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Cite This: ACS Appl. Mater. Interfaces 2017, 9, 42566−42576

A Dual Macrophage Targeting Nanovector for Delivery of Oligodeoxynucleotides To Overcome Cancer-Associated Immunosuppression Xiao-Yan He, Bo-Ya Liu, Jin-Long Wu, Shu-Lun Ai, Ren-Xi Zhuo, and Si-Xue Cheng* Key Laboratory of Biomedical Polymers of Ministry of Education, College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072, People’s Republic of China S Supporting Information *

ABSTRACT: To overcome cancer-associated immunosuppression, we prepared a dual-targeting vector to deliver CpG oligodeoxynucleotides (ODN) to macrophages. The dualtargeting system composed of mannosylated carboxymethyl chitosan (MCMC)/hyaluronan (HA) for macrophage targeting and protamine sulfate for ODN complexation was prepared by self-assembly. The effects of ODN delivery on immune cells was studied in J774A.1 cells. Due to the enhanced delivery efficiency, the dual-targeting delivery system exhibits a higher immune stimulatory activity compared with the monotargeting delivery system containing either MCMC or HA, resulting in a dramatically enhanced secretion of proinflammatory cytokines and a successful shift to activated macrophages (M1). Besides macrophages, the influence of the delivery system on tumor cells (MCF-7) was also investigated. In MCF-7 cells, the increased expressions of nuclear transcription factor-κB (NF-κB), PIK3R3, and phosphorylated protein kinase B (p-Akt) caused by activated NF-κB and phosphoinositide 3-kinase/Akt signalings were observed. Nevertheless, upregulated Fas as well as Fas ligand (FasL) may induce Fas/FasL-mediated apoptosis, which results in the increased expressions of caspases in tumor cells. KEYWORDS: gene delivery, macrophage targeting, self-assembly, CpG ODN, immunotherapy, cancer treatment

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pression.9−11 Through the recognition by toll-like receptor 9 (TLR9), CpG ODN may induce the activation of particular signal pathways including nuclear transcription factor-κB (NFκB) and mitogen-activated protein kinase (MAPK), inducing antitumor immunological responses.12,13 Although CpG ODN can overcome cancer-associated immunosuppression and initiate antitumor immune responses, the potential side effects of ODN on tumor cells cannot be ignored. According to previous studies, CpG ODN may result in the activation of phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) signaling.14 As we know, activated NF-κB and PI3K/Akt signalings are regarded as the important promoters for tumor development.15,16 Inhibition of NF-κB and PIK3/Akt activation in tumor cells has been investigated as an antitumor strategy.17−19 To address these issues, the purpose of current investigation is to optimize the delivery efficiency of ODN to targeted immune cells using a dual-targeting nanovector and thus to minimize the side effects of ODN on tumor cells.

INTRODUCTION By using the body’s immune system, immunotherapy holds great promise in cancer treatments due to its less toxic effects, lower recurrence rate, and decreased long-term mortality.1 Among various immune cells, macrophