Vinblastine-Loaded Nanoparticles with Enhanced Tumor-Targeting

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Vinblastine-loaded nanoparticles with enhanced tumor targeting efficiency and decreasing toxicity: Developed by one-step molecular imprinting process Yongyan Zhu, Ruixuan Liu, Haoji Huang, and Quanhong Zhu Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.9b00243 • Publication Date (Web): 03 May 2019 Downloaded from http://pubs.acs.org on May 6, 2019

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Molecular Pharmaceutics

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

1

Vinblastine-loaded nanoparticles with enhanced tumor targeting

2

efficiency and decreasing toxicity: Developed by one-step molecular

3

imprinting process

4

Yongyan Zhu a, Ruixuan Liu a, Haoji Huang a, Quanhong Zhu a*

5

a

6

China

7

* Corresponding Author

School of Traditional Chinese Medicine, Southern Medical University, Guangzhou 510515,

8 9

Corresponding author information:

10

Prof. Quanhong Zhu

11

Address: School of Traditional Chinese Medicine, Southern Medical University, 1023 South

12

Shatai Road, Guangzhou 510515, China.

13

Tel.: +86-20-6164-8770

14

Fax: +86-20-6164-8770

15

E-mail address: [email protected] (Q. -h. Zhu)

16 17

Abstract

18

Molecularly imprinted polymers have exhibited good performance as carriers on drug

19

loading and sustained release. In this paper, vinblastine (VBL) loaded polymeric nanoparticles

20

(VBL-NPs) were prepared by one-step molecular imprinting process, avoiding the waste and

21

incomplete removal of template, and evaluated as targeting carriers for VBL delivery after

22

modification. Using acryloyl amino acid co-monomers and disulfide cross-linkers, VBL-NPs were

23

synthesized and then conjugated with polyethylene glycol-folate. The dynamic size of obtained

24

VBL-NPs-PEG-FA was 258.3nm (PDI=0.250), and the encapsulation efficiency was

25

45.82±1.45%. The nanoparticles of VBL-NPs-PEG-FA were able to completely release VBL

26

during 48h under mimic tumor intracellular condition (pH4.5, 10mM glutathione (GSH)),

27

displaying significant redox responsiveness, while the release rates were much slower in mimic


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1

body liquid (pH7.4, 2μM GSH) and tumor extracellular environment (pH6.5, 2μM GSH).

2

Furthermore, the carriers NPs-PEG-FA, prepared without VBL, showed the satisfactory intrinsic

3

haemocompatibility, cellular compatibility and tumor targeting properties: it could rapidly and

4

efficiently accumulate to folate receptor (FR) positive Hela cells and then internalized via

5

receptor-mediated endocytosis, and the retention in tumor tissues could last for over 48h.

6

Interestingly, VBL-NPs-PEG-FA could evidently increase the accumulation of VBL in tumor

7

tissue while decrease the distribution of VBL in organs, exert the similar anticancer efficacy

8

against Hela tumors in xenograft model of nude mice to VBL injection, and significantly improve

9

the abnormality of liver and spleen observed in VBL injection. VBL-NPs-PEG-FA is potential to

10

be delivery carrier for VBL with enhancing tumor targeting efficacy of VBL and decreasing

11

toxicity to normal tissues.

12 13

Keywords: Vinblastine, amino acid monomers, one-step molecular imprinting polymers, redox

14

responsiveness, tumor targeting, decreasing toxicity

15 16

1

Introduction

17

Vinblastine (VBL) is the first chemotherapeutic agent from plants and has been widely used

18

in clinical for treatment Hodgkin’s disease, testicular cancer, ovarian cancer, breast cancer, head

19

and neck cancer, non-Hodgkin’s lymphoma, etc.

20

assembly and then to interfere the formation of the mitotic spindle resulting in mitotic arrest

21

and still considered to be an efficacious and superb drug and listed on the World Health

22

Organization's List of Essential Medicines 4. However, patients have to suffer from the

23

systemically delivery and unpleasant side effects 5, such as myelosuppression (Leucopenia and

24

anemia), inappropriate antidiuretic hormone secretion, ileus, mucositis, neuropathy, and

25

Raynaud’s phenomenon when taking VBL medication 2, 6-7.

