Novel Curcumin Liposome Modified with Hyaluronan Targeting CD44

May 10, 2017 - Novel Curcumin Liposome Modified with Hyaluronan Targeting CD44 Plays an Anti-Leukemic Role in Acute Myeloid Leukemia in Vitro and in ...
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Novel Curcumin Liposome Modified with Hyaluronan Targeting CD44 Plays an Anti-Leukemic Role in Acute Myeloid Leukemia in Vitro and in Vivo Dan Sun,† Jian-Kang Zhou,† Linshu Zhao,‡ Zhe-Yu Zheng,† Jiao Li,† Wenchen Pu,† Shaoyang Liu,† Xue-Sha Liu,† Shu-Jun Liu,§ Yu Zheng,*,† Yun Zhao,*,† and Yong Peng*,† †

State Key Laboratory of Biotherapy, West China Hospital, College of Life Sciences, Sichuan University and Collaborative Innovation Center of Biotherapy, Chengdu 610064, China ‡ Division of Biosciences, Faculty of Life Sciences, University College London, London WC1E 6BT, United Kingdom § The Hormel Institute, University of Minnesota, Austin, Minnesota 55912, United States S Supporting Information *

ABSTRACT: Curcumin has been widely used as a food additive for centuries and has been recently explored for its anti-inflammatory and antitumor properties. Although curcumin is pharmacologically safe and efficacious to certain cancers, its role against acute myeloid leukemia (AML) still remains unclear, and it lacks clinical application due to low water solubility and low in vivo bioavailability. To address these issues, we developed a novel curcumin liposome modified with hyaluronan (HA-CurLPs) to specifically deliver curcumin to AML by targeting CD44 on AML cell surface. When compared with free curcumin and nontargeted liposome (Cur-LPs), the HA-Cur-LPs exhibited good stability, high affinity to CD44, increased cellular uptake, and more potent activity on inhibiting AML cell proliferation. The KG-1 cell implanted AML mice had significantly delayed, or even prevented, AML progression following treatment with 50 mg/kg of curcumin dose in the HA-Cur-LPs every 2 days for 2 weeks. Mechanistically, the anti-AML effects of HA-Cur-LPs were achieved by inhibiting Akt/ERK pathways and activating caspase-dependent apoptosis. Moreover, HA-Cur-LPs played a critical role in downregulation of DNMT1 expression in AML, leading to DNA hypomethylation and reactivation of tumor suppressor genes such as miR-223. The development and assessment of the HA-CurLPs in this study provide another potential choice for AML therapy, using HA-Cur-LPs as either a single treatment agent or in combination with other treatments. KEYWORDS: curcumin, liposome, hyaluronan, acute myeloid leukemia, CD44 curcumin was associated with decreased WT1 expression.11 In our previous study, we demonstrated that curcumin downregulated expression of DNA methyltransferase 1 (DNMT1) gene in acute myeloid leukemia (AML), a heterogeneous clonal disorder marked by accumulation of undifferentiated myeloid blasts.12 Despite the aforementioned exciting findings, the clinical application of curcumin in cancer treatment is hampered due to its extremely low aqueous solubility and instability, particularly under alkaline conditions, which lead to poor in vivo bioavailability and limited therapeutic effects.13−16 An approach to improve the poor biopharmaceutical properties is to deliver drugs using nanocarriers, such as liposomes, polymer nanoparticles, and biodegradable microspheres. Systemic admin-

1. INTRODUCTION Curcumin, a yellow phenolic antioxidant extracted from the rhizome of Curcuma longa L.,1 was extensively used for enhancing the color and flavor of foods and as a component of many traditional Asian medicines to treat inflammation.2 The structure of curcumin was first elucidated by Milobedeska et al.3 and synthesized by Lampe et al.4 Recently, curcumin was widely discovered to exhibit pharmacological activities against inflammation, chronic diseases, and cancers. Dietary curcuminoids have shown the cytotoxic effects on human leukemic cells such as K562, Jurkat, and HL-60 cells.5,6 William et al. also found that curcumin was effective against leukemic cells expressing p210 BCR-ABL or T315I BCR-ABL, showing promise to treat BCR-ABL induced B-cell acute lymphoblastic leukemia.7 It was generally believed that curcumin induced cell cycle arrest and apoptosis through regulating certain tumor suppressor genes or oncogenes.8−10 Anuchapreeda et al. observed that the inhibition of K562 cell proliferation by © XXXX American Chemical Society

