Cell Permeable NBD Peptide-Modified Liposomes by Hyaluronic Acid

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Cell permeable NBD peptide-modified liposomes by hyaluronic acid coating for the synergistic targeted therapy of metastatic inflammatory breast cancer Yuqing Sun, Xuqian Li, Lili Zhang, Xiao Liu, Baohong Jiang, Zhiguo Long, and Yanyan Jiang Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/ acs.molpharmaceut.8b01123 • Publication Date (Web): 22 Jan 2019 Downloaded from http://pubs.acs.org on January 24, 2019

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Molecular Pharmaceutics

Cell permeable NBD peptide-modified liposomes by hyaluronic acid coating for the synergistic targeted therapy of metastatic inflammatory breast cancer

Yuqing Sun a, Xuqian Li a, Lili Zhang c, Xiao Liu a, Baohong Jiang b, Zhiguo Long d Yanyan Jiang a,*

a

Key Laboratory of Smart Drug Delivery, Ministry of Education (Fudan University),

Department of Pharmaceutics, School of Pharmacy, Fudan University, Shanghai, China. b

Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai.

201203, China. c

School of Pharmacy, Shanghai University of Traditional Chinese Medicine, Shanghai

201203, China d Department

of Hematology, Shanghai Pudong Hospital, Fudan University, Shanghai,

201399, China

Corresponding Author

Name: Yanyan Jiang Postal address: No.826, zhangheng road, pudong new district, Shanghai, China Tel. & Fax: +86-21-51980077 *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

Cell permeable NBD peptide-modified liposomes by hyaluronic acid coating for the synergistic targeted therapy of metastatic inflammatory breast cancer Yuqing Sun a, Xuqian Li a, Lili Zhang c, Xiao Liu a, Baohong Jiang b, Zhiguo Long d, Yanyan Jiang a,*

a

Key Laboratory of Smart Drug Delivery, Ministry of Education (Fudan University),

Department of Pharmaceutics, School of Pharmacy, Fudan University, Shanghai, China. b

Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai,

201203, China. c School

of Pharmacy, Shanghai University of Traditional Chinese Medicine, Shanghai,

201203, China d Department

of Hematology, Shanghai Pudong Hospital, Fudan University, Shanghai,

201399, China

Keywords: TAT-NBD peptide; Curcumin; Celecoxib; anti-inflammation; antimetastasis

Abstract Chronic inflammation is closely related to the development, deterioration, and metastasis of tumors.

Recently, many studies have shown that down-regulating the

expression of inflammation by blocking nuclear factor-κB (NF-κB) and signal transducer and activator of transcription 3 (STAT3) pathways could significantly inhibit tumor growth and metastasis.

The combined application of curcumin (CUR)


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and celecoxib (CXB) has been proven to exert a synergistic anti-tumor effect via inhibiting the activation of NF-κB and STAT3.

TAT-NBD (TN) peptide, a fusion

peptide of NF-κB essential modulator (NEMO)-binding domain peptide (NBD) and cell-penetrating peptide (TAT), can selectively block NF-κB activating pathway resulting in tumor growth inhibition.

In the present study, a novel TN-modified

liposome co-loading both CXB and CUR (TN-CCLP) at a synergistic ratio was firstly constructed with the property of synchronous release, then hyaluronic acid (HA) as CD44 targeting moiety was coated on the surface of the cationic liposome via electrostatic interaction to prepare the anionic HA/TN-CCLP.

In vitro results of

cytotoxicity, macrophage migration inhibition, and anti-inflammation efficacy revealed that TN-CCLP and HA/TN-CCLP were significantly superior to TN-LP and CCLP, while TN-CCLP exhibited better effects than HA/TN-CCLP due to higher cellular uptake ability.

Different from in vitro data, after systematically treating 4T1 breast

tumor-bearing mice, HA/TN-CCLP exerted the most striking effects on antiinflammation, inhibition of macrophage recruitment, and anti-tumor, because of the longest circulation time and maximum tumor accumulation.

