and Cur-TPP/MS to salvage cell viability under cytotoxic levels of ROS was evaluated. For a various concentration of free drug and drug-loaded microspheres in Figure 5A, it was found that Cur, Cur-TPP, Cur/MS and Cur-TPP/MS showed a significant therapeutic benefit at 3 μM and 5μM (p < 0.05) under 250 μM H2O2 injury, and Cur-TPP, Cur-TPP/MS showed remarkably better therapeutic effect (p < 0.01). With the increase of drug concentration, only Cur/MS and Cur-TPP/MS up-regulated the cell viability (p

Sialic Acid-Functionalized PEG-PLGA Microspheres Loading

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