Targeting Delivery of Oligodeoxynucleotides to Macrophages by

Subscriber access provided by Stockholm University Library

Article

Targeting Delivery of Oligodeoxynucleotides to Macrophages by Mannosylated Cationic Albumin for Immune Stimulation in Cancer Treatment Shu-Lun Ai, Xiao-Yan He, Bo-Ya Liu, Ren-Xi Zhuo, and Si-Xue Cheng Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.9b00184 • Publication Date (Web): 23 Apr 2019 Downloaded from http://pubs.acs.org on April 24, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 34 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

Targeting

Molecular Pharmaceutics

Delivery

of

Oligodeoxynucleotides

to

Macrophages by Mannosylated Cationic Albumin for Immune Stimulation in Cancer Treatment Shu-Lun Ai, Xiao-Yan He, Buo-Ya Liu, Ren-Xi Zhuo and Si-Xue Cheng* Key Laboratory of Biomedical Polymers of Ministry of Education, Department of Chemistry, Wuhan, University, Wuhan 430072, People’s Republic of China

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

Page 2 of 34

ABSTRACT: To efficiently deliver CpG oligodeoxynucleotides (ODN) to macrophages for the reversal of cancer-induced immunosuppression, nanoparticles ODN@MCBSA with mannosylated cationic albumin (MCBSA) as a macrophage targeting vector were constructed. Compared with ODN@CBSA with cationic albumin (CBSA) as a vector, ODN@MCBSA exhibited significantly improved cellular uptake mediated by mannose moieties, resulting in significantly enhanced secretion of pro-flammatory cytokines including IL-12, IL-6, TNF-α and iNOS. The modulation of macrophages toward the favorable M1 phenotype was confirmed by the up-regulated CD80 expression after being treated by ODN delivery systems. In addition to immune cells, the effects of the ODN delivery system on cancerous HeLa cells were also investigated. The results showed ODN@MCBSA did not affect the overall tumor cell viability. However, enhanced NF-κB, p-Akt, PIK3R3, Fas and FasL, as well as upregulated caspases were observed in tumor cells, implying the pleiotropic effects on tumor cells. Our study provides a more in-depth understanding on the immunotherapeutic effects of CpG ODN, and highlights the importance of macrophage targeting delivery to minimize the effects on tumor cells. These results indicate MCBSA could serve as a promising delivery vector of CpG ODN to macrophages for cancer immunotherapy.

KEYWORDS. gene delivery, macrophage targeting, CpG ODN, immunotherapy, cancer treatment


Page 3 of 34 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


INTRODUCTION By triggering immune responses of the immune system to treat cancers, immunotherapy holds great promise owing to the advantages of enhanced long-term survival and lowered risk of recurrence with minimized side effects.1-4 Among diverse immune cells, macrophages with the phagocytic capacity play a critical role in innate immunity and adaptive immunity.5,6 In tumor environments, due to tumor induced immunosuppression, tumor-associated macrophages (TAMs) are mainly featured with M2 phenotype characteristics, which favor tumor cell proliferation and tumor progression.6 As a promising strategy in tumor immunotherapy, re-modulating TAMs to M1 phenotype can activate anti-tumor immunological responses to inhibit tumor growth.6,7 Among diverse immunostimulating agents, oligodeoxynucleotides containing CpG moieties (CpG ODN) can elicit useful immune responses through various cell pathways including MyD88-dependent nuclear factor-κB (NF-κB) pathway, and thus shift the polarity of macrophages to the anti-cancer phenotype.6 Due to the lack of cell-penetrating ability and nuclease resistance, naked CpG ODN cannot achieve satisfactory therapeutic efficiency. An effective strategy for solving these problems is to develop efficient ODN delivery vectors, which can enhance the cellular internalization and protect ODN from nuclease degradation.8 So far, diverse nanomaterials based on lipids,9 polymers,10 biomacromolecules,11-16 exosomes,17 metals,18 inorganics,19 metal-organic frameworks,20,21 and hybrids22-24 have been reported as the carriers of CpG ODN. However, the vectors with deal biocompatibility, good biodegradability, and high delivery efficacy are still highly desirable.


3


Page 4 of 34

Despite the extensive studies on the effects of CpG ODN on immune cells, the studies on the effects of CpG ODN on tumor cells are very limited.15,25 As is well known, CpG ODN results in the activation of NF-kB and PI3K/Akt signaling.6,26 Activation of NF-kB and PI3K/Akt signaling pathways plays an important role on promoting tumor development and progression.27-29 The influence of CpG ODN on tumor cells should not be overlooked and further study is warranted. To address these issues, in this investigation, we incorporated mannose as a targeting moiety to cationic bovine serum albumin (CBSA) to obtain mannosylated cationic bovine serum albumin (MCBSA) for macrophage targeting delivery of CpG ODN. CBSA prepared via modification of bovine serum albumin (BSA) possesses the advantages of good biodegradability and biocompatibility.30-32 Mannose moieties endow macrophage targeting capability to the vector owing to the over-expression of mannose receptors on macrophages.33,34 To identify the optimized ligand amount, a series of MCBSA samples with different mannose amounts were synthesized and the influence of mannose content on cellular uptake and immune stimulating capacity was investigated. Furthermore, the effect of the CpG ODN delivery system on cancerous cells was also studied to provide a more comprehensive understanding on the functions of CpG ODN in tumor treatments.

MATERIALS AND METHODS Materials Bovine serum albumin (BSA) (Sigma Aldrich), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) (Sigma Aldrich), ethylenediamine (EDA) (Shandong Freda Biochem


4

Page 5 of 34 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


Co., Ltd., China), α-D-mannopyranosylphenyl isothiocyanate (Toronto Research Chemicals), lipopolysaccharide (LPS) (Sigma Aldrich), and Lipofectamine 2000 (Lip2000) (Invitrogen) were used as received. CpG ODN 1826 (abbreviated as ODN) and Cy3 labeled CpG ODN 1826 were purchased from Sangon Biotech Co., Ltd. (Shanghai, China).

Synthesis of Cationic Albumin (CBSA) and Mannosylated Cationic Albumin (MCBSA) CBSA was synthesized according to a literature procedure.31 In brief, the solution of EDA was added to BSA solution slowly under continuous stirring. Subsequently, EDC was added and the mixture was stirred for 2 h. Finally, the reaction was quenched by the addition of acetate buffer (4 M, 400 μl, pH 4.75) and the mixture was dialyzed against deionised water. The product CBSA was obtained after freeze-drying. To synthesize MCBSA, CBSA was dissolved in 10 ml of PBS (0.1 M, pH 9.0). Subsequently, the DMSO solution containing a particular mount (5 mg, 10 mg, and 20 mg) of α-D-mannopyranosylphenyl isothiocyanate (Man) was added. After stirring for 24 h at room temperature, the reaction mixture was dialyzed against deionized water for 72 h and then lyophilized to obtain MCBSA samples (MCBSA 1, MCBSA 2 and MCBSA 3) with particular amounts of mannose moieties.

Fabrication of ODN Delivery Systems ODN, CBSA, MCBSA and Lipofectamine 2000 were dispersed in deionized water.


5


Page 6 of 34

The solution (50 µl) containing CBSA (25 µg) was added into the solution (50 µl) containing ODN (1 μg), and mixed gently for 10 min to obtain nanoparticles ODN@CBSA. The solution (50 µl) containing MCBSA (25 µg) was added into the solution (50 µl) containing ODN (1 μg), and mixed gently for 10 min to obtain nanoparticles ODN@MCBSA. The solution (50 µl) containing Lipofectamine 2000 (1 µg) was added into the solution (50 µl) containing ODN (1 μg), and incubated for 30 min to obtain Lip2000/ODN complexes.

Characterizations of ODN Delivery Systems The size and polydispersity index (PDI) of nanoparticles were measured by dynamic light scattering (DLS) (Zetasizer Nano ZS, Malvern). All measurements were carried out in triplicate. The morphology of nanoparticles was observed by a JEM-100CXII transmission electron microscope at an acceleration voltage of 100 kV. The solution containing nanoparticles was dropped on a copper grid with formvar film. Subsequently, the sample was stained by fresh 0.2% (w/v) solution of phosphotungstic acid, and dried. The encapsulation of ODN in nanoparticles was studied by the agarose gel retardation assay as detailed in SI. To determine the encapsulation efficiency of ODN, the solution containing nanoparticle was centrifuged at 4 °C for 1 h at 10000 rpm. After that, the supernatant of solution containing unprecipitated free ODN was measured using a Quant-iT OliGreen


6

Page 7 of 34 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


ssDNA Assay Kit (Molecular Probes) according to the manufacturer’s protocol using a spectrofluorophotometer (RF-5301 PC, Shimadzu). The ODN encapsulation efficiency was calculated by: Encapsulation efficiency = (WT - WF)/WT ×100% where WT was the total weight of ODN fed and WF was the weight of unencapsulated free ODN.

In Vitro Cytotoxicity In vitro cytotoxicity was determined by MTT assay as detailed in SI.

Study on Cellular Uptake of ODN Delivery Systems The cellular uptake of Cy3 labeled ODN loaded nanoparticles was studied by confocal laser scanning microscopy (CLSM) and flow cytometry as detailed in SI.

Study on Immune Stimulation of ODN Delivery Systems The secretion of IL-12 (p40), IL-6, iNOS, TNF-α, and IL-10 in RAW264.7 cells was determined by Enzyme Linked Immunosorbent Assay (ELISA) as detailed in SI. mRNA of iNOS and Arg-1 was detected by quantitative polymerase chain reaction (qPCR) analysis. CD80 and CD206 expression in RAW264.7 cells was studied by flow cytometry as detailed in SI. The protein expression in RAW264.7 cells was determined by Western blot analysis as detailed in SI.


7


Page 8 of 34

Study on Effects of ODN Delivery Systems on Cancerous Cells The protein expression in HeLa cells after being treated by ODN delivery systems was determined by Western blot analysis as detailed in SI.

RESULTS AND DISCUSSION Preparation and Characterizations of ODN Delivery Systems. To form complexes with CpG ODN with negative charges, cationic albumin (CBSA) was synthesized by the condensation reaction between BSA and ethylenediamine. Subsequently, to achieve the macrophage targeting ability, mannose moieties were incorporated to CBSA through the reaction between CBSA with amino groups and α-D-mannopyranosylphenyl isothiocyanate with isothiocyanate groups. To achieve the optimal targeting efficacy, the amount of mannose moieties incorporated to CBSA was varied to obtain three MCBSA samples. Correspondingly, three kinds of CpG ODN delivery systems (ODN@MCBSA 1, ODN@MCBSA 2 and ODN@MCBSA 3) were obtained by encapsulating CpG ODN in these MCBSA samples via electrostatic interaction (Scheme 1). Nanoparticles without the targeting ligand (ODN@CBSA) were prepared for comparison.


8

Page 9 of 34 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


Scheme 1. Structure of the CpG ODN delivery system and targeting delivery of CpG ODN to macrophages for immune stimulation

The size and zeta potential of these nanoparticles measured by dynamic light scattering (DLS) are shown in Table 1 and Figure 1. Three types of mannosylated nanoparticles show slightly larger hydrodynamic sizes than ODN@CBSA. All ODN loaded nanoparticles possess a size less than 220 nm with a polydispersity index (PDI) lower than 0.20, indicating the nanoparticles are suitable for cellular internalization by macrophages. TEM images (Figure 2) show both ODN@CBSA and ODN@MCBSA 2 are spherically shaped. The size of dried ODN loaded nanoparticles visualized by TEM is smaller than the hydrodynamic size measured by light scattering technique. As shown in Table 1, the zeta potentials of the three types of mannosylated nanoparticles are lower than that of ODN@CBSA due to the fact that some amino groups of CBSA are replaced by mannose ligands after mannosylation.


9


Page 10 of 34

The ODN encapsulation efficiencies are higher than 95% for all nanoparticles. Agarose gel electrophoresis retardation assay confirms that ODN can be effectively loaded in the ODN delivery systems we prepared (Figure S1).

Table 1. Morphology and encapsulation efficiency of ODN delivery systems Delivery system

Particle (nm）

Size

Polydispersity

index

(PDI)

Zeta (mV)

potential

Encapsulation Efficiency of ODN (%)

ODN@CBSA

181±6

0.18

34.7±1.8

99.4

ODN@MCBSA 1

196±9

0.19

33.8±1.6

98.7

ODN@MCBSA 2

205±11

0.20

33.1±0.9

98.5

ODN@MCBSA 3

218±5

0.20

32.9±1.1

98.4

Figure 1. Size distributions of ODN delivery systems.


10

Page 11 of 34 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


Figure 2. TEM images of (A) ODN@CBSA and (B) ODN@MCBSA 2.

To evaluate the biocompatibility of different nanoparticles, the in vitro cytotoxicity of ODN loaded nanoparticles was determined by MTT assay. The viability of RAW264.7 cells after being treated by all ODN loaded nanoparticles is higher than 98% at the ODN concentration of 5 μg/ml, whereas the cell viability after the treatment by Lip2000/ODN complexes is around ~80% (Figure 3). These results indicate that all ODN loaded nanoparticles do not show apparent cytotoxicity against RAW264.7 cells, and the good biocompatibility of MCBSA is favorable for biological applications.

Figure 3. The cell viability of RAW264.7 cells after being treated by different agents.

Cell Uptake of ODN Delivery Systems


11


Page 12 of 34

To study the delivery capability of diverse ODN delivery systems to macrophages, RAW264.7 cells (mannose receptor-positive) were co-incubated with the delivery systems loaded with Cy3 labeled ODN and observed by confocal laser scanning microscopy (CLSM). As shown in Figure 4, red ﬂuorescence can hardly be observed in RAW264.7 cells treated with naked ODN due to the weak cell penetrating ability of naked ODN. Red fluorescence can be observed in the cells treated with ODN@CBSA and ODN@MCBSA. The cells treated by mannosylated nanoparticles show more intense red fluorescence as compared with unmannosylated nanoparticles, which indicates that the introduction of mannose moieties could enhance the cellular uptake efficiently since the mannose moiety can bind specifically to lectin on RAW264.7 cells. In addition, the red fluorescence intensity is much higher after ODN@MCBSA 2 treatment as compared with ODN@MCBSA 1 treatment since more mannose ligands are presented in ODN@MCBSA 2, facilitating the rapid entry of the nanoparticles into the cells. Whereas no obvious difference in the fluorescence intensities can be detected between ODN@MCBSA 3 treated cells and ODN@MCBSA 2 treated cells. These results demonstrate that the mannose amount in ODN@MCBSA 2 is enough for binding with the receptors on RAW264.7 cells, and further increased mannose ligands do not enhance cellular internalization apparently. The flow cytometry analysis is in consistent with the CLSM observation.


12

Page 13 of 34 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


Figure 4. Confocal microscopy observation (A) and flow cytometry analysis (B) on the delivery of ODN (Cy3 labeled) to RAW264.7 cells. The cells were treated by (a) naked ODN, (b) ODN@CBSA, (c) ODN@MCBSA 1, (d) ODN@MCBSA 2, and (e) ODN@MCBSA 3 for 4 h at an ODN concentration of 5 μg/ml. Scale bar: 12 μm. Untreated RAW264.7 cells were used as the control.

In addition, to further confirm the macrophage targeting of ODN@MCBSA is caused by the presence of mannose moieties in the vectors, we carried out cell uptake studies in the presence of free mannose. The result indicates the addition of free mannose in the cell culture medium results in obviously decreased cell uptake of ODN@MCBSA due to the competitive effect of free mannose, confirming the macrophage targeting is mediated by mannose moieties (Figure S2). The cellular uptake of diverse nanoparticles in cancerous HeLa cells was also studied. The incorporation of mannose moieties to the delivery vector does not result in enhanced cell uptake


13


Page 14 of 34

(Figure 5). According to previous studies, mannose moieties do not lead to enhanced cell internalization for cancerous MCF-7 cells,15 which is in consistent with our results.

Figure 5. Confocal microscopy observation (A) and flow cytometry analysis (B) on the delivery of ODN (Cy3 labeled) to HeLa cells. The cells were treated by (a) naked ODN, (b) ODN@CBSA, (c) ODN@MCBSA 1, (d) ODN@MCBSA 2, and (e) ODN@MCBSA 3 for 4 h at an ODN concentration of 5 μg/ml. Scale bar: 24 µm.

Immunostimulatory Properties of ODN Delivery Systems To assess the immunostimulatory activity, the secretion levels of pro-inflammatory (IL-12, IL-6, TNF-α, and iNOS) and anti-inflammatory (IL-10) cytokines from RAW264.7 cells were determined by enzyme linked immunosorbent assay (ELISA).


14

Page 15 of 34 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


As shown in Figure 6, all secreted cytokine levels of macrophages treated by blank carriers (CBSA and Lip2000) do not enhanced obviously, suggesting the effects form the delivery carrier can be ignored. All ODN loaded nanoparticles result in significantly enhanced pro-inflammatory cytokines and increased anti-inflammatory IL-10. Three types of mannose-bearing nanoparticles induce significantly enhanced cytokine secretion as compared with unmannosylated nanoparticles. Being consistent with the cell uptake study, obviously enhanced cytokine release from the cells after being treated ODN@MCBSA 2 could be detected as compared with ODN@MCBSA 1. However, the increase in the cytokine level after ODN@MCBSA 3 treatment is limited as compared with ODN@MCBSA 2 treatment. These results indicate the secretion of the cytokine is strongly related to the ODN delivery efficiency of vectors. Besides, the amounts of secreted cytokines in RAW264.7 cells treated by the three types of mannosylated nanoparticles are obviously higher as compared with Lip2000/ODN complexes.


15


Page 16 of 34

Figure 6. Cytokine secretion levels of RAW264.7 cells after being treated by different agents for 24 h. The ODN concentration for the cell treatment was 5 μg/ml.


16

Page 17 of 34 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


The enhanced secretion of inflammatory markers implies the successful regulation of macrophages by our ODN delivery systems, which favors anti-cancer immune responses. It is worthy of attention that, IL-10, an anti-inflammatory cytokine, is also up-regulated after the treatment of ODN loaded nanoparticles. Based on previous studies, CpG ODN would lead to up-regulated secretion of pro-inflammatory cytokines as well as anti-inflammatory IL-10 in treated macrophages.15,16 Our results are in accordance with the previous reports. As we know, IL-10 is a pleiotropic cytokine. On the one hand, IL-10 suspends adaptive immune responses to favor tumor development. On the other hand, IL-10 also suppresses tumor progression through inhibition of NF-κB activation. Herein, lipopolysaccharide (LPS) was used as a positive control. As compared with LPS, the targeting ODN delivery systems result in a dramatically higher secretion level of IL-12 and lower secretion levels of IL-6, TNF-α, and iNOS. It should be noted that LPS induces greatly increased anti-inflammatory IL-10, and ODN delivery systems only lead to slightly enhanced IL10, implying that ODN delivery systems possess better specificity in inducing cytokine secretion to activate immune responses. PCR was further used to determine mRNA of iNOS (a M1 maker) and Arg-1 (a M2 maker). PCR analysis shows a similar trend, i.e., all ODN loaded nanoparticles result in enhanced mRNA of studied cytokines (Figure 7), and mannosylated nanoparticles exhibit an enhanced efficiency in stimulating the cytokine production as compared with unmannosylated nanoparticles. Compared with mRNA of Arg-1, mRNA of iNOS increases more obviously.


17


Page 18 of 34

Figure 7. PCR analysis on mRNA of iNOS and Arg-1 in RAW264.7 cells after being treated by different agents for 24 h. The ODN concentration for the cell treatment was 5 μg/ml.

Regulation of Polarity of Macrophages by ODN Delivery Systems As is well known, the polarity of macrophages can be converted to anti-tumor M1 phenotype or anti-inflammatory M2 phenotype in response to local environmental stimuli. M1 macrophages and M2 macrophages can express some unique markers and secrete various characteristic cytokines. Herein, in order to explore the polarization of macrophages, the expression of CD80


18

Page 19 of 34 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


as a M1 marker35 and CD206 as a M2 marker36 was determined by Western blot as well as flow cytometry. Compared with the cells treated by unmodified ODN@CBSA, obviously increased expression of CD80 in RAW264.7 cells after incubation with mannose modified nanoparticles was observed due to the macrophage targeting ability of mannose moieties (Figure 8 and Figure 9), which is in accordance with the cellular uptake study. ODN@MCBSA 2 and ODN@MCBSA 3 with high amounts of mannose moieties lead to higher CD80 expression compared with ODN@MCBSA 1. For the M2 marker (CD206), a slightly increased expression was observed in the macrophages co-incubated with ODN loaded nanoparticles. As compared with the great enhancement of CD80 expression, the increase in CD206 expression is very limited after the same treatment. Taken together, these results demonstrate that the ODN delivery mainly favors M1-like polarization in macrophages. These results are consistent with previous studies on other ODN delivery systems.15,16

Figure 8. Western blot analysis on the expression of CD80 and CD206 in RAW264.7 cells after different treatments for 24 h. a. Untreated control. b. ODN@CBSA. c. Lip2000/ODN complexes. d. ODN@MCBSA 1. e. ODN@MCBSA 2. f. ODN@MCBSA 3. The ODN concentration for the cell treatment was 5 μg/ml.


19


Page 20 of 34

More importantly, ODN delivery systems exhibit much better efficiency in shifting the macrophage phenotype to anti-tumor M1 phenotype as compared with LPS since LPS also unfavorably induces dramatically increased CD206 expression. It can be concluded that ODN delivery systems possess much better specificity in regulating the macrophage polarity.


20

Page 21 of 34 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


Figure 9. Flow cytometric analysis on CD 80 (labeled by PE) and CD206 (labeled by APC) in RAW264.7 cells after being treated by diverse agents for 24 h. The ODN concentration for the cell treatment was 5 μg/ml.

Analysis of Signal Transduction Pathways in Macrophages After being delivered to macrophages, CpG ODN binds to toll-like receptor 9 (TLR9) and activates particular signaling pathways such as NF-κB pathway and mitogen-activated protein kinase (MAPK) pathways, leading to the secretion of pro-inflammatory cytokines. To analyze the signal transduction pathways in macrophages, the proteins involved in NF-κB and MAPK pathways in macrophages were investigated by Western blot assay.

Figure 10. Western blot analysis on proteins involved in NF-κB and MAPK signal pathways in RAW264.7 cells after different treatments at an ODN concentration of 5 μg/ml for 24 h. a. Untreated control. b. ODN@CBSA. c. Lip2000/ODN complexes. d. ODN@MCBSA 1. e. ODN@MCBSA 2. f. ODN@MCBSA 3.

As we know, without the stimulation of CpG ODN, NF-κB remains in a quiescent state through complexation with inhibitor of NF-κB (IκB). Upon activation by CpG ODN, IκBα is


21


Page 22 of 34

phosphorylated and dissociated from NF-κB, resulting in free NF-κB which initiates gene transcription after nuclear translocation.6 The decay of IκBα is used as an indicator of NF-κB activation. Herein, the significantly decrease of IκBα and the obvious enhancement of NF-κB after the treatments by diverse ODN delivery systems confirm the activation of the NF-κB pathway (Figure 10). The increase in the expression level of NF-κB is dependent on the delivery efficiency of the carrier. Compared with unmodified ODN@CBSA, modified ODN@MCBSA results in stronger stimulation with higher NF-κB expression. Besides NF-κB pathway, the MAPK signaling can be also activated by CpG ODN stimulation.37,38 Herein, we studied the expression of the proteins evolved in extracellular regulatory protein kinase (ERK) and p38 signaling pathways in macrophages stimulated by ODN delivery systems. Enhanced phosphorylated ERK (p-ERK) and phosphorylated p38 (p-p38) in RAW264.7 cells treated by ODN delivery systems confirm the activation of these pathways in the macrophages stimulated by CpG ODN (Figure 10). In addition, mannosylated nanoparticles up-regulate p-ERK and p-p38 more significantly as compared with unmodified ODN@CBSA. The analysis on the signal pathways in macrophages is coincided with the cytokine production measured by the ELISA and PCR.

Effects of ODN Treatment on Tumor Cells In the tumor microenvironment, immune cells co-exist with tumor cells. The influences of ODN delivery systems on the tumor cells should be an important concern. Since cancerous HeLa cells do not overexpress mannose receptors, no difference in the intracellular accumulation of ODN in HeLa cells among three types of mannosylated nanoparticles could be observed because the presence of mannose ligands does not result in enhanced tumor cell uptake as detailed above.


22

Page 23 of 34 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


Figure 11. Western blot analysis of related protein expression in HeLa cells after different treatments at an ODN concentration of 5 μg/ml for 24 h. a. Untreated control. b. Naked ODN. c. ODN@CBSA. d. ODN@MCBSA 2.

The expression of related proteins in HeLa cells after the treatment of ODN delivery system was analyzed by Western blot. Since three types of mannosylated nanoparticles result in almost the same cellular uptake for tumor cells, we used ODN@MCBSA 2 as a representative to study the effect of ODN delivered by mannosylated nanoparticles on cancerous cells. As shown in Figure 11, the expression of NF-κB, p-Akt, PIK3R3, Fas, FasL, caspase 8, caspase 3 and caspase 7 in tumorous HeLa cells is significantly up-regulated by ODN delivery systems. Mannosylated nanoparticles and unmannosylated nanoparticles play a similar regulatory role on HeLa cells since HeLa cells do not overexpress mannose receptors.


23


Page 24 of 34

The enhanced NF-κB protein in HeLa cells is resulted from the activation of NF-κB pathway. PIK3R3 is a regulatory subunit of phosphoinositide 3-kinase (PI3K). The up-regulated expression of PIK3R3 and phosphorylated protein kinase B (p-Akt) in treated cancer cells implies the activation of PI3K/Akt signaling pathway. As it is well known these pathways boost proliferation of tumor cells and promote cancer metastasis.39 In this regard, ODN has an unfavorable effect on tumor cells. Nevertheless, previous studies indicate that NF-κB may also have potential antioncogenic and proapoptotic functions.39 In addition, NF-κB activation is responsible for the up-regulated Fas and Fas ligand (FasL), resulting in cancer cell apoptosis mediated by Fas/FasL pathway.40 In this study, Fas and FasL are apparently up-regulated after the delivery of ODN, which is favorable for the elimination of cancer cells by immune cells such as natural killer cells and cytotoxic T cells through FasL-induced apoptosis. In addition, the interaction between Fas expressed tumor cell and FasL expressed tumor cells may induce tumor cell death through Fas/FasL-mediated apoptosis.41,42 The increased caspase expression in cancer cells after the ODN delivery is most likely to be induced by the Fas/FasL mediated apoptosis between Fas and FasL expressed tumor cells. There is no doubt that the effects of CpG ODN on cancer cells are pleiotropic. Stimulated by CpG ODN, the changes in protein expression may have contradictory effects on the growth and proliferation of cancer cells. As an overall result, our ODN delivery systems do not significantly affect the viability of HeLa cells (Figure S3). Our research provides a more extensive view on CpG ODN in cancer immunotherapy.

CONCLUSIONS


24

Page 25 of 34 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


In this study, we synthesized mannosylated cationic albumin for macrophage targeting delivery of CpG ODN. Incorporation of mannose ligands to cationic albumin can enhance intracellular uptake of macrophages obviously and endow the carrier with improved immune stimulation activity. CpG ODN delivered by mannosylated cationic albumin can stimulate macrophages to secrete more pro-flammatory cytokines (IL-12, IL-6, TNF-α and iNOS) and enhance CD80 expression in macrophages, indicating the polarization of macrophages to the anti-tumor M1 phenotype. Besides, ODN delivery systems do not result in obvious changes in cancerous cell viability. However, the activation of NF-κB and PI3K/Akt signaling pathways in tumor cells implies the pleiotropic effects of CpG ODN delivery on tumor cells. Our study provides a more in-depth understanding on the immunotherapeutic effects of CpG ODN, and highlights the importance of macrophage targeting delivery to minimize the effects in tumor cells.

ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website. Experimental details. Agarose gel electrophoresis retardation assay. Delivery of ODN to RAW264.7 cells in the presence of free mannose. Viability of HeLa cells after being treated by different agents.

AUTHOR INFORMATION Corresponding Author * [email protected]


25


Page 26 of 34

ORCID Si-Xue Cheng: 0000-0001-9611-4421 Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT The financial support from National Natural Science Foundation of China (51533006 and 21875169) is gratefully acknowledged.

ABBREVIATIONS ODN, oligodeoxynucleotides; BSA, bovine serum albumin; CBSA, cationic albumin; MCBSA, mannosylated cationic albumin; NF-kB, nuclear factor kB; PI3K, posphoinositide 3-kinase; Akt, protein kinase B;

MAPK, mitogen-activated protein kinase; ERK, extracellular regulatory

protein kinase; IL-12, interleukin 12; IL-6, interleukin 12; IL-10, interleukin 10; TNF-α, tumor necrosis factor; iNOS, inducible nitric oxide synthase; Arg-1, Arginase 1; DLS, dynamic light scattering; CLSM, confocal laser scanning microscopy; ELISA, enzyme linked immunosorbent assay; PCR, polymerase chain reaction.

REFERENCES (1) Mellman, I.; Coukos, G.; Dranoff, G. Cancer Immunotherapy Comes of Age. Nature 2011,


26

Page 27 of 34 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


480, 480–489. (2) Binnewies, M.; Roberts, E. W.; Kersten, K.; Chan, V.; Fearon, D. F.; Merad, M.; Coussens, L. M.; Gabrilovich, D. I.; Ostrand-Rosenberg, S.; Hedrick, C. C.; Vonderheide, R. H.; Pittet, M. J.; Jain, R. K.; Zou, W.; Howcroft, T. K.; Woodhouse, E. C.; Weinberg, R. A.; Krummel, M. F. Understanding the Tumor Immune Microenvironment (TIME) for Effective Therapy, Nat. Med. 2018, 24, 541–550. (3) Chen, Q; Wang, C.; Zhang, X.; Chen, G.; Hu, Q.; Li, H.; Wang, J.; Wen, D.; Zhang, Y.; Lu, Y.; Yang, G.; Jiang, C.; Wang, J.; Dotti, G.; Gu, Z. In Situ Sprayed Bioresponsive Immunotherapeutic Gel for Post-Surgical Cancer Treatment, Nature Nanotech. 2019, 14, 89–97. (4) Ruan, H.; Bu, L.; Hu, Q.; Cheng, H.; Lu, W.; Gu, Z. Strategies of Combination Drug Delivery for Immune Checkpoint Blockades, Adv. Healthcare Mater. 2019, 8, 1801099. (5) Gordon, S. Alternative Activation of Macrophages. Nat. Rev. Immunol. 2003, 3, 23–35. (6) Klinman, D. M., Immunotherapeutic Uses of CpG Oligodeoxynucleotides. Nat. Rev. Immunol. 2004, 4, 249–258. (7) Kim, S. Y.; Heo, M. B.; Hwang, G. S.; Jung, Y.; Choi, D. Y.; Park, Y. M.; Lim, Y. T. Multivalent Polymer Nanocomplex Targeting Endosomal Receptor of Immune Cells for Enhanced Antitumor and Systemic Memory Response. Angew. Chem. Int. Ed. 2015, 54, 8139–8143. (8) Fontana, F.; Liu, D.; Hirvonen, J.; Santos, H. A. Delivery of Therapeutics with Nanoparticles: What's New in Cancer Immunotherapy? WIREs Nanomed. Nanobiotechnol. 2016, 9, 1421. (9) Lollo, G.; Vincent, M.; Ullio-Gamboa, G.; Lemaire, L.; Franconi, F.; Couez, D.; Benoit, J.


27


Page 28 of 34

P. Development of Multifunctional Lipid Nanocapsules for the Co-Delivery of Paclitaxel and CpG-ODN in the Treatment of Glioblastoma. Int. J. Pharm. 2015, 495, 972–980. (10)Silva, J. M.; Zupancic, E.; Vandermeulen, G.; Oliveira, V. G.; Salgado, A.; Videira, M.; Gaspar, M.; Graca, L.; Preat, V.; Florindo, H. F. In Vivo Delivery of Peptides and Toll-Like Receptor

Ligands

by

Mannose-Functionalized

Polymeric

Nanoparticles

Induces

Prophylactic and Therapeutic Anti-Tumor Immune Responses in a Melanoma Model. J. Control. Release 2015, 198, 91–103. (11)Wang, C.; Sun, W.; Wright, G.; Wang, A. Z.; Gu, Z. Inflammation-Triggered Cancer Immunotherapy by Programmed Delivery of CpG and Anti-PD1 Antibody. Adv. Mater. 2016, 28, 8912–8920. (12)Nishida, Y.; Ohtsuki, S.; Araie, Y.; Umeki, Y.; Endo, M.; Emura, T.; Hidaka, K.; Sugiyama, H.; Takahashi, Y.; Takakura, Y.; Nishikawa, M. Self-Assembling DNA Hydrogel-Based Delivery of Immunoinhibitory Nucleic Acids to Immune Cells. Nanomed. Nanotech. Bio. Med. 2016, 12, 123–130. (13)Li, W.; Luo, L.; Huang, J.; Wang, Q.; Liu, J.; Xiao, X.; Fang, H.; Yang, X.; Wang, K. SelfAssembled DNA Nanocentipedes as Multivalent Vehicles for Enhanced Delivery of CpG Oligonucleotides. Chem. Commun. 2017, 53, 5565–5568. (14)Miyamoto, N.; Mochizuki, S.; Fujii, S.; Yoshida, K.; Sakurai, K. Adjuvant Activity Enhanced by Cross-Linked CpG-Oligonucleotides in β-Glucan Nanogel and Its Antitumor Effect. Bioconjugate Chem. 2017, 28, 565–573. (15)He, X. Y.; Liu, B. Y.; Wu, J. L.; Ai, S. L.; Zhuo, R. X.; Cheng, S. X., A Dual Macrophage Targeting Nanovector for Delivery of Oligodeoxynucleotides To Overcome CancerAssociated Immunosuppression. ACS Appl. Mater. Interfaces 2017, 9, 42566–42576.


28

Page 29 of 34 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


(16)He, X. Y.; Liu, B. Y.; Ai, S. L.; Xu, L.; Zhuo, R. X.; Cheng, S. X. Functional Polymer/Inorganic Hybrid Nanoparticles for Macrophage Targeting Delivery of Oligodeoxynucleotides in Cancer Immunotherapy. Mater. Today Chem. 2017, 4, 106–116. (17)Morishita, M.; Takahashi, Y.; Matsumoto, A.; Nishikawa, M.; Takakura, Y. Exosome-Based Tumor Antigens-Adjuvant Co-Delivery Utilizing Genetically Engineered Tumor CellDerived Exosomes with Immunostimulatory CpG DNA. Biomaterials 2016, 111, 55–65. (18)Tao, Y.; Zhang, Y.; Ju, E.; Ren, H.; Ren, J. Gold Nanocluster-Based Vaccines for DualDelivery of Antigens and Immunostimulatory Oligonucleotides. Nanoscale 2015, 7, 12419– 12426. (19)Chen, S.; Zhang, Q.; Jia, L.; Du, X.; Hanagata, N. A Facilely Controlled Length, Cytotoxicity, Length-Dependent and Cell Type-Dependent Cellular Uptake of Silica Nanotubes and Their Applications in the Delivery of Immunostimulatory CpG oligodeoxynucleotides. J. Mater. Chem. B 2015, 3, 7246–7254. (20)Zhang, H.; Chen, W.; Gong, K.; Chen, J. Nanoscale Zeolitic Imidazolate Framework-8 as Efficient Vehicles for Enhanced Delivery of CpG Oligodeoxynucleotides. ACS Appl. Mater. Interfaces 2017, 9, 31519–31525. (21)Zhang, Y.; Liu, C.; Wang, F.; Liu, Z.; Ren, J.; Qu, X. Metal-Organic-Framework-Supported Immunostimulatory Oligonucleotides for Enhanced Immune Response and Imaging. Chem. Commun. 2017, 53, 1840–1843. (22)Zhang, H.; Yan, T.; Xu, S.; Feng, S.; Huang, D.; Fujita, M.; Gao, X. D. Graphene OxideChitosan Nanocomposites for Intracellular Delivery of Immunostimulatory CpG oligodeoxynucleotides. Mater. Sci. Eng. C Mater. Biol. Appl. 2017, 73, 144–151.


29


Page 30 of 34

(23)Zhang, H.; Chen, S.; Zhi, C.; Yamazaki, T.; Hanagata, N. Chitosan-Coated Boron Nitride Nanospheres Enhance Delivery of CpG Oligodeoxynucleotides and Induction of Cytokines. Inter. J. Nanomed. 2013, 8, 1783–1793. (24)Zhang, H.; Yamazaki, T.; Zhi, C.; Hanagata, N. Identification of a Boron Nitride Nanosphere-Binding Peptide for the Intracellular Delivery of CpG Oligodeoxynucleotides. Nanoscale 2012, 4, 6343–6350. (25)Pourianazar, N. T.; Gunduz, U. Changes in Apoptosis-Related Gene Expression and Cytokine Release in Breast Cancer Cells Treated with CpG-Loaded Magnetic PAMAM Nanoparticles. Int. J. Pharm. 2016, 515, 11–19. (26)Gao, M.; Ha, T.; Zhang, X.; Wang, X.; Liu, L.; Kalbfleisch, J.; Singh, K.; Williams, D.; Li, C. The Toll-Like Receptor 9 Ligand, CpG Oligodeoxynucleotide, Attenuates Cardiac Dysfunction in Polymicrobial Sepsis, Involving Activation of Both Phosphoinositide 3 Kinase/Akt and Extracellular-Signal-Related Kinase Signaling. J. Infect. Dis. 2013, 207, 1471–1479. (27)Mantovani, A.; Allavena, P.; Sica, A.; Balkwill, F. Cancer-Related Inflammation. Nature 2008, 454, 436–444. (28)Wang, C. Y.; Mayo, M. W.; Baldwin Jr., A. S. TNF- and Cancer Therapy Induced Apoptosis Potentiation by Inhibition of NF-κB. Science 1996, 274, 784–787. (29)Liu, P.; Cheng, H.; Roberts, T. M.; Zhao, J. J. Targeting the Phosphoinositide 3-Kinase Pathway in Cancer. Nat. Rev. Drug Discov. 2009, 8, 627–644. (30)Kesharwani, P.; Jain, A.; Jain, A.; Jain, A. K.; Garg, N. K.; Tekade, R. K.; Raj Singh, T. R.; Iyer, A. K. Cationic Bovine serum Albumin (CBA) Conjugated Poly Lactic-co-Glycolic


30

Page 31 of 34 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


Acid (PLGA) Nanoparticles for Extended Delivery of Methotrexate into Brain Tumors. RSC Adv. 2016, 6, 89040–89050. (31)Han, J.; Wang, Q.; Zhang, Z.; Gong, T.; Sun, X. Cationic Bovine Serum Albumin Based Self-Assembled Nanoparticles as siRNA Delivery Vector for Treating Lung Metastatic Cancer. Small 2014, 10, 524–535. (32)Lu, W.; Zhang, Y.; Tan, Y. Z.; Hu, K. L.; Jiang, X. G.; Fu, S. K. Cationic AlbuminConjugated Pegylated Nanoparticles as Novel Drug Carrier for Brain Delivery. J. Control. Release 2005, 107, 428–448. (33)Jiang, H. L.; Kang, M. L.; Quan, J. S.; Kang, S. G.; Akaike, T.; Yoo, H. S.; Cho, C. S. The Potential of Mannosylated Chitosan Microspheres to Target Macrophage Mannose Receptors in an Adjuvant-Delivery System for Intranasal Immunization. Biomaterials 2008, 29, 1931–1939. (34)Zeng, Z.; Dai, S.; Jiao, Y.; Jiang, L.; Zhao, Y.; Wang, B.; Zong, L. Mannosylated Protamine as a Novel DNA Vaccine Carrier for Effective Induction of Anti-Tumor Immune Responses. Int. J. Pharm. 2016, 506, 394–406. (35)Tran, T. H.; Rastogi, R.; Shelke, J.; Amiji, M. M. Modulation of Macrophage Functional Polarity towards Anti-Inflammatory Phenotype with Plasmid DNA Delivery in CD44 Targeting Hyaluronic Acid Nanoparticles. Sci. Rep. 2015, 5, 16632–16647. (36)Spiller, K. L.; Nassiri, S.; Witherel, C. E.; Anfang, R. R.; Ng, J.; Nakazawa, K. R.; Yu, T.; Vunjak-Novakovic, G. Sequential Delivery of Immunomodulatory Cytokines To Facilitate the M1-to-M2 Transition of Macrophages and Enhance Vascularization of Bone Scaffolds. Biomaterials 2015, 37, 194–207.


31


Page 32 of 34

(37)He, H.; Kogut, M. H. CpG-ODN-Induced Nitric Oxide Production Is Mediated through Clathrin-Dependent Endocytosis, Endosomal Maturation, and Activation of PKC, MEK1/2 and p38 MAPK, and NF-κB Pathways in Avian Macrophage Cells (HD11). Cell. Signal. 2003, 15, 911–917. (38)Krieg, A. M. CpG Motifs in Bacterial DNA and Their Immune Effects. Annu. Rev. Immunol. 2002, 20, 709–760. (39)Baldwin, A. S. Control of Oncogenesis and Cancer Therapy Resistance by the Transcription Factor NF-κB. J. Clin. Invest. 2001, 107, 241–246. (40)Arunkumar, N.; Liu, C.; Hang, H.; Song, W. Toll-Like Receptor Agonists Induce Apoptosis in Mouse B-Cell Lymphoma Cells By Altering NF-κB Activation. Cell Mol. Immunol. 2013, 10, 360–372. (41)Yamada, A.; Arakaki, R.; Saito, M.; Kudo, Y.; Ishimaru. N. Dual Role of Fas/FasLMediated Signal in Peripheral immune Tolerance. Front. Immunol. 2017, 8, 403. (42)Li, D. C.; Zheng, S. Y.; Zhao, J.; Ge, J. F. Adenovirus-Mediated FasL Gene Transfer into Human Gastric Carcinoma. World J. Gastroenterol. 2005, 11, 3446–3450.


32

Page 33 of 34 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


For Table of Contents Use Only

Targeting Delivery of Oligodeoxynucleotides to Macrophages by Mannosylated Cationic Albumin for Immune Stimulation in Cancer Treatment Shu-Lun Ai, Xiao-Yan He, Buo-Ya Liu, Ren-Xi Zhuo and Si-Xue Cheng*


33


TOC graphic 400x348mm (96 x 96 DPI)


Page 34 of 34

Targeting Delivery of Oligodeoxynucleotides to Macrophages by

Recommend Documents