In particular, HA/TN-

CCLP could availably block the lung metastasis of breast cancer.

Taken together, the

novel CD44 targeted TN-CCLP exhibited the potential for inhibiting tumor development and metastasis through improving inflammatory infiltration of tumor tissue. 1. Introduction Chronic

"non-resolving

inflammation"

pathogenesis of malignant tumors 1.

contributes

significantly

to

the

It is involved in different stages of tumor

development, including initiation, promotion, deterioration, invasion, and metastasis 2.



Page 4 of 49

Some evidence has indicated nuclear factor-κB (NF-κB), signal transducer and activator of transcription 3 (STAT3) are two major intrinsic pathways in cancerassociated inflammation

3-6.

Activation of NF-κB and STAT3 in tumor cells is

triggered by inflammatory factors (TNF-α, IL-6, etc.) in tumor inflammatory microenvironment, which in turn activates NF-κB and STAT3 of stromal cells, creating a vicious circle and leading to persistent activation of tumor cells and tumor-associated stromal cells 6.

Stromal cells, including tumor-associated macrophages (TAM),

myeloid-derived suppressor cells (MDSC), tumor endothelial cells (TEC), etc., are generally considered to be necessary for invasion, migration, and metastasis 4-7. In brief, inhibition of the two transcription factors can make significant contribution to antiinflammatory therapy, thereby impede the growth and metastasis of tumor. Recent studies have shown that some anti-inflammation drugs display interesting anti-cancer properties. Among them, curcumin (CUR), the natural product of sesquiterpenoids, attracts increasing attention due to the remarkable anti-inflammation, anti-oxidant, and anti-cancer activities 8.

Numerous studies have indicated that the

anti-cancer properties of CUR are related to transcription factors NF-κB and STAT3 in many cancers, including lung, cervical, prostate, breast, osteosarcoma, and liver cancers 9.

Celecoxib (CXB), a non-steroidal anti-inflammation drug, can down-regulate the

level of prostaglandins (PGE2), decrease the expression of vascular endothelial growth factor C (VEGF-C), and block the activation of STAT3 10, 11. CXB has been proven an inhibitor of macrophage recruitment as well as tumor metastasis at high dose

12, 13.

However, these chemical inhibitors often have toxic side effects and non-selectively


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result in the detrimental effects on normal cellular function, because NF-κB plays an indispensable role in many biological processes. (NEMO)-binding

domain

(NBD)

peptides

NF-κB essential modulator

include

the

major

sequences,

TTLDWSWLQME and TALDWSWLQME, which can prevent the activation of the IκB kinase (IKK) complex and have provided an opportunity to selectively block the inflammation-induced activation of NF-κB by targeting the NBD-NEMO interaction. To facilitate cellular uptake, NBD peptide is often fused with cell-penetrating peptide (TAT) 14-18.

Recent studies have shown that compared with other NF-κB inhibitors,

the fusion peptide TAT-NBD (TN) can effectively ameliorate inflammatory responses activities without overt signs of toxicity for maintaining the basal activity of the IKK 15.

Although these anti-inflammation agents have numerous pharmacological activities, their poor aqueous solubility, low bioavailability, rapid metabolism, and rapid systemic elimination are barriers to their clinical application. In previous decades, the drug delivery system (DDS) using nano-carriers, such as liposomes, nanoparticles, solid dispersion, complex, emulsion, micelles, gels, and microparticles, were employed to overcome these limitations.

Among drug carriers, liposomes have shown rather

promising prospects for in vivo drug delivery.

Liposomal CUR could enhance the

anti-tumor and pharmacological activities of CUR by improving pharmacokinetics and pharmacodynamics and reducing the dosage required for targeting tumor 9.

Similarly,

liposomes can be used to increase the therapeutic efficacy of CXB while minimizing its severe cardiovascular toxicity.

Erdoğ et al

19

reported the encapsulation of CXB



Page 6 of 49

within the long-circulating liposomes and the effect of these formulations against colorectal cancer cell lines.

In regard to the NBD peptides, though many

investigations have revealed its broad range of anticancer activity, including adenocarcinoma, melanoma, prostate, and breast cancers 17, 20, 21, direct administration of free peptides are not suitable for clinical application due to its instability and rapid clearance in vivo.

It is reasonably predictable that the nano-carriers could enhance the

efficiency of NBD peptide by improving in vivo biological behavior and targeting to tumor tissues, though the carrier-mediated NBD peptide delivery system has not been reported yet. Considering tumor microenvironment heterogeneity, combined administration of anti-inflammation drugs with different mechanism could achieve synergistic therapeutic effects by regulating multiple signaling pathways in abnormal cells.

A

study aimed at treating ulcerative colitis had demonstrated that nanoparticles of CURCXB combination were superior to those of either agent alone along with reducing the dose of CXB and overall toxicity

22.

Another study also confirmed that CUR

synergistically potentiated the growth-inhibitory and pro-apoptotic effects of CXB in colorectal cancer cells, and enabled the use of CXB at lower and safer concentration 23. Our preliminary study also proved that the combination of CUR and CXB at appropriate ratio shows synergistic inhibition effects against breast cancer cells.

Although the

superiorities of combined drugs and their co-delivery by nano-carriers are prominent, no research involving anti-inflammation drugs co-delivery system has been found yet to explore the potential for targeted anti-tumor therapy.


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In the present study, the anti-tumor effect of CUR, CXB, and TN peptide was first investigated, and the combined ratio of three agents was optimized.

Then, for the first

time, an anti-inflammation tumor-targeted liposome was constructed by co-loading TN, CXB, and CUR. As illustrated in Scheme 1, hydrophobic CXB and CUR (CC) were simultaneously entrapped into TN-modified cationic liposomes to form TN-modified liposome co-loading both CXB and CUR (TN-CCLP), hyaluronic acid (HA) was coated on the surface of the cation liposome via electrostatic interaction forming an HA-coated, TN-modified anti-inflammation liposome (HA/TN-CCLP).

After

intravenous injection, in vivo fate of HA/TN-CCLP could be reasonably predicted.

In

blood circulatory system, negatively-charged HA on the face of liposome could protect TN peptide from its degradation, improve drugs’ plasma half-life, slow down the process of elimination, and play a similar role to polyethylene glycol (PEG)

24, 25.

Subsequently, HA/TN-CCLP might preferentially accumulate at the tumor tissues via the enhanced permeability and retention (EPR) effect and active targeted CD44 overexpressing cells, including tumor cells, tumor stem cells, and some tumorassociated stromal cells

26.

In addition, concentrated hyaluronidase (HAase) in

malignant tumor environment could degrade or partially degrade HA to expose TNCCLP, and thus cellular uptake of liposomes might be enhanced due to the cellpenetrating mechanism of TN peptide and cation-mediated endocytosis.

As a result,

internalized cationic liposome induced lysosomal escape by proton sponge effect

27,

followed by the release of anti-inflammation drugs to block the activation of cytoplasmic NF-κB and STAT3, and ultimately down-regulate the level of



Page 8 of 49

inflammation factors, modulate the recruitment of macrophages, inhibit the growth and metastasis of tumor cells.

In order to investigate the potential of TN-CCLP with or

without HA coating, the three-drug co-loaded liposomes were evaluated in cellular uptake, inhibition of macrophage migration, anti-inflammation, and anti-tumor activity, while TN-LP and CCLP were used as controls.

Further, we evaluated the HA-coated

liposomes for active targeting on breast cancer, by in vivo assays of pharmacokinetics, biodistribution, levels of inflammatory factors, inhibition of tumor growth and metastasis.

We believe the information is significant in the development of novel anti-

inflammation drug co-delivery system and will facilitate the use of CD44 targeting for cancer therapy. 2. Materials and methods 2.1. Materials (2,3-Dioleoyloxy-propyl)-trimethylammonium

(DOTAP),

glycero-3-phosphoethanolamine-N-[methoxy(polyethylene

1,2-distearoyl-sn-

glycol)-2000]

(DSPE-

PEG2000), DSPE-PEG2000-Mal and cholesterol were purchased from Shanghai AVT Pharmaceutical

Technology

Co.,

Ltd.

(Shanghai,

phosphatidylcholine (S100PC) was from Lipoid (Germany).

China).

Soybean

Celecoxib (CXB) and

curcumin (CUR) were purchased from Dalian Meilun Biotechnology Co., Ltd. (Dalian, China).

TAT-NBD (YGRKKRRQRRRGTTLDWSWLQMEC) was obtained from

GL Biochem Co., Ltd., (Shanghai, China).

Hyaluronic acid (Mw = 36 kDa) was

offered by Shandong Freda Biochem Co., Ltd., (Shandong, China).

Hyaluronidase

(HAase), Poly-L-Lysine (PLL), Annexin V-FITC/PI staining kit and LysoTracker Red


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were obtained from Sigma–Aldrich (St. Louis, MO, USA).

Anti-CD44 antibody,

Anti-CD68 antibody, and Anti-IKB alpha antibody were purchased from Abcam (Cambridge, MA, USA).

TNF alpha and IL-6 ELISA Kit were obtained from Boster

Biological Technology Co., Ltd. (Wuhan, China). 2.2 Synthesis and characterization of TN-PEG2000 -DSPE The TN and DSPE-PEG2000-MAL (1.2 : 1, molar ratio) were added into deionized water at room temperature.

After 12 h of stirring, the resulting solution was

dialyzed (MWCO = 3500 Da) in deionized water for 48 h to remove the excess peptides. The final purified product was lyophilized and analyzed by 1H-NMR (400MHz, Varian, America) and MALDI-TOF/TOF (AB Sciex, USA). 2.3 Preparation of HA/TN-CCLP and other Liposomes HA/TN-CCLP composed of SPC/ DOTAP/ cholesterol/ TN-PEG2000-DSPE/ DSPE-PEG2000= 60: 15: 20: 2.5: 2.5 (molar ratio) were prepared by a film hydration method.

The CC to phospholipid weight ratio was 9.2 %.

To obtain a thin lipid film,

these phospholipids and CC were dissolved in a solution of chloroform/ methanol= 4: 1 (v/ v) and dried at 45 °C under vacuum.

The film was then hydrated with pH 5.7

PBS (0.3% Tween 80) at 45 °C for 40 min, then treated by miniature ultrasonic probe for 3 min at 80 W, and the prepared suspension was purified by centrifugation at 10000 rpm for 1 h to remove the insoluble CC.

HA/TN-CCLP were prepared by dispersing

HA solution (100 mg/mL) in TN-CCLP at a HA/ lipid mass ratio of 1: 1 and then vortex for 5 min at room temperature.

The liposomal formulation was ultrafiltrated

(MWCO= 100k Da) to separate the free HA molecules.



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Coumarin 6 (Cou-6) or DiR-loaded LP, TN-LP, and HA/TN-LP were prepared according to the above procedure for the research of uptake and in vivo imaging.

The

CCLP had a lipid composition of SPC/ DOTAP/ cholesterol/ DSPE-PEG2000= 60: 15: 22.5: 2.5 (mole ratio). 2.4 Characterization 2.4.1 Size distribution and zeta potential Size, PDI, and ζ-potential of samples were measured using a Zetasizer Nano ZS (Malvern, UK) at 25 °C.

Prior to measurement, liposomes were diluted 10 times in

distilled water. 2.4.2 Encapsulation efficiency and drug loading The CXB and CUR in HA/TN-CCLP were quantitatively determined with an LC10 AT HPLC system (Shimadzu, Japan) at a UV absorption wavelength of 254 nm. Aliquots of liposomes were dried under vacuum and dissolved in methanol by vigorous vortex mixing. Drug encapsulation efficiency (EE) was calculated as: weight of CC in liposomes(mg)

EE(%)＝weight of CC initially added(mg)×100 Drug loading was calculated as: weight of CC in liposomes(mg)

Drug Loading(%)＝

weight of Lipids (mg)

×100

2.4.3 Transmission electron microscopy The morphology of samples was observed with a TEM (JEM-1400 PLUS, Japan) at an acceleration voltage of 200 kV.

Prior to TEM examination, these samples were

stained with 2 % (v/ v) phosphotungstic acid for 5 min, a drop of the stained sample


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was deposited on the carbon mesh, and then the excess sample was removed with filter paper and dried at 25 °C. 2.4.4 Stability in serum and HAase To estimate the stability in physiological conditions, the size distributions and zeta potential of TN-LP and HA/TN-LP were measured in PBS that contained 1mg/mL HAase or 10% FBS at 37 ℃ for 24 h.

At specific time intervals, the samples were

withdrawn to measure. 2.5 In vitro drug release The release behavior of CXB and CUR from different formulations were measured at 37 ℃ under various conditions.

Samples were suspended in different medium and

then transferred into dialysis bags (MWCO= 14,000 Da) against the same solution that contained 2 % SDS to meet the sink condition.

At determined time points, 2 mL of

the external solution was withdrawn and the same volume of fresh solution was added to the dialysate.

The amount of the released CXB and CUR was measured using

HPLC as described above. 2.6 Uptake and penetrability of liposomes 2.6.1 Cell lines and Mammospheres 4T1 cells (mouse breast carcinoma), RAW264.7 (mouse monocyte macrophages) and HUVEC (human umbilical vein endothelial cells) were purchased from Cell Bank of Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China).

RAW264.7, HUVEC and 4T1 cells were cultured in DMEM and

RPMI supplemented with 10 % (v/ v) FBS and 1 % (v/ v) penicillin-streptomycin in a



Page 12 of 49

humidified atmosphere of 95 % air and 5 % CO2 at 37 ℃, respectively. A kind of multi-cell lines mammospheres model co-incubated with 4T1, HUVEC and RAW (4: 1: 1, ratio of cells number), which is more closer to the growth of tumor in vivo, was established by hanging drop cultural according to our previous method with slight modification 28.

The mammospheres model was used to evaluate in vitro

penetrability and tumor inhibition ability of liposome formulations. 2.6.2 Expression of CD44 on cells CD44 expression on 4T1, RAW and HUVEC cells was examined by flow cytometry.

Cells (1×106) were washed with blocking buffer, incubated with 2 μg of

FITC-labeled anti-mouse CD44 antibody at 4 °C for 1 h, rinsed with PBS for three times, and resuspended in 1 mL of PBS, analyzed by FACS (Beckman, America). 2.6.3 In vitro cellular uptake and uptake inhibition After 24 h incubation, the growth medium of cells was replaced with fresh growth medium containing free cou-6, cou-6-loaded LP, TN-LP and HA/TN-LP with the final concentration of 0.3 μg/mL.

Then further incubated for 2 h, the medium was removed

and cells were washed with PBS for two times to stop the cellular uptake.

Finally,

they were trypsinized and a FACS was used to measure the cellular uptake. uptake inhibition studies, HA and PLL were used as competitive inhibitor.

For

Briefly,

cells were pre-incubated with HA (10 mg/mL) or PLL (1 mg/mL) for 1 h, and then incubated with cou-6-loaded TN-LP and HA/TN-LP for another 2 h. were processed as described above. 2.6.4 Intracellular location of HA/TN-CCLP


Finally, the cells

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The intracellular location of liposomes was studied in 4T1 cells with confocal imaging.

Cells (2×105) were seeded on 12 mm round glass coverslip in advance, then,

incubated with cou-6-loaded TN-LP and HA/TN-LP with the final concentration of 0.3 μg/mL in serum-free medium at 37 °C.

After washed with cold PBS for three times,

the cells were treated with 1 mL of Lysotracker red for 1 h to stain lysosomes.