Received: February 27, 2017 Accepted: April 26, 2017

A

DOI: 10.1021/acsami.7b02863 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

Research Article

ACS Applied Materials & Interfaces Table 1. Characteristics of Conventional Curcumin Liposomes with Varied Lipid Compositions lipids composition (M) DMPC/DMPG/CHOL/DSPE-PEG2000 37.5:7.5:50:5 DMPC/DMPG/CHOL/DSPE-PEG2000 22.5:22.5:50:5 DMPC/DMPG/CHOL/DSPE-PEG2000 22.5:22.5:50:5 DMPC/DMPG/CHOL/DSPE-PEG2000/DSPE 22.5:22.5:50:5:5 DMPC/DMPG/CHOL/DSPE-PEG2000/DSPE 22.5:22.5:50:5:5

curcumin/lipids (w/w) 10% 10% 20% 20%

mean diameter (nm)

polydispersity (PDI)

± ± ± ±

5.5 3.4 10.2 13.1

0.266 0.231 0.200 0.228

−12.6 −11.8 −14.1 −12.9

90.73 ± 0.18

0.093

−13.0 ± 0.25

210.7 124.7 200.9 203.6

0%

istration of liposomal curcumin has shown an increase in bioavailability and suppression effect on certain tumors. For example, Li et al. disclosed that liposome-encapsulated curcumin was efficient at inhibiting colorectal cancer cell growth and had the antitumor and antiangiogenesis effects on the xenograft animal tumor model.17,18 While liposomal delivery is capable of addressing the issue of low aqueous solubility and the poor bioavailability, the selective delivery of curcumin to target tissues is called urgent attention to further improve efficacy and reduce side effects. One strategy to achieve target delivery is “active” targeting, which could be ligand-directed or stimuli-responsive.19,20 The adhesion molecule CD44 is a transmembrane glycoprotein and a key signaling receptor involved in myelopoiesis.21 Abnormal expression of different isoforms of CD44 has been found to characterize many types of malignant cells. Ghaffari et al. identified that the expression of CD44 variant exons in AML is much more common than that in normal cells, which not only suggests a potential anti-CD44 AML therapy22−24 but also implies that CD44 is an ideal receptor for actively targeted delivery of antiAML drugs. Hyaluronan (HA), also known as hyaluronic acid, is an active component in many postsurgical adhesion barriers and is unique in its protein-repellent behavior.25 Importantly, in view of the ability to specifically recognize HA receptors, such as CD44 and RHAMN which are overexpressed in certain cancer cells, HA is widely employed in cancer treatment to direct drug or nanocarrier to the tumor tissues.26,27 Therefore, HA was considered in this study as a targeting ligand to deliver curcumin to the CD44-overexpressing AML cells. The modification of curcumin liposomes with HA is believed to further enhance the delivery, increase cellular uptake, reduce nontargeted drug toxicity, and promote anti-AML efficacy.28 In the present study, we designed and formulated a novel nanosized liposomal carrier encapsulating curcumin and modified with HA (HA-Cur-LPs) for targeted delivery to human AML cells. The high binding affinity of HA-Cur-LPs to CD44-Fc in vitro heralded its targeting effects. HA-Cur-LPs showed significant inhibition of human AML cell proliferation in vitro and improved pharmacokinetics (PK) in mice, implying enhanced anti-AML effects. When administered into xenogeneic AML mice, which were built by implanting human KG-1 cells, HA-Cur-LPs significantly delayed, or even prevented, AML progression. Mechanistically, the anti-AML effects of HACur-LPs were achieved by inhibiting Akt/ERK pathways and activating caspase-dependent apoptosis. Moreover, HA-CurLPs plays a critical role in downregulation of DNMT1 expression in AML, leading to DNA hypomethylation and reactivation of tumor suppressor genes (TSGs) such as miR223. Overall, this study developed novel nanoparticle HA-CurLPs to treat AML patients by administration of HA-Cur-LPs

zeta potential (mV) ± ± ± ±

1.23 0.28 2.2 1.7

EE (%) 68.4 75.8 70.6 69.10

± ± ± ±

5.2 4.3 3.7 2.8

either alone or in combination with other clinically effective agents.