1-2.

It binds tubulin to disrupt microtubule 2-3,

26

Researchers have managed to develop intelligent delivery carriers for improving the safety

27

and toxicological profiles of chemotherapeutic agents 8. Up to now, polymeric nanoparticles (NPs)

28

have played an important role in the promising drug delivery systems 9, and about 30% of total

29

nanomedicines approved by FDA from the mid-1990s to 2016 belong to NPs, including

30

Copaxone® and Neulasta®, the top 10 best-selling drugs in the US in 2013 10. The research of



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1

VBL on NPs are much rarer, although poly(lactide-co-glycolide) 6, poly(caprolactone) grafted

2

dextran 11, and liposomes 12-13, or natural polymeric materials cyclodextrin and chitosan 14-18 have

3

been used to construct VBL delivery systems for acquiring sustained release, improving

4

bioavailability or targeting tumors 12, 14, 19. However, these carriers all didn’t pay attention to the

5

stimuli-responsive release of VBL under the tumor intracellular environment where the

6

physicochemical properties are special and different from the surrounding tissues. To achieve the

7

smart release of VBL in the tumor microenvironment and hence decrease the systemic toxicity is

8

still struggling.

9

Due to specific recognition cavities complementary in shape, size and chemical functionality

10

to the template molecule 20-21, molecular imprinted polymers (MIPs) are with large surface areas

11

and strong affinities to the template via non-covalent interactions, such as hydrogen bonds, ionic

12

bonds, electrostatic interaction and hydrophobic effect

13

loading and sustained drug release when serving as drug delivery systems

14

reported for the delivery of targeting anticancer agents 25-26, ocular peptide drugs 27, oral insulin 28,

15

and enantioselective controlled release of racemic drugs 29, etc.

22-23,

and thus resulting in high drug 24.

MIPs have been

16

However, the template molecule should be removed from the polymers for obtaining

17

matched imprints in the process of MIPs’ synthesis, which certainly results in the waste of

18

template molecule and hence is infeasible for the delivery of expensive agents. In recent years,

19

MIPs, with one-step molecular imprinting process, omitting template elution step, have displayed

20

good performance as drug containing delivery carriers. The paclitaxel imprinted polymeric

21

nanoparticles without template removal were modified with PEG and folate to serve as drug

22

carriers for controlled release and targeting delivery of paclitaxel

23

molecular imprinting technique was also used to synthesize propranolol imprinted polymers for

24

sustained release

25

25.

26

Although

31

30.

In addition, the one step

and doxorubicin imprinted nanoparticles for pH and thermo-sensitive release

VBL

imprinted

nanoparticles,

using

methacrylate

as

monomers

and

27

trimethylolpropane trimethacrylate as cross-linkers via conventional molecular imprinting process

28

with removal of the template, showed good encapsulation efficacy and sustained release behavior

29

in our group’s previous study26, the waste of template in conventional molecular imprinting and

30

biocompatibility of MIPs should be seriously concerned. In this paper, we further developed one


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1

step molecular imprinting process to construct the delivery carriers for VBL, and acrylated natural

2

amino acids, N-acryloyl-phenylalanine (APA) and N-acryloyl-lysine (ALys), were introduced as

3

monomers for improving both the interaction with VBL and the biocompatibility

4

N,N’-bis(acryloyl)cystamine (BACy) with reducible disulfide bonds, commonly incorporated into

5

drug carriers for smart responsive release under internal microenvironment of tumor cells with

6

high concentrations of glutathione (GSH)

7

process, which could be “switch on” triggered and dramatically release drug molecules inside

8

tumor cells.

9

33-35,

32.

Meanwhile,

was employed as cross-linkers in the imprinting

After grafted with the polyethylene glycol-folate (PEG-FA) on the surface of VBL loaded

10

polymeric nanoparticles (VBL-NPs) for prolonging the circulation time in bloodstream

11

achieving tumor-specific targeting

12

VBL-loaded nanoparticles (VBL-NPs-PEG-FA) were evaluated through the in vitro and in vivo

13

assays, including the drug loading properties, drug release profile, cellular compatibility and

14

haemocompatibility assay, cell uptake assay, bio-distribution and antitumor efficiency assay.

30

and high-efficiency cellular uptake

37,

36

and

the designed

15 16

2

Materials and methods

17

2.1 Materials, cells and animals

18

Poly (ethylene glycol)-bis-amine (MW=2000, NH2-PEG-NH2) and anhydrous dimethyl

19

sulfoxide were purchased from Shanghai Aladdin biological technology co., Ltd (China).

20

Cetyltrimethylammonium Bromide (CTAB), Folic Acid Hydrate (FA), L-phenylalanine (L-Phe), ,

21

L-lysine (L-Lys), acryloyl chloride, cystamine dihydrochloride, N-Hydroxysuccinimide (NHS),

22

carbodiimide hydrochloride (EDC), glutathione (GSH, reduced form) and Fluorescein

23

5-Isothiocyanate (isomer I) (FITC) were from TCI (Shanghai) Development Co., Ltd (China).

24

Azobisisobutyronitrile (AIBN) was obtained from Fuchen Chemical Reagent factory (Tianjin,

25

China) and recrystallized with methanol. Vinblastine sulfate (VBL) was from Yueyang

26

Biomedical Science and Technology Co., Ltd (Hainan, China). Dimethyl sulfoxide (BioReagent,

27

suitable for hybridoma), 0.25% Trypsin-EDTA, methyl thiazolyl tetrazolium (MTT) and Triton

28

X-100 were from Sigma Chemical Company (USA). Roswell Park Memorial Institute (RPMI)

29

1640 medium, Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum (FBS),

30

penicillin and streptomycin were purchased from Gibco (USA). 4% paraformaldehyde was from



1

Google biotechnology co. LTD (Wuhan, China). 4', 6-diamidino-2-phenylindole (DAPI) was from

2

Xingzhi biotechnology co. LTD (Guangzhou, China). Sulfo-Cyanine7 NHS ester was purchased

3

from Xi’an ruixi biological technology Co.,Ltd (China). Human PF4 ELISA Kit (CXCL4), human

4

Thrombin-Antithrombin Complex ELISA Kit (TAT) and Complement C5a Human ELISA Kit

5

were purchased from Abcam (Cambridge, UK). TUNEL Kit was from Roche Molecular Systems,

6

Inc. (USA). Hematoxylin and Eosin (H&E) Staining Kit was from Google biotechnology co. LTD

7

(Wuhan, China).

8 9

Hela human cervical cancer cells, A549 human lung cancer cells and L929 mouse fibroblast cells were purchased from Cell Bank of Chinese Academy of Sciences (Shanghai, China).

10

BALB/c nude mice (female, 5−6 weeks old, 18 ± 2 g) were supplied by Laboratory Animal

11

Center of Southern Medical University (Guangzhou, China) (No. SCXK Yue 2006-0015), and

12

maintained in an animal laboratory under specific pathogen-free (SPF) conditions. All animal

13

procedures were performed in compliance with the protocol evaluated and approved by the ethics

14

committee of Southern Medical University.

15

2.2 Preparation and Characteristics of VBL loaded polymers

16

The cross-linker with disulfide bond N,N’-bis(acryloyl)cystamine (BACy) and two

17

N-acryloyl-L-amino acid monomers, including N-Acryloyl-L-phenylalanine (APA) and

18

N-Acryloyl-L-lysine (ALys) were synthesized. The detailed synthesis processes were described in

19

“Supporting information” and their 1H-NMR spectra were shown in Fig.S1, Fig.S2, Fig.S3 in

20

“Supporting information”.

21

The VBL loaded polymers, APA/ALys@MIP (VBL-NPs), were prepared in deionized water

22

by one-step molecular imprinted process omitting template elution 30 according to Table 1 and the

23

detail synthesis procedure was described in “Supporting information”. The corresponding

24

non-imprinted polymers (NIP) were synthesized using the same methods but without VBL to

25

obtain drug-free nanoparticles, APA/ALys@NIP (NPs). And these polymers were conjugated with

26

PEG-FA to obtain the drug loaded APA/ALys@MIP-PEG-FA (VBL-NPs-PEG-FA) and the

27

empty APA/ALys@NIP-PEG-FA (NPs-PEG-FA) respectively. The structures of these polymers

28

were evaluated by FT-IR and 1H-NMR (see Fig.S4, Fig.S5, Fig.S6 and Fig.S7 in “Supporting

29

information”). And the zeta potentials, average hydrodynamic diameters and polydispersity index

30

of the polymers were determined by dynamic light scattering (DLS, Nano S90, Malvern, UK)


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1

after suspended in deionized water. The surface morphology and the pore size of the polymers

2

were measured by Transmission electron microscopy (TEM, JEM-2100HR, JEOL, Japan) and

3

Surface Area and Porosimetry analyzer (NOVA4200e, Quantachrome, USA) at 77K with the

4

Brunauer–Emmett–Teller (BET) method respectively.

5

2.3

6

Encapsulation and drug releasing assay The synthesized non-modified VBL-NPs were dialyzed with fresh deionized water, and the

7

dialysate was collected to determine the amount of VBL

8

conditions: the HPLC system (1200, Agilent, USA) equipped with a reverse-phase C18 column

9

(Agilent, 250×4.60 mm, 5μm) and ultraviolet-visible detector at the detection wavelength of

10

269nm; 1.4% (v/v) triethylamine aqueous solution (adjusted to pH 7.2 with phosphoric acid) and

11

acetonitrile (40:60, v/v) were as the mobile phase; the flow rate was 1.0 ml/min at ambient

12

temperature. The amount of VBL encapsulated inside polymers was calculated by subtracting the

13

concentration of free VBL in dialysate from the initial feed amount of VBL.

14

38

via HPLC under the following

The encapsulation of VBL by NPs-PEG-FA was measured as literatures with some

15

modifications

16

suspended in 10 mL PBS (pH4.5) containing 10mmol/L glutathione (GSH) at 37 ℃

17

oscillation for 3days to collapse drugs loaded in the polymers. And then the medium was

18

centrifuged at 10,000rpm for 10 min. The supernatant was collected was analyzed by HPLC under

19

the above condition to obtain the amount of VBL encapsulated by polymers. The entrapment

20

efficiency and drug loading capacity was calculated as the equation 1 and 2:

21

Entrapment efficiency (%) =

22

Drug loading capacity (%) =

30.

1mg of lyophilized VBL-NPs-PEG-FA were placed into dialysis bag and

The amount of VBL encapsulated by polymers The initial amount of VBL The amount of VBL encapsulated by polymers The amount of the drug loaded polymers

× 100% × 100%

with

(1) (2)

23 24

The VBL release profiles from VBL-NPs and VBL-NPs-PEG-FA in vitro were determined

25

by dialysis method 39. Briefly, the lyophilized drug-loaded polymers (containing 1mg VBL) were

26

put in dialysis bag (molecular weight cutoff 1000) and immersed in 25 mL buffer solution at

27

various pH values (4.5, 6.5 and 7.4) with various concentration of GSH (0μM, 2μM and 10mM) in

28

dark at 37 °C respectively. At the determined time points, 1 mL of the release medium was

29

withdrawn and replaced with the same volume of fresh medium. The samples were centrifuged at



Page 8 of 45

1

10,000rpm for 10 min, and the supernatant was transferred to the UV-Star®Microplate (96 well,

2

Greiner, Germany) and measured the amount of VBL released at 269nm by multiskan spectrum

3

microplate spectrophotometer (Multiskan Go, Thermo scientific, USA). The cumulative release

4

percentage was calculated as the equation 3:

5

Cumulative release percentage(%) 

Cn  V1 

 (C

n - 1 

M

Vn

)

- 1

(3)

 100%

6

Here, C represents the concentration of free drug in the sample solution; V is the volume of

7

sample solution; V1 is the total volume of release medium, and M is the amount of drug in the

8

drug loaded polymers.

9

2.4 In vitro biocompatibility evaluation

10

Cellular compatibility 40

2.4.1

11

L929 cells were maintained in DMEM supplemented with 10% fetal bovine serum and 1%

12

antibiotics at 37℃ in a 5% CO2 incubator (Revco EliteⅡ, Thermo Scientific, USA). Cells were

13

subcultured by disaggregating with Trypsin (0.25%, w/v)-EDTA (0.02%, w/v). L929 cell line in

14

logarithmic growth phase was seeded in 96-well plate at 3000 cells/well and cultured for 18~20h.

15

Then cells were incubated with drug-free NPs or NPs-PEG-FA for 24 h, 48h and 72h respectively

16

to evaluate the intrinsic toxicity of polymers to normal cells. The concentrations of drug free

17

polymers suspended in culture medium were 120, 12, 1.2, 0.12, 0.012 and 0.0012μg/mL

18

respectively. The cells in the normal control were only treated with the culture medium without

19

polymers. And the negative control, containing culture medium but no cells, was set to obtain the

20

absorbance of MTT reagent and culture medium. Each group was repeated in six wells. After

21

incubating for determined time, the cell viability was determined by measuring the absorbance

22

(490 nm) via microplate spectrophotometer (Multiskan FC, Thermo scientific, USA). The cell

23

viabilities were evaluated via calculating the relative growth rate (RGR) according to the equation

24

4. Each experiment was performed in triplicate.

25

Relative growth rate (%) = 𝑂𝐷 𝑜𝑓 𝑛𝑜𝑟𝑚𝑎𝑙 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 𝑔𝑟𝑜𝑢𝑝 ― 𝑂𝐷 𝑜𝑓 𝑛𝑒𝑔𝑎𝑡𝑖𝑣𝑒 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 𝑔𝑟𝑜𝑢𝑝 ∗ 100%

26

2.4.2

27

𝑂𝐷 𝑜𝑓𝑒𝑥𝑝𝑒𝑟𝑖𝑚𝑒𝑛𝑡𝑎𝑙 𝑔𝑟𝑜𝑢𝑝 ― 𝑂𝐷 𝑜𝑓 𝑛𝑒𝑔𝑎𝑡𝑖𝑣𝑒 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 𝑔𝑟𝑜𝑢𝑝

(4)

Hemocompatibility assay The haemocompatibility of intravenous delivery systems is considered to be of decisive

28

significance.

29

ethylenediaminetetraacetic acid-K2 (EDTA-K2), sodium citrate, sodium heparin as anticoagulant

Human

blood

was

respectively

drawn


into

vacutainers

containing

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1

respectively. We incubated the empty NPs or NPs-PEG-FA with blood to evaluate the influence of

2

polymers on erythrocytes, coagulation, and complement system.

3

2.4.2.1 Haemolysis

4

Hemolytic assay was performed as literatures with some modifications

41-42.

The drug-free

5

polymers were suspended in normal saline (NS) at 0.5, 1, 2.5 and 5mg/mL respectively. Red blood

6

cells (RBCs) were harvested from EDTA-K2 anti-coagulated human blood by centrifugation at

7

3000 rpm for 10 min. After washed 4 times with NS, RBCs were diluted 1:10 with NS. 200μL

8

polymer suspension was mixed with 200μL of the above described RBC suspension and the final

9

volume was made up to 1 mL with NS. In case of positive and negative control, RBC cells were

10

suspended in triton X-100 (1%, w/v) and NS respectively. All samples were incubated for 60 min

11

at 37 ℃

12

centrifugation at 3000 rpm for 10 min, the supernatants were taken and measured for the release of

13

haemoglobin at 541nm via ELISA reader (Multiskan FC, Thermo scientific, USA). Each

14

experiment was performed in triplicate. % Haemolysis was calculated as the equation 5 43-44.

15

% Haemolysis 

16

2.4.2.2 Plasma clotting time

with an intermittent mixing by inversion of the microcentrifuge tubes. After

Absorbance of test sample - Absorbance of negative control  100% Absorbance of positive control - Absorbance of negative control

(5)

17

Using sodium citrate as anticoagulant, blood was centrifuged at 1000rpm for 5min to give

18

platelet-rich plasma (PRP) and then the remaining blood was centrifuged at 3000 rpm for 10 min

19

to obtain platelet-poor plasma (PPP). The empty NPs or NPs-PEG-FA were suspended in NS at

20

5mg/mL, and 100μL suspension was mixed with 500μL of PPP to incubate for 30min at 37℃. NS

21

was used as negative control. The activated partial thromboplastin time (APTT), thromboplastin

22

time (TT), prothrombin time (PT) and the fibrinogen concentration (Fbg C) were measured

23

immediately on automatic blood coagulation analyzer CA-7000 (Sysmex Corporation, Kobe,

24

Japan), and the data were averaged from measurements on three specimens45-46.

