Cationic Polymeric Nanoparticle Delivering CCR2 siRNA to

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Cationic Polymeric Nanoparticle Delivering CCR2 siRNA to Inflammatory Monocytes for Tumor Microenvironment Modification and Cancer Therapy Song Shen, Yue Zhang, Kai-Ge Chen, Ying-Li Luo, and Jun Wang Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b00997 • Publication Date (Web): 16 Jan 2018 Downloaded from http://pubs.acs.org on January 18, 2018

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Molecular Pharmaceutics

1

Cationic Polymeric Nanoparticle Delivering CCR2 siRNA to Inflammatory Monocytes for

2

Tumor Microenvironment Modification and Cancer Therapy

3

Song Shenb,c,#, Yue Zhanga,#, Kai-Ge Chena, Ying-Li Luoa, and Jun Wanga,b,c,d,*

4 5

a

6

R. China

7

b

8

Guangzhou, Guandong 510006, P. R. China

9

c

School of Life Sciences, University of Science & Technology of China, Hefei, Anhui 230027, P.

Institutes for Life Sciences and School of Medicine, South China University of Technology,

National Engineering Research Center for Tissue Restoration and Reconstruction, Guangzhou,

10

Guangdong 510006, P. R. China

11

d

12

Guangzhou 510641, P. R. China

13

#

14

*Corresponding author:

15

E-mail: [email protected] (Jun Wang)

Research Institute for Food Nutrition and Human Health, South China University of Technology,

These authors contribute equally to this work

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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 Accumulating

evidence

has

confirmed

that

malignant

tumors

have

a

complex

3

microenvironment, which consists of a heterogeneous collection of tumor cells and other cell

4

subsets (including the full gamut of immune cells). Tumor-associated macrophages (TAMs),

5

derived from circulating Ly6Chi monocytes, constitute the most substantial fraction of

6

tumor-infiltrating immune cells in nearly all cancer types and contribute to tumor progression,

7

vascularization, metastasis, immunosuppression and therapeutic resistance. Interrupting monocyte

8

recruitment to tumor tissues by disturbing pivotal signaling pathways (such as CCL2-CCR2) is

9

viewed as one of most promising avenues for tumor microenvironment manipulation and cancer

10

therapy. One critical issue for monocyte-based therapy is to deliver therapeutic agents into

11

monocytes efficiently. In the present study, we systematically investigated the relationship

12

between the surface potential and the bio-distribution of polymeric nanoparticles in monocytes in

13

vivo, aiming to screen and identify an appropriate delivery system for monocyte targeting, and we

14

found that cationic nanoparticles have a higher propensity to accumulate in monocytes compared

15

with their neutral counterparts. We further demonstrated that siCCR2-encapsulated cationic

16

nanoparticle (CNP/siCCR2) could modify immunosuppressive tumor microenvironment more

17

efficiently and exhibit superior antitumor effect in an orthotopic murine breast cancer model.

18 19

KEYWORDS: tumor microenvironment, tumor-associated macrophages, monocyte, cationic

20

nanoparticle, siCCR2.

21 22


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1

1. INTRODUCTION

2

Traditional non-surgical oncology treatment strategies (e.g., chemotherapy, radiation and

3

molecular targeted therapy) have historically focused on inhibiting the proliferation of tumor cells

4

or inducing apoptosis.1-3 Although these strategies can suppress or postpone tumor growth in some

5

patients, a large proportion of tumor-bearing patients do not experience therapeutic benefits due to

6

serious side effects, drug resistance or tumor recurrence. In the past decades, researchers from

7

multidisciplinary areas have made great inroads into understanding the underlying etiology of

8

cancer, and it is now evident that the malignant tumors are complex masses containing not only

9

neoplastic cells but also non-transformed cellular elements, including the full gamut of immune

10

cells.4-7 Increasing investigation has indicated that certain tumor-infiltrating immune cells, such as

11

regulatory T cells (Treg cells), myeloid-derived suppressor cells (MDSCs) and tumor-associated

12

macrophages (TAMs), are major contributors to tumor progression and therapeutic tolerance and

13

are widely viewed as attractive and promising therapeutic targets.8-12

14

TAMs, which are derived from circulating Ly6Chi (lymphocyte antigen 6C) inflammatory

15

monocytes, constitute the most substantial fraction of tumor-infiltrating immune cells in nearly all

16

types of cancer.13-15 TAMs have been shown to participate in the construction of an

17

immunosuppressive tumor microenvironment and to protect tumor cells against chemotherapy by

18

releasing abundant growth factors, cytokines, and proteases.15 Studies have suggested that TAMs

19

accumulation in tumors serves as an independent prognostic indicator of multiple cancers.16, 17

20

With a better understanding of TAMs, it is thought that therapeutic targeting of TAMs holds

21

considerable clinical promise, and multiple approaches for macrophage targeting (e.g., blocking

22

inflammatory monocyte recruitment, re-educating M2-like TAMs, interfering tumor-promoting

23

function or depleting TAMs directly) have been put forward.18-21 Several elegant studies revealed

24

that bone marrow and spleen are the primary source and reservoir of monocytes that generate

25

TAMs.13, 22 Recruitment of monocytes to tumor tissues requires the interaction of a tumor-derived

26

chemoattractants (such as CCL2, CSF-1, and CXCL12) with a specific chemokine receptor that is

27

highly expressed on monocytes (such as CCR2), and CCL2-CCR2 chemokine axis is viewed as a

28

direct and predominant mediator of monocyte recruitment in multiple tumors.23, 24Therefore,

29

disruption of the CCL2-CCR2 signaling axis could theoretically decrease the abundance of TAMs

30

from the source, and this strategy represents one of the most promising avenues for tumor



1

microenvironment manipulation and cancer prevention. The effectiveness of this strategy have

2

been verified in preclinical and clinical trials using small molecular inhibitors and antibodies

3

against CCR2 (or CCL2).23, 25 Compared with these therapeutic drugs, small interference RNA

4

(siRNA) possesses a powerful ability to specifically suppress the expression of target genes, and

5

they can be produced far more efficiently and quickly.26, 27 Therefore, systemically transporting

6

CCR2 siRNA to monocytes with an optimal delivery system is a strategy with great prospects for

7

cancer therapy.

8

Nanotechnology-based delivery systems (e.g., lipid-based, polymeric, and inorganic

9

nanoparticles) have gained considerable commercial and translational attention28, 29 and achieved

10

some success in immuno-oncology therapies.30-32 Different from traditional nanomedicines, which

11

must overcome multiple biological barriers upon intravenous administration to transport

12

therapeutic agents into tumor tissues and tumor cells,33 monocyte-targeted nanomedicines can be

13

effectively taken up by circulating and resident monocytes in the peripheral blood, spleen and

14

bone marrow. A growing number of studies have revealed that physiochemical properties (e.g.,

15

size, shape, and surface chemistry) of nanomedicines significantly affect their in vivo behavior.34

16

However, reports on the bio-distribution of nanoparticles within immune cells are rare, and

17

insufficient effort has been made to manipulate the in vivo performance of immune cells as a

18

delivery system for disease therapy.35 It is worth investigating the relationship between the

19

physiochemical properties and immune cell accumulation of nanoparticles and to identify

20

appropriate delivery systems to manipulate the in vivo performance of immune cells for cancer

21

immunotherapy.

22

In the present study, the correlations between the surface potentials of nanoparticles, one of the

23

key physiochemical properties, with their bio-distribution in monocytes and therapeutic efficacy

24

of monocyte-targeted nanomedicines were systematically studied. Firstly, we constructed a series

25

of polymeric nanoparticles with varied surface potentials and analyzed the enrichment of different

26

nanoparticles in monocytes within peripheral blood, spleen and bone marrow. Our results showed

27

that positively charged PEG-PLA nanoparticles have a higher propensity to accumulate in

28

monocytes, compared to their neutral counterparts. Furthermore, we confirmed that

29

siCCR2-encapsulated cationic nanoparticles (CNP/siCCR2) could more efficiently suppress CCR2

30

expression in monocytes. More importantly, by blocking monocyte recruitment to tumor tissues,


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1

CNP/siCCR2 could modify the tumor immune microenvironment, inhibit growth of primary

2

tumors, decrease tumor metastasis and improve the antitumor effects of chemotherapeutics (Figure

3

1). Our present work indicated that interfering the in vivo fate of tumor-associated immune cells

4

using nanoscale delivery systems with proper physiochemical properties presents a potential and

5

promising strategy for tumor microenvironment and cancer therapy.

6 7

Figure 1. Schematic illustration of CNP/siCCR2-mediated tumor microenvironment modification and

8

cancer therapy. (A) Construction of nanoparticles with varied surface potentials and evaluation of their

9

bio-distribution in monocytes in vivo. (B) CCR2 siRNA-encapsulated cationic nanoparticles (CNP/siCCR2) could

10

more efficiently interrupt monocyte recruitment from peripheral blood to tumor tissues and decrease TAMs

11

abundance, resulting in tumor microenvironment modification and tumor growth suppression. Spleen and bone

12

marrow are the main reservoirs of circulating monocytes.

13



1

2. EXPERIMENTAL SECTION

2

2.1 Materials

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The block copolymer poly(ethylene glycol)-block-polylactide (PEG5K-b-PLA11K) and

3 4

cationic

lipid

N,N-bis(2-hydroxyethyl)-N-methyl-N-(2-cholesteryloxycarbonyl

5

ammonium bromide (BHEM-Chol) were synthesized according to a previously reported method.36

6

Cy5-labeled siRNA and unlabeled negative control siRNA (siN.C.) were provided by Suzhou Ribo

7

Life Science Co., Ltd. (Kunshan, China) and CCR2 siRNA was purchased from Guangzhou

8

RiboBio Co., Ltd. (Guangzhou, China). The sequence of Cy5-siRNA and siN.C. is scrambled

9

(antisense strand, 5′-ACGUGACACGUUCGGAGAAdTdT-3′), and the sequence of CCR2 siRNA

10

is

5′-UUUGcAGAGACGUUuAGcAdTsdT-3′

(antisense

strand).

11

hydrochloride was purchased from Aladdin Industrial Corporation (Shanghai, China).

12

2.2 Cell line and animals

aminoethyl)

Doxorubicin

(DOX)

13

Murine breast cancer cell line 4T1, obtained from American Type Culture Collection

14

(Manassas, USA), was cultured in Dulbecco's Modified Eagle Medium (DMEM, Life

15

Technologies, Eggenstein, Germany) containing 10% (v/v) fetal bovine serum (FBS, MesGen

16

Biotech, Shanghai, China) at 37 °C with 5% CO2. Female BALB/c mice were obtained from

17

Beijing HFK Bioscience Co., Ltd. (Beijing, China) and used at 6-8 weeks of age. All animals

18

received care in compliance with the guidelines outlined in the Guide for the Care and Use of

19

Laboratory Animals, and the procedures were approved by the USTC Animal Care and Use

20

Committee.

21

2.3 Flow cytometry

22

Cell suspensions were incubated with anti-mouse CD16/32 (BioLegend, San Diego, CA) to

23

block the Fc receptor before cell surface staining. The antibodies used for flow cytometry in this

24

study included PE-Cy7-CD45, FITC-CD11b, FITC-CD3, APC-Cy7-CD19, PercP-Cy5.5-Gr-1,

25

APC-F4/80,

26

APC-Cy7-CD4, PE-CD25 (Biolegend, San Diego, CA) and APC-CCR2 (BD Biosciences, San

27

Jose, CA). The stained cells were collected using a BD Verse flow cytometer (BD Biosciences)

28

and analyzed using FlowJo 7.6 software (TreeStar, Inc., Ashland, Oregon).