2. MATERIALS Curcumin was purchased from Yuanye, Shanghai; 1,2-dimyristoly snglycero-3-phosphocholine (DMPC), 1,2-dimyristoyl-sn-glycero-3[phospho-rac-(1-glycerol)] (DMPG), N-(carbonyl-methoxypolyethylene glycol-2000)-1, 2-distearoyl-sn-glycero-3-phosphoethanolamine (mPEG-DSPE), and 1,2-distearoyl-sn-glycero-3-phospho-rac-glycerol (DSPE) were purchased from Lipoid, Germany. Cholesterol (CHOL) and Sephadex G-25 were obtained from Sigma; high-molecular-weight HAs were provided by Acros Organics. N-Hydroxysuccinimide (NHS) and 1-ethyl-3-(3-(dimethylamino)propyl)-carbodiimide hydrochloride (EDCI) were purchased from Alfa Aesar. The anti-CD44 antibody was purchased from Abcam; allophycocyanin labeled anti-human CD45 antibody (hCD45-APC), and fluorescein isothiocyanate isomer I labeled anti-human CD33 antibody (hCD33-FITC) were obtained from BioLegend. Recombinant human CD44-Fc Chimera Protein (CD44-Fc) was purchased from R&D system; sensor Chip CM3 was provided from GE Healthcare. DNMT1 antibody was purchased from New England Biolabs; antibodies against Akt, phospho-Akt, ERK, and phosphor-ERK and the apoptosis antibody sampler kit were purchased from Cell Signaling Technology. All other reagents were of analytical reagent grade; “water” refers to ultrapurified Milli-Q water (Millipore, France) with a resistivity of ≥18 MΩ·cm 2.1. Cell Lines and Animals. K562, KG-1, and MV4-11 leukemia cells were cultured in RPMI 1640 medium supplemented with 10% (v/v) fetal bovine serum (Gibco Australia origin) and 100 IU/mL penicillin/streptomycin (Invitrogen, USA) at 37 °C in a humidified incubator containing 5% CO2. BALB/c mice and severe combined immunodeficient NPG mice were purchased from Vitalstar Laboratory Animal Technology Co., Ltd. (Beijing, China). All animal experiments were approved by the Animal Care and Use Committee of Sichuan University (Chengdu, China).

3. METHODS 3.1. Preparation of Curcumin Liposomes. 3.1.1. Preparation of Conventional Curcumin Liposomes and Characterization. Conventional curcumin liposomes (Cur-LPs) were prepared by thinfilm evaporation.29,30 The lipids consists of DMPC/DMPG/CHOL/ DSPE-PEG2000/DSPE (22.5:22.5:50:5:5, molar ratio) (Table 1). Curcumin was dissolved in chloroform and methanol mixture (2:1, v/v) with a drug/lipids ratio of 20% (w/w). The mixture was evaporated at 40 °C and 125 rpm in a rotary evaporator for 1.5 h to form a dry lipid film, hydrated for 1.5 h with 1 × PBS (pH7.4) and ultrasonicated in an ice−water bath for 10 min to decrease the particle size. The final lipid concentration was 10 mg/mL. The unentrapped curcumin was removed by Sephadex G-25. The whole preparation process is protected from light. Empty liposomes (LPs) were prepared using the same protocol but excluding curcumin. Single factor method was adopted for prescription screening. The encapsulation efficiency (EE) and loading efficiency (DL) of curcumin were calculated according to the following equations.

Encapsulation efficiency (%) = Ce/ C t × 100% B

DOI: 10.1021/acsami.7b02863 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces where Ct stands for the initial drug amount added and Ce stands for the drug amount encapsulated in the liposomes.

time intervals and replaced with the same volume of PBS containing 0.2% Tween-80 each time. The samples were analyzed by highperformance liquid chromatography (HPLC). 3.5. Hemolysis Assay. Healthy rabbit erythrocytes were collected, washed, and resuspended in normal saline (NS) (2% final concentration). The LPs and HA-LPs were added to the erythrocyte suspension at the final concentration of 1.25, 2.5, 3.75, 5, and 6.25 mg/ mL, respectively, and incubated at 37 °C for 3 h. Erythrocytes treated with purified water and NS were used as positive and negative controls, respectively. Hemoglobin release in the supernatant was measured by UV spectrophotometry at 545 nm. The mixtures were centrifuged at 1500 rpm for 10 min before analysis. The percentage of hemolyzed cells was calculated, and less than 5% hemolysis represented good hemocompatibility and nontoxicity against erythrocytes membranes. 3.6. Safety Study of the Liposomes in Vivo. Safety of the liposomes in vivo was evaluated by hematoxylin and eosin (H&E) staining of major tissues. Briefly, after sacrifice of the BALB/c mice treated by i.v. administration of curcumin formulation, the excised tissue samples were fixed in 10% phosphate-buffered formalin and embedded in paraffin, then cut in to 5 μm thick paraffin sections and subsequently placed on a glass slide. The slides were stained with hematoxylin and eosin (H&E). The histopathological alterations were observed and imaged with a light microscope (Olympus BX43). 3.7. Study of CD44 Receptor Affinity by Surface Plasmon Resonance Spectroscopy. The interactions of LPs and HA-LPs with CD44 receptors were monitored by surface plasmon resonance spectroscopy (SPR) spectroscopy using a BIAcore T100 instrument (GE Healthcare Life Sciences). Human recombinant CD44-Fc receptors were immobilized on a carboxyl methylated dextran sensor chip (Sensor Chip CM3) using amine coupling. Carboxylic groups were activated by a mixture of EDC/NHS for 10 min at 10 μL/min followed by an injection of 10 μg/mL CD44-Fc in a 10 mM acetate buffer at pH 4.0 at 10 μL/min until the value of response ligand (RL) reached 400. The remaining groups were blocked by an injection of ethanolamine. A flow channel blocked by ethanolamine was used as a reference surface. The specific interaction of HA-lipoplexes with the immobilized CD44-Fc was assessed. All samples were analyzed at a flow rate of 30 μL/min with 10 mM HEPES running buffer and contact time of 120 s. The surface was washed and regenerated with a 10 mM glycine-HCl buffer at pH 3.0 for 30 s followed by a 60 min waiting time for dissolution after each experiment. Free HA (MW = 320 K) solution was systematically passed through the channel to verify the integrity of the CD44-Fc receptors. The analyses were performed in triplicate using BIAcore T100 evaluation software, version 2.0.2 (GE Healthcare). 3.8. Pharmacokinetics of HA-Cur-LPs in Mice. The pharmacokinetic study was assessed in BALB/c mice. Following overnight fasting, nine mice were randomized into three groups and received i.v. injection of free curcumin in DMSO, Cur-LPs, or HA-Cur-LPs in normal saline solution, respectively, at 10 mg/kg curcumin dose. At the preset sampling time points, 20 μL blood samples were collected through caudal vein bleeding into heparinized centrifuge tube, followed by the centrifugation to obtain plasma, which was then stored at −80 °C until determination of the drug concentration. For curcumin quantitation, plasma samples were thawed to room temperature, and curcumin was extracted using acetonitrile and quantified by HPLC with tandem mass spectrometry (LC/MS/MS) method.33 3.9. In Vitro and in Vivo Antitumor Efficacy. 3.9.1. In Vitro Antitumor Efficacy. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was carried out to estimate cell viability. Briefly, KG-1 cells were seeded in a 96-well plate (10 000 cells per well in 100 μL), then treated with PBS, DMSO, free curcumin, LPs, HALPs, Cur-LPs, or HA-Cur-LPs. After 96 h, 10 μL of MTT solution (0.5 mg/mL) (Sigma) was added to each well and incubated at 37 °C for 4 h. Then 100 μL of joint cracking liquid (10% SDS, 5% isobutanol, 0.012 mol/L HCl) was then added to dissolve formazan, and a spectrophotometer was used to measure absorbance at 570 nm.

Loading efficiency (%) weight of encapsulated curcumin = × 100% weight of lipid amount in the liposomes DiD, a lipophilic near-infrared fluorescent membrane dye, was used to label the liposomes. To prepare DiD-loaded liposomes without curcumin (DiD-LPs), DiD was dissolved with lipids in organic solvent. After evaporation and hydration, DiD amount encapsulated in the liposomes was assessed by deducting the unloaded DiD in the supernatant from the amount of drug added initially. The nanoparticle size distribution and zeta potential were measured by a laser particle size analyzer (Malvern Nano-ZS 90, UK) utilizing dynamic light scattering (DLS) technique. Samples were diluted using water and measured in triplicate. The morphology was observed by high-resolution transmission electron microscopy(TEM). The samples were prepared by placing diluted liposomes on a copper grid followed by drying under vacuum pressure and staining with 3% phosphotungstic acid for 30 s. 3.1.2. Preparation of HA-Conjugated Liposome and Characterization. HA with different molecular weight (117, 320, or 660 K) was covalently conjugated to the surface of Cur-LPs, LPs, and Did-LPs using EDC and NHS coupling agents.31,32 Briefly, HA was dissolved in sodium acetate buffer to a final concentration of 2 mg/mL and preactivated by incubation with EDCI and NHS at pH 4.5 and 37 °C with stirring for 2 h. The activated HA was added to a suspension of the Cur-LPs, pH 7.0, and stirred at 37 °C for 16 h. At the end of the incubation, the HA-conjugated liposomes (HA-Cur-LPs) were separated from excessive reagents and byproducts by centrifugation at 50 000 rpm for 1 h at 4 °C and suspended again with 1× PBS, pH 7.0. HA-conjugated empty liposomes (HA-LPs) and DiD-loaded liposomes (HA-DiD-LPs) as controls were prepared using the same protocol. HA-LPs, HA-Cur-LPs, and HA-DiD-LPs were also characterized as described above. The amount of HA conjugated to the liposomes was determined by CTAB precipitation assay.31 Briefly, 50 μL of HA standard solutions (0.1−2 mg/mL) or diluted supernatants were added to 96-well plate. The samples were incubated with 50 μL of 0.2 M sodium acetate buffer at 37 °C for 10 min. Then, 100 μL of 10 mM CTAB solution was added to the mixture, and the absorbance of the precipitated complex was read within 10 min against the blank at 570 nm using a microplate reader (BioTek Instruments). The conjugated HA amount was calculated by subtracting the amount in the supernatant fraction from the amount added initially. 3.2. In Vivo Imaging of Mice after HA-DiD-LPs Treatment. For in vivo fluorescence imaging, BALB/c mice were treated with HADiD-LPs, which were conjugated with different molecular weight HAs at different concentrations, through tail intravenous (i.v.) injection at 0.2 mg/kg of DiD. After 12 h, the mice were anesthetized for in vivo fluorescence imaging by a Quick View 3000 Bio-Real. The fluorescence filters were set at excitation = 655 nm and emission = 714 nm. 3.3. Differential Scanning Calorimetry (DSC) Assay. DSC measurements were performed using NETZSCH DSC instrument. For DSC measurement, curcumin (Cur), HA, LPs, HA-LPs, Cur-HALPs, the mechanical mixtures of LPs and HA (LPs + HA), and the mechanical mixtures of HA-LPs and Cur (HA-LPs + Cur) were used. All powder or lyophilized liposomes samples were weighed into the aluminum pans and sealed. Samples were scanned from 10 to 400 °C with 10 °C/min incremental increases in temperature. 3.4. In Vitro Drug Release. In vitro release of HA-Cur-LPs was investigated in a simulated blood pH environment (pH 7.4). Briefly, 1 mL of HA-Cur-LPs was diluted with 9 mL of 1× PBS. Then, 1 mL of this suspension was used to determine the initial curcumin concentration, and the remaining 9 mL was dialyzed in a dialysis bag (3500 Da) in 50 mL of PBS (pH 7.4) containing 0.2% Tween-80. The experiment was performed in triplicate at 37 °C with shaking at 100 rpm for 1 week. Samples (1 mL each) were withdrawn at preset C

DOI: 10.1021/acsami.7b02863 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

Figure 1. In vivo imaging of HA-DiD-LPs in mice. (A) Representative in vivo fluorescent images of mice 12 h postinjection of normal saline (NS) and different HA-DiD-LPs, respectively. (B) Representative ex vivo fluorescent images of organs 12 h postinjection of normal saline (NS) and different HA-DiD-LPs, respectively. (C) Physicochemical characteristics of different HA-DiD-LPs. The HA concentration was measured by CTAB precipitation assay. (D) Quantification analysis of the mean fluorescence intensity in the mouse organs (N = 3). 3.9.2. Human AML Mice Model and Treatment. To build a human AML mice model, 5 × 106 KG-1 cells were i.v. injected into NPG mice. After injection, mice were inspected daily and sacrificed as soon as they developed disease-symptoms or after a maximum observation period of 10 weeks. To validate the successful building of the AML mice model, human AML KG-1 cells were detected in peripheral blood samples, which were extracted every week by flow cytometry using hCD45-APC and hCD33-FITC, two human monoclonal antibodies against KG-1 specific surface markers. At 1 week post-transplantation, the successfully built AML mice were randomized to five groups and received normal saline, free curcumin, HA-LPs, Cur-LPs, or HA-Cur-LPs, respectively, at 50 mg/ kg dose level of curcumin every 2 days for 2 weeks (n = 5). Body weight of mice was measured daily. Event-free survival was defined as survival without symptoms of disease. 3.10. Flow Cytometry Analysis. Peripheral blood samples, ripped erythrocyte in NH4Cl method, or AML cell lines were passed through a cell strainer to generate single-cell suspensions in PBS and stained with hCD45-APC and hCD33-FITC. Following staining, cells were washed once in staining medium and then analyzed on a FACS Calibur (BD Biosciences).

3.11. Quantitative Real-Time PCR (RT-PCR) Assays. Quantitative RT-PCR was used to assess the expression levels of miR-223. Total RNA was extracted by TRIzol reagent (Life Technologies) from KG-1 cells treated with HA-LPs and HA-Cur-LPs and transcribed by reverse transcriptase (Life Technologies). Quantitative RT-PCR reactions were performed in triplicate using TaqMan gene expression assay (Life Technologies) with an ABI prism 7700 detector (Life Technologies). miR-223 expression was normalized to U6 internal control. 3.12. Protein Preparation and Immunoblotting. The cells were lysed on ice for 20 min in lysis buffer (20 mM HEPES, pH 7.0, 150 mM NaCl, 0.1% NP-40, 1 mM b-glycerophosphate, 1 mM Na3VO4, 1 mM NaF, 1 mM benzamide, and 1 mM phenylmethylsulfonyl fluoride) with protease inhibitors cocktail (Beyotime Biotechnology), followed by centrifugation at 12 000 g for 10 min at 4 °C. Proteins in the supernatants were resolved on SDS polyacrylamide gels, then transferred onto PVDF membranes and incubated with appropriate antibodies. The signals were detected using ECL reagents (Life Technologies). 3.13. Dual Luciferase Reporter Assay. In a dual luciferase reporter assay, the promoter of DNMT1 was cloned into the luciferase D

DOI: 10.1021/acsami.7b02863 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces Table 2. Characteristics of the final curcumin liposome formation liposomes

curcumin/lipids (w/w)

mean diameter (nm)

polydispersity (PDI)

zeta potential (mV)

EE (%)

DL (%)

HA-LPs HA-Cur-LPs

0% 20%

101.3 ± 1.5 236.4 ± 5.2

0.124 0.232

−24.7 ± 0.24 −36.8 ± 1.9

65.8 ± 3.3

13.2 ± 0.7

Figure 2. Characterization of the final curcumin formation. (A) Representative TEM photograph of Cur-LPs (left) and HA-Cur-LPs (right). (B) Change of entrapment efficiency of curcumin in Cur-LPs and HA-Cur-LPs over storage time at 4 °C. (C) Drug release from the Cur-LPs and CurHA-LPs. (D) Change of particle size and PDI of the final formation (Cur-HA-LPs) during storage at 4 °C. reporter vector pGL4.21 (Promega) and transfected into HEK293T cells. The empty vector pGL4.21 was used as a negative control. The dual luciferase reporter assay was performed according to the manufacturer’s protocol of dual luciferase assay kit (Promega). The relative luciferase activities were calculated as the ratio of firefly/renilla activities and normalized to those of the negative control. 3.14. Statistical Methods. The data were presented as the mean ± standard error of at least three independent experiments. The results were tested for statistical significance using Student’s t test (twotailed); p-value < 0.05 was considered statistically significant. The noncompartmental pharmacokinetic parameters are from GraphPad Prism v6.01 software in one-phase decay analysis.

In addition, HAs with MW = 117, 320, or 660 K were covalently conjugated to the liposome surface. The particle properties of DiD-labeled and HA-conjugated empty liposomes (HA-DiD-LPs) are presented in Figure 1C. The in vivo imaging of mice after treatment with HA-DiD-LPs was shown in Figure 1A,B,D. The average fluorescence signal values revealed that HA-DiD-LPs with medium grafting density showed higher hepatic and splenic uptake (red spot region) in the lower molecular weight HA groups (117 and 320 K HA groups). However, in the highest molecular weight HA group (660 K HA group), the low grafting density showed higher hepatic and splenic uptake. Therefore, the Cur-LPs with embellishment of 55 μg of 320 K HA per μmol of lipids was chosen to investigate pharmacology and efficacy in vitro and in vivo. 4.2. Characterization of Curcumin Liposomes. The mean diameter, zeta potential, EE, and DL of both HA-Cur-LPs and HA-LPs formulations were described in Table 2. Both liposomes were negatively charged and retained low PDIs (