The

CLSM (Carl Zeiss LSM710, Germany) examination was conducted after the cells were fixed with 4 % paraformaldehyde and stained with DAPI for 10 min. 2.7 Cytotoxicity and apoptosis 2.7.1 In vitro cytotoxicity The cells were seeded in 96-well plates at a density of 5× 103 cells/well and grew for 24 h.

Then, cells were treated with free drugs, blank liposomes and drug-loaded

liposomes at various concentrations at 37 °C.

After 48 h incubation, the growth

medium was removed and 200 μL of 0.5 mg/mL MTT solution was added. incubation continued for another 4 h.

The

Finally, the resultant formazan salt crystals were

dissolved by adding 200 μL of DMSO to each well, the absorbance of each well was measured at 570 nm using a Microplate reader (BioTek, America). The combination index (CI) used to evaluate the synergistic effect of drugs was calculated by CompuSyn software according to the Chou-Talalay isobologram equation: CI= Dm1/ D1+ Dm2/ D2 where Dm is the ICx values of the combined drug, and D1 and D2 are ICx values of single drugs.

CI < 1, CI = 1 and CI > 1 indicate synergistic, additive and

antagonistic effects of drug combinations, respectively 29, 30.



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2.7.2 In vitro cell apoptosis Cell apoptosis was detected by flow cytometry, using an Annexin V-FITC/PIstaining kit.

In brief, 4T1 cells were seeded in a 24-well plate at a density of 1×105

per well and treated with different drug-loaded formulations for 48 h at total drug concentration of 20 μM.

After the culture medium was removed, the cells were

washed with cold PBS, then trypsinized and stained with Annexin V-FITC and PI for 10 min at room temperature in the dark.

Thereafter, binding buffers were added and

the samples were analyzed by FACS. 2.7.3 Growth inhibition of mammosphere Mammospheres with a diameter of 200 μm were selected and incubated with drugloaded liposomes at total drug concentration of 20 μM.

The size of the mammosphere

was monitored by an optical microscope during the 9-day incubation.

The major (dmax)

and minor (dmin) diameters of each mammosphere were measured, and the volume was calculated by the following formula: V= 0.5×Dmax×Dmin2 The relative volume (Vr) of the mammosphere after treatment was calculated with the following formula: Vr= (Vn/V0) × 100%, where Vn is the volume of the mammosphere at day n after treatment, and V0 is the volume of the mammosphere prior to treatment. 2.8 In vitro synergistic anti-migration and anti-inflammation 2.8.1 Migration inhibition of macrophages The migratory ability of RAW cells towards 4T1 cells was firstly evaluated by scratch assay using a 2-chamber Culture-Insert (Ibidi, USA).


RAW and 4T1 cells

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were separately laid on the left and right sides of the Culture-inserts at a density of 1× 105 cells per well and cultured to 80 % confluence. free drugs and drug-loaded liposomes for 8 h. removed using sterile tweezers.

4T1 cells were incubated with Afterward, Culture-Insert were

And cells were cultured with serum-free medium, the

distances between growth frontiers of two clusters were monitored and photographed under an inverted fluorescence microscope (DMI 4000D, Leica, Germany). The migratory number of RAW towards 4T1 was investigated by transwell assay. 4T1 cells were grown to 70 % to 80 % confluence in completed culture media.

The

medium was replaced with free drugs or drug-loaded liposomes in serum-free media, and cells were cultured for an additional 48 hours, then the supernatant was used as conditioned medium (CM).

The 8-μm pore Transwell polycarbonate membrane

chambers (Corning, Pittston, PA) were used.

RAW cells (2× 105) in 100 μL of serum-

free medium were added to the upper chamber, and CM from 4T1 was added to the lower chamber.

The migration process lasted for 6 h at 37 °C.

Then the cells

remained in upper chamber was removed and those migrated to the bottom chamber were stained with DAPI.

The number of cells was counted in five randomly selected

fields under an inverted fluorescence microscope. 2.8.2 In vitro inflammation factors levels 4T1 cells (1×106) were seeded in 6-well plate for 24 h, and then incubated with free drugs or drug-loaded formulations at various concentrations for 24 h at 37 °C.

To

collect protein, the harvested 4T1 cells in ice PBS containing protease inhibitor cocktail were centrifuged at 12 000 rpm for 5 min at 4 °C.



For western blot assay, total protein was measured using a BCA protein assay kit and separated on 10 % sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).

The proteins were then transferred to a nitrocellulose membrane,

which was incubated in blocking solution containing 5 % skim milk powder at 37 °C for 2 h and rinsed with phosphate buffer solution containing Tween 20 (TBST) two to three times.

Subsequently, the membrane was incubated with primary antibodies,

anti-IKB alpha antibody, at 4 °C for 2 h and a secondary antibody at 4 °C overnight. Finally, immunoreactive proteins were visualized, and images were acquired with an Odyssey Infrared Imaging System (BioRed, America) after staining with a chemiluminescence kit. For ELISA assay, the resultant supernatants were collected for detection of IL-6 and TNF-α with ELISA kits according to the manufacturer’s instruction. 2.9 Tumor targeting properties of liposomes 2.9.1 Animals model Female BALB/c mice (6 weeks old, 18–22 g) were purchased from Shanghai Laboratory Animal Center (Shanghai, China).

The orthotopic tumor-bearing mouse

model of breast cancer was established by subcutaneous injection of 4T1 cells (1×106 cells/mouse) in the mammary fat pad.

In vivo study protocol was performed in

compliance with the guidelines of the Ethics Committee of Animal Center of Fudan University. 2.9.2 In vivo distribution Tumor-bearing mice with the tumor size reached around 200 mm3 were used to


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investigate the in vivo distribution of liposomes.

DiR-labeled preparations were

injected into the mice via the tail vein at a fixed DiR dose of 0.5 mg/kg based on the animal’s body weight.

At 1, 6, 12, and 24 h after injection, the treated mice were

sacrificed to harvest the major organs (heart, liver, spleen, lung, kidney, brain and tumor) and blood, and fluorescence images were acquired with IVIS Spectrum (Perkin Elmer, America). The pharmacokinetics was studied in tumor-bearing mice. The pharmacokinetic parameters were calculated by non-compartmental analysis using DAS 2.0. 2.10 In vivo anti-tumor and anti-metastasis efficacy 2.10.1 In vivo tumor growth inhibition 4T1 tumor xenograft nude mice were weighted, randomly separated into 7 groups (n=8).

When the tumor volume reached around 80 mm3, the nude mice were

intravenously administrated with 0.2 mL of different preparations at a total dose of 20 mg/kg at Day 9, 12, 15, 18. 21.

The body weight and tumor volume were calculated

every 3 days, and the survival period was monitored throughout the study. 2.10.2 In vivo analysis of biological samples After 21 days treatment according to the above dosage regimen, tumor-bearing mice were sacrificed to collect tumors, lung and bone tissues for H&E and TUNEL analysis. For immunofluorescence analysis, monoclonal mouse anti-mouse CD68 was applied to the embedded tumor and lung tissue. The harvested lungs were fixed in Bouin’s solution for 72 h at 25 °C, and the



Page 18 of 49

number of metastatic nodules appearing on the surface of the lungs was counted to assess lung metastases from breast tumor. To investigated the in vivo inflammation factors levels, the tumors were homogenized on ice in PBS containing protease inhibitor cocktail and centrifuged at 12 000 rpm for 5 min at 4 °C.

Then, the expression of IKB-α, IL-6 and TNF-α were

measured by western blot and ELISA as described above. 2.11 Statistical analysis All the values were presented as mean± standard deviation (SD) and comparison among the different groups was performed by one-way ANOVA, and t-test was used to analyze the data. individual groups.

Kaplan–Meier method was used to compare the lifespan of The level of statistical significance in all statistical analyses was

denoted as *p

Cell Permeable NBD Peptide-Modified Liposomes by Hyaluronic Acid

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