29

2.4 Preparation and characterization of siRNA-encapsulated nanoparticles

30

APC-Cy7-F4/80,

PE-Ly6C,

BV510-CD11c,

PercP-Cy5.5-CD8,

FITC-CD3,

siRNA solution (200 µg in 25 µL RNase-free water) was emulsified in 500 µL chloroform


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1

containing 25 mg PEG5K-b-PLA11K and different amounts of BHEM-Chol (1, 2, 3 and 4 mg) for 1

2

min at 80 W over an ice bath using Vibra-Cell™ VCX 130 (Sonics & Materials, Inc., Newtown,

3

USA). Subsequently, 5 mL of RNase-free water was added to the primary emulsion and further

4

emulsified for 1 min at 80 W. Chloroform was then removed using a Rotavapor® R-3 evaporator

5

(BUCHI Co., New Castle, USA). The diameter and zeta potential of nanoparticles were

6

characterized with a Malvern Zetasizer Nano ZS90 (Worcestershire, UK) and analyzed in

7

triplicate at a concentration of 1 mg/mL. The encapsulation efficiency of the various nanoparticles

8

was examined by HPLC according to methods described previously.36

9

2.5 Bio-distribution of different nanoparticles in monocytes in vivo

10

Cy5-siRNA-loaded nanoparticles with various surface potentials were prepared using the

11

aforementioned method and administered i.v. into BALB/c mice at an equivalent dose of 40 µg of

12

Cy5-siRNA per mouse (n = 3 per group), and mice injected with PBS were used as controls. Mice

13

were euthanized at predetermined time-points post-injection (2 h, 12 h and 24 h). Peripheral blood

14

was taken from the retro-orbital plexus of mouse eyes and added into a tube containing heparin

15

anticoagulant, then lysed with ACK lysis buffer (STEMCELL Technologies, Vancouver, Canada).

16

Spleens were removed and disrupted between two microscope glass slides, and the pellets were

17

resuspended and filtered through a 200-mesh sieve. The cell suspension was centrifuged (1,500

18

rpm for 10 min) and lysed with ACK lysis buffer, and then the splenocytes were resuspended in

19

PBS supplemented with 0.2% BSA (w/v). For isolation of bone marrow cells, femurs were flushed

20

with PBS, and cells were collected for ACK lysis and cell counting. The viability of cells was

21

confirmed by trypan blue dye exclusion.

22

To analyze the bio-distribution of Cy5-siRNA-loaded nanoparticles in monocytes, the isolated

23

immune cells were labeled with FITC-CD11b and PE-Ly6C antibodies, and then Cy5 signal

24

intensity was detected using a BD FACSVerse™ flow cytometer (BD Biosciences, Bedford, USA).

25

In detail, cells were resuspended in FACS buffer (1 × PBS, 2% BSA, 1 mM EDTA and 0.1%

26

sodium azide) at 5 × 106 cells/mL, then 100 µL cell suspensions were distributed into plastic tubes

27

and incubated with TruStain FcX™ (anti-mouse CD16/32) at 1.0 µg/106 cells for 20 min on ice

28

prior to immunostaining. Cell suspensions were incubated with a cocktail of monoclonal

29

antibodies on ice for 20 min in the dark, and then washed with 2 mL FACS buffer by



1

centrifugation at 1,500 rpm for 5 min. Finally, cell pellets were resuspended in 300 µL FACS

2

buffer, and 5 µL DAPI (4',6-Diamidino-2-Phenylindole, Dilactate) staining solution was added

3

before examination to exclude dead cells. The percentage of Cy5-positive CD11b+ Ly6Chi

4

monocytes was examined by a BD FACSVerse™ flow cytometer, and data were analyzed using

5

FlowJo software (Tree Star, Inc., Ashland, Oregon).

6

2.6 Monocyte ratio in total immune cells isolated from peripheral blood, spleen and bone

7

marrow in tumor-bearing mice

8

The orthotopic murine breast cancer models were generated by subcutaneous injection of 4T1

9

cells (1.0 × 106 cells diluted in 100 µL PBS) into the second mammary fat pad of BALB/c mice

10

(female, 6-8 weeks old). Mice were sacrificed 5 days and 10 days post-model establishment,

11

immune cells were isolated from peripheral blood, spleen and bone marrow were stained with

12

APC-CD45, FITC-CD11b and PE-Ly6C antibodies, and the percentages of CD11b+Ly6Chi

13

monocytes were detected using a BD FACSVerse™ flow cytometer.

14

2.7 CCR2 expression in different immune cells

15

Spleens were collected from 4T1 tumor-bearing mice (10 days post tumor models

16

establishment), and splenic cells were collected using the same procedure as described above. The

17

isolated cells were stained with an antibody cocktail containing PE-Cy7-CD45, FITC-CD3,

18

APC-Cy7-CD19 and APC-CCR2 (or PE-Cy7-CD45, FITC-CD11b, PerCP-Cy5.5-Ly6G,

19

APC-Cy7-F4/80, PE-Ly6C, BV510-CD11c and APC-CCR2) according to the Cell Surface

20

Immunofluorescence Staining Protocol provided by BioLegend, Inc. Then, CCR2 expression of

21

different immune cells was detected using BD FACSVerse™ flow cytometer (BD Biosciences,

22

Bedford, USA).

23

2.8 Nanoparticle-mediated down-regulation of CCR2 expression in vivo

24

The orthotopic murine breast cancer models were generated according to the using the method

25

previously published literature.31 When tumors reached a volume of approximately 50 mm3, mice

26

were treated with PBS, siRNA-encapsulated neutral nanoparticles (denoted as NNP/siN.C. and

27

NNP/siCCR2) and siRNA-encapsulated cationic nanoparticles (CNP/siN.C. and CNP/siCCR2) i.v.

28

every other day. The injection dose of siRNA was 1.0 mg per kilogram of body weight. Peripheral

29

blood, spleen and bone marrow from tumor-bearing BALB/c mice were collected 24 h after the

30

fifth injection. The isolated immune cells were stained with FITC-CD11b and PE-Ly6C antibodies,


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1

and then CD11b+Ly6Chi monocytes were sorted by MoFlo Astrios (Beckman Coulter, Brea, USA).

2

The CCR2 expression of the sorted CD11b+Ly6Chi monocytes was examined by quantitative

3

real-time PCR analysis. Total RNA isolation and reverse transcription were conducted using a

4

method described previously.37 The mRNA expression of CCR2 and glyceraldehyde-3-phosphate

5

dehydrogenase (GAPDH) were analyzed by qPCR using the FastStart Universal Probe Master

6

(Roche Applied Science, Indianapolis). All measurements were performed in duplicate, and the

7

arithmetic means of the cycle threshold (Ct) values were used for calculations: target gene mean

8

Ct values were normalized to the respective housekeeping gene (GAPDH), and then to the

9

PBS-treated control. The values obtained were exponentiated 2(-△△Ct) to be expressed as n-fold

10

changes in regulation compared with the experimental control 2(-△△Ct) by the method of relative

11

quantification.

12

Meanwhile, western blotting was applied to analyze knock-down of CCR2 in monocytes

13

post-treatemnt. In detail, isolated cells were washed with PBS (0.01 M, pH 7.4) and lysed with

14

RIPA Lysis Buffer (Beyotime Biotechnology, Shanghai, China). Protein concentrations were

15

quantified using the Pierce BCA Protein Assay Kit (Thermo Scientific, Waltham, USA). Volumes

16

containing 15 µg protein were loaded onto SDS-PAGE, then transferred to PVDF membranes

17

(Millipore, Bedford, USA) and probed with anti-CCR2 (Novus Biologicals, Littleton, USA) and

18

anti-GAPDH (Proteintech Group, Chicago, USA) antibodies. Membranes were imaged using an

19

ImageQuant™ LAS 4000 (GE Healthcare Life Sciences, Fairfield, USA).

20

2.9 Analysis of tumor-infiltrating immune cells after treatment

21

Animals were sacrificed after being treated with the different formulations, and tumor tissues

22

were excised. Then, tumor tissues were transferred to a dish and cut into small pieces. The

23

fragments were suspended in 10 mL of RPMI-1640 medium and collected by centrifugation at 600

24

rpm for 5 min. The pellets were resuspended in 10 mL of digestion solution (1 mg/mL type I

25

collagenase, 100 µg/mL type IV hyaluronidase and 100 µg/mL DNase I in RPMI-1640 medium

26

containing 10% FBS) and incubated at 37 ℃ for 2 h with persistent agitation. Digested cells were

27

filtered through a 200-mesh sieve and collected by centrifugation at 1,500 rpm for 10 min at 4 °C.

28

After ACK lysis and cell counting, total cells were incubated with an antibody cocktail of

29

PE-Cy7-CD45, FITC-CD11b, PercP-Cy5.5-Gr-1 and APC-F4/80 (or PE-Cy7-CD45, FITC-CD11b,

30

PercP-Cy5.5-CD8, APC-Cy7-CD4 and PE-CD25) according to manufacturer's protocol and



1

examined by a BD FACSVerse™ flow cytometer. The above-mentioned antibodies were

2

purchased from BioLegend, Inc. (San Diego, USA).

3

2.10 Expression of TAMs-associated genes

4

Tumor tissues were transferred to a dish and cut into small pieces, and then total RNA

5

isolation and reverse transcription were conducted using the method described in a previous

6

publication.37 The mRNA expressions of VEGFA, MMP9 and IL10 were examined using real-time

7

PCR.

8

2.11 Tumor suppression in the murine breast cancer model

9

The orthotopic murine breast cancer models were generated according to previously described

10

method,31 and when tumors reached an average volume of 50 mm3, mice were divided into 5

11

groups (n = 5) for the tumor suppression study. Mice were treated with different formulations by

12

intravenous injection every other day for 20 days; the injection dose of siRNA was 1.0 mg per

13

kilogram of body weight. Tumor growth was monitored by measuring the perpendicular diameter

14

by caliper every three day. The estimated tumor volume was calculated using the formula: length

15

× width2/2.

16

2.12 Examination of pulmonary metastasis after treatment

17

4T1 tumor-bearing mice were sacrificed 24 h after the last treatment, and lung tissues were

18

excised. The tissues were fixed in 4% formaldehyde (Sigma Aldrich, St. Louis, USA) and

19

embedded in paraffin. Paraffin-embedded 8 µm lung sections stained with hematoxylin-eosin

20

(H&E) were prepared for immunohistochemical analysis. The metastatic clusters were examined

21

under a Nikon TE2000 microscope (Tokyo Prefecture, Japan).

22

2.13 CCL2 expression in tumor tissues after treatment with doxorubicin

23

4T1 tumor-bearing mice were treated with PBS and doxorubicin-loaded PEG-PLA

24

nanoparticles (NPDOX 2.5 mg per kilogram of body weight) every other day. NPDOX was prepared

25

according to previously reported methods.38 Twenty-four hours after the third injection, tumor

26

tissues were collected, and the expression of CCL2 in total cells was examined by real-time PCR

27

according to methods previously described.37

28

2.14 Immunohistochemical evaluation

29

Mice were sacrificed 24 h after the last treatment, and tumor tissues were excised. The tissues


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1

were fixed in 4% formaldehyde and embedded in paraffin for analysis. Paraffin-embedded 8 µm

2

tumor sections were prepared for immunohistochemical analysis. The proliferation of tumor cells

3

was detected using an antibody against proliferating cell nuclear antigen (PCNA). Deparaffinized

4

slides were boiled for 5 min in 0.01 M sodium citrate buffer (pH 6.0) in a pressure cooker for

5

antigen retrieval. Subsequently, slides were allowed to cool for another 5 min in the same buffer.

6

After several rinses in PBS and pretreatment with blocking medium for 5 min, slides were

7

incubated with the PCNA antibody (Maxin Biotech, Fuzhou, China), which had been diluted to

8

1:300 in antibody diluent solution, for 20 min at room temperature and then at 4 ℃ overnight.

9

After washing slides in Tris-buffered saline, a streptavidin-biotin system was used according to the

10

manufacturer's instructions (BioGenex, San Ramon, CA). The slides were counterstained using

11

Aquatex (Merck, Gernsheim, Germany). Apoptosis of tumor cells following the treatments was

12

determined using the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling

13

(TUNEL) method according to the manufacturer's instructions (Roche, Basel, Switzerland). All

14

sections were examined under a Nikon TE2000 microscope (Tokyo Prefecture, Japan).

15

2.15 Statistical analysis

16

The level of significant differences between group means was determined by Student’s t-test

17

for parametric data sets. All statistical analyses were performed using Prism 4 (GraphPad Software,

18

San Diego, CA). A P value of 0.05 was considered significant in all analyses (95% confidence

19

level).



1

3. RESULTS AND DISCUSSION

2

3.1 Preparation of nanoparticles with varied surface potentials

3

Manipulating the in vivo fate of immune cells (e.g., inflammatory monocytes) represents a

4

novel and attractive therapeutic strategy for the treatment of multiple diseases including malignant

5

tumors. An increasing amount of work has indicated that the physiochemical properties of

6

nanomedicines significantly affect their in vitro and in vivo behavior.34, 39 In the present project,

7

we first assessed how surface potential, a key physiochemical property of nanoparticles,

8

influences the bio-distribution of nanoparticles in monocytes, to identify nanoparticles with proper

9

surface potentials for the delivery of monocyte-targeting therapeutics. Cationic lipid-assisted

10

nanoparticles, which have been shown to be able to function as siRNA carriers,36, 37 were selected

11

to investigate this relationship. siRNA-encapsulated nanoparticles were fabricated with

12

poly(ethylene glycol)-b-poly(d,l-lactide) (PEG-b-PLA) and cationic lipid (BHEM-Chol) using a

13

double emulsion-solvent evaporation technique, and different nanoparticles with varied surface

14

potential were obtained by regulating the amount of BHEM-Chol during preparation (prepared

15

nanoparticles were denoted as NP-1, NP-2, NP-3 and NP-4). As shown in Table 1, neutral

16

nanoparticles (zeta potential is approximately 2.7 mV) were generated when the mass ratio of

17

BHEM-Chol : siRNA was 5:1. With increasing lipid to RNA ratios (10:1, 15:1 and 20:1), a series

18

of positive nanoparticles were obtained (zeta potentials were 11.8 mV, 18.4 mV and 25.2 mV,

19

respectively). Although the prepared nanoparticles had different surface potentials, no significant

20

difference was observed with respect to diameter or loading efficiency (Table 1), and all these

21

nanoparticles could keep stable after treated with RNase A in the absence and presence of serum

22

(Figure S1). We also attempted to prepare nanoparticles with higher zeta potential, however, when

23

the ratio of BHEM-Chol : siRNA was 25:1, we did not observe any further increase in surface

24

potential, in addition, excessive cationic lipid formed nanoparticles by self-assembly. In present

25

study, siRNA-encapsulated PEG-PLA with varied surface charge (from 2.7 mV to 25.2 mV) were

26

used as models to investigate the correlation between surface charge, a fundamental property of a

27

nanoscale delivery system, and its bio-distribution in inflammatory monocytes. We attempt to

28

identify nanoparticles with proper surface potentials for monocyte targeting and cancer therapy.

29

Noted that strong positive nanoparticles and negative nanoparticles were not considered in this


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1

work, and maybe further investigated using other nanoparticulate systems.

2

Table 1. The components, encapsulating efficiency, diameter and surface potential of nanoparticles

Feeding Weight Formulation

siRNA (mg)

BHEMChol

PEG-PLA

E.E. (%)

(mg)

Diameter

Zeta

(nm)

Potential (mV)

(mg)

3

NP-1

0.2

1

25

96.7 ± 1.2

128.3 ± 18.1

2.7 ± 0.7

NP-2

0.2

2

25

97.4 ± 3.4

123.5 ± 13.8

11.8 ± 2.3

NP-3

0.2

3

25

98.1 ± 4.8

120.9 ± 12.2

18.4 ± 4.8

NP-4

0.2

4

25

97.5 ± 1.7

126.8 ± 15.6

25.2 ± 6.9

3.2 Bio-distribution of nanoparticles in monocyte cells in vivo

4

Although the distribution of nanoparticles in different immune organs (e.g., liver, spleen) has

5

been widely investigated, the distribution in certain immune cell subpopulations has been rarely

6

reported.40 In this study, we found that RAW264.7 cells (a murine monocyte cell line) uptake more

7

PEG-PLA nanoparticles with higher surface potential in vitro (Figure S2), we believed that the

8

relationships of surface potentials and bio-distribution of nanoparticles in inflammatory

9

monocytes worth further investigation. Firstly, the in vivo safety of four nanoparticles with varied

10

surface charge were assessed. As shown in Figure S3 that the nanoparticles treatment did not

11

induce body weight loss, and the spleen of mice did not look enlarged or different between the

12

controls and NP-treated mice. In addition, the ratios of CD8+ T cell and CD11b+Ly6Chi monocytes

13

in splenic cells did not exhibit significant difference after treatments (Figure S4), suggesting these

14

PEG-PLA nanoparticles have no obvious toxicity at the selected injection dosage. The in vivo

15

pharmacokinetic profiles of different nanoparticles were also evaluated, as shown in Figure S5

16

that the half-lives of circulation decrease slightly with the increase of surface charges. Then, 4T1

17

tumor-bearing mice were treated with Cy5-siRNA loaded nanoparticles (2 OD siRNA per mouse),

18

and then sacrificed at 2 h, 12 h and 24 h post i.v. administration. Immune cells isolated from

19

peripheral blood, spleen and bone marrow were stained with FITC-CD11b and PE-Ly6C

20

antibodies to examine the signal intensity of Cy5 within CD11b+Ly6Chi monocytes. To identify the

21

percentage of Cy5+ monocytes after nanoparticle injection, gates for Cy5 events were established

22

in PBS-treated mice, and the ratio of Cy5+ was compared between nanoparticle-treated and



1

PBS-treated mice for the CD11b+Ly6C+ monocyte population. All percentages were significantly

2

different from the control group, showing that the Cy5+ events were from the injected

3

nanoparticles. Next, we evaluated the Cy5-siRNA distribution within the monocytes at

4

predetermined time points (Figure 2A).

5

We determined the percentage of monocytes in the CD11b+Ly6Chi population that were

6

Cy5-positive 12 h post-injection. As shown in Figure 2B, 2C and 2D, the ratio of Cy5+ monocytes

7

to total monocytes in the blood, spleen and bone marrow were 33.2%, 49.3% and 23.0%,

8

respectively, in mice injected with Cy5-siRNA-loaded neutral nanoparticles (denoted as NP-1).

9

With the increasing of surface potential, the percentage of Cy5+ monocytes also increased. The

10

Cy5+ monocytes in mice treated with Cy5-siRNA-loaded cationic nanoparticles with the highest

11

zeta potential (denoted as NP-4) were 79.5%, 86.2% and 50.5%; these values were much higher

12

than other groups. The percentages of Cy5+CD11b+Ly6Chi monocytes at 2 h and 24 h also

13

indicated that monocytes prefer to uptake cationic nanoparticles in vivo (Figure S6). We noted the

14

distinct behavior of neutral and cationic nanoparticles, however, the underlying mechanism is

15

complicated. As one prominent phagocyte, monocytes are key cellular participants determining

16

the in vivo fate of nanomaterials, increasing researches revealed that various mechanism were

17

involved in the monocyte-mediated uptake and clearance, such as ligand/receptor interaction and

18

particle opsonization.41 In addition, the surface charges influence the formation of protein corona

19

on nanoparticles, which significantly affect the bio-distribution and uptake process of

20

nanoparticles.42 The higher propensity of cationic nanoparticles to accumulate in inflammatory

21

monocytes, encouraged us to deliver therapeutic agents to monocytes for tumor microenvironment

22

modification.


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1 2

Figure 2. Biodistribution of nanoparticles in CD11b+Ly6Chi monocytes in vivo. (A) Flow cytometry gating

3

strategy for the analysis of the percentage of Cy5+CD11b+Ly6Chi monocytes, mice injected with PBS solution

4

were set as the blank controls. The percentages of CD11b+Ly6ChiCy5+ monocytes compared to total monocyte

5

within peripheral blood (B), spleen (C) and bone marrow (D) 12 h post injection of different nanoparticles

6

encapsulating Cy5-labeled siRNA (Cy5-siRNA). Gates for Cy5 events were established in untreated mice. ** P

Cationic Polymeric Nanoparticle Delivering CCR2 siRNA to

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