25

2.4.2.3 Coagulation activation, thrombogenicity and complement system activation

26

The influence of polymers to the activation markers for coagulation activation

27

(thrombin-antithrombin complex, TAT), thrombogenicity (platelet factor 4, PF4) and complement

28

system activation (human complement fragment C5a) were studied through ELISA using human

29

blood using sodium heparin as anticoagulant. The empty NPs or NPs-PEG-FA were suspended in



1

NS at 0.5, 1, 2.5 and 5mg/mL respectively. 100μL of polymers suspension was incubated with

2

500μL of blood at 37 ℃ for 30min and then centrifuged at 3000rpm/min for 10min. The

3

concentration of TAT in plasma was measured by ELISA. The PF4 and C5a were evaluated

4

without the centrifugation step after incubated with PRP and PPP respectively.

5

2.5 Cellular uptake

6

FITC (58.41mg) of anhydrous DMSO solution was dropwise added into the empty

7

NPs-PEG-FA (34.58mg) of anhydrous DMSO solution under vigorous stirring in 10min and the

8

mixture was reacted for 24h at room temperature in dark. The mixture was dialyzed against

9

phosphate buffered saline (PBS) (pH7.4, 0.01M) and deionized water, and then lyophilized to

10

obtain fluorescence labeled polymers NPs-PEG-FA-FITC 47.

11

The intracellular distribution of NPs-PEG-FA-FITC was evaluated with two cancer cell lines,

12

the folate-receptor positive Hela cells and the folate-receptor negative A549 cells 48-49, cultured in

13

folic acid-deficient RPMI 1640 medium with 10% FBS and penicillin (100 U/ml)/streptomycin

14

(100 U/ml) at 37 ℃ in a 5% CO2 incubator. Exponentially growing cells were adherent on

15

confocal dish (35mm) with a density of 3× 105 cells/ well for 20 h. The original medium was

16

discarded and cells were incubated with 1.5ml of folic acid-deficient culture medium containing

17

NPs-PEG-FA-FITC (0.2mg/mL). After incubated for determined time, the culture medium was

18

removed and cells were gently rinsed three times with PBS (pH7.4, 0.01M) containing 0.05% v/v

19

tween 20 (TPBS), fixed with 4% paraformaldehyde for 15min, and again rinsed with TPBS three

20

times. DAPI (10mg/mL) was added to visualize the nuclei for 10 min before imaging. After

21

washed with TPBS for three times, the antifade mounting medium was dropped to prevent the

22

fluorescence quenching and the cellular uptake was observed via confocal laser scanning

23

microscopy (LSM 880 with Airyscan, ZEISS, Germany).

24

The quantification of cellular uptake was performed via flow cytometry. Exponentially

25

growing Hela cells and A549 cells were seeded in six-well culture plates at the density of 6.5×105

26

cells per well respectively and incubated at 37 ℃ for 20 h. The cells were then incubated with

27

2mL of folic acid-deficient culture medium containing NPs-PEG-FA-FITC (2mg/mL) for 12h.

28

The cells were washed with cold PBS (4°C) three times, harvested using trypsin–EDTA, and

29

centrifuged at 1000 rpm for 5 min at 4°C. And the pellets were washed with cold PBS and

30

centrifuged for three times, and then re-suspended into cold PBS to quantify the cellular uptake by


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1

flow cytometry immediately (CytoFLEX，Beckman Coulter, USA).

2

2.6 In Vivo imaging of NPs-PEG-FA

3

Sulfo-Cyanine7 NHS ester (1.28mg) was conjugated to the drug-free NPs-PEG-FA (31.8mg)

4

anhydrous DMSO solution and stirred vigorously for 24h in dark. The mixture solution was

5

dialyzed by deionized water and then lyophilized to give NPs-PEG-FA-Cy7 50.

6

0.1 mL of Hela cells (1×107 cells) normal saline suspension was injected subcutaneously into

7

the back neck of mice. Tumor volumes were measured every other day via vernier caliper and

8

calculated according to the formula 6

9

mm3, the tumor bearing mice were randomly assigned to four groups. 0.1mL of NPs-PEG-FA-Cy7

10

(5mg/mL) was injected into mice through tail vein. At predetermined time points (4h, 12h, 24h

11

and 48h), one group of mice underwent the in vivo fluorescence imaging via the in vivo imaging

12

system (FX Pro, Bruker, USA) with excitation wavelength of 760 nm and emission wavelength of

13

790nm. Then mice were sacrificed by cervical dislocation and the major organs including heart,

14

liver, spleen, lung, kidney and tumor were resected and imaged.

15

Tumor volume =

16

2.7 The in vivo drug distribution assay

πlength × width2 6

51-52.

When the tumor volume reached approximately 80

(6)

17

The mice bearing Hela tumor with the volume approximately 80 mm3, obtained via

18

subcutaneously injecting Hela cells (1×107 cells) normal saline suspension into the back neck of

19

mice, was used for drug accumulation studies. When the mean volume of tumor reached

20

approximately 80 mm3, VBL-NPs-PEG-FA or VBL injection were administrated intravenously

21

via tail vein at an equivalent dose of VBL 5mg /kg . Mice were respectively sacrificed

22

respectively at 1, 3, 6, 12, 24, 48h and the samples of blood, organs and tumor were collected. The

23

organs and tumor were rinsed with cold saline, dried by filter paper and frozen at -20℃. 0.1mL of

24

blood was mixed with 0.1mL NS containing 10mM GSH and vortexed with 0.1 mL of

25

acetonitrile. 0.1g of frozenthawed tissues were homogenized in 0.3mL NS containing 10mM

26

GSH. The homogenate were extracted with 0.3mL of acetonitrile by vortex. After centrifugation

27

(10000rpm, 10 min), the organic phase was separated and dried under a nitrogen gas stream. The

28

residue was dissolved in 200μL of mobile phase and filtered with ultrafiltration membrane (220

29

nm)

26.

The filtrate was employed for HPLC analysis under above described conditions. The



Page 12 of 45

1

standard curve of VBL was prepared by adding free VBL to the tumor tissue homogenate from

2

untreated mice. The linear detection range was 0.1−50 μg/mL.

3

2.9 In Vivo Antitumor Effect

4

The mice bearing Hela tumor with the volume approximately 80 mm3, obtained via the

5

methods described above, were randomly assigned to three groups, including the

6

VBL-NPs-PEG-FA, VBL injection and the normal saline groups, eighteen mice each group.

7

0.1mL of various formulations was administered intravenously through tail vein every three days

8

at the first three times and then every four days at the next four times. The VBL-NPs-PEG-FA and

9

VBL groups were given at the same VBL dose of 1 mg/kg. The body weight and tumor volume

10

were measured and recorded at the next day after each administration. Six mice per group were

11

sacrificed on the 10th, 18th and 26th via cervical dislocation euthanasia respectively. The tumors of

12

mice undergoing the third time of euthanasi were collected and carried out terminal

13

deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay to detect DNA damage and

14

fragmentation, the marker of cells apoptosis, and then imaged via inverted fluorescence

15

microscopy (NIKON ECLIPSE TI-SR, Japan). At the same time, the major organs of liver, spleen,

16

kidney, lungs and heart were weighed and the organ coefficients (relative organ weight) of each

17

organ were calculated via the equation 7

18

sections of each organ were stained with hematoxylin and eosin (H&E) and observed via an

19

optical microscope (NIKON ECLIPSE CI, Japan).

20

Organ coefficient = 𝑇ℎ𝑒 𝑏𝑜𝑑𝑦 𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑚𝑜𝑢𝑠𝑒 × 100%

21

2.10 Statistical Analysis.

53.

After suffering from cryotomy, the frozen tissue

𝑇ℎ𝑒 𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑜𝑟𝑔𝑎𝑛

(7)

22

Data are shown as the mean ± standard deviation (SD). Statistical evaluation was performed

23

by the unpaired Student’s t test and one-way analysis of variance (ANOVA) via IBM SPSS

24

Statistics 20.0 software. If the variances were unequal, Welch or Brown-Forsythe would be used

25

to amend. And Tamhane’s T2 or Dunnett’s T3 would be used to Post Hoc multiple comparisons

26

for the unequal variances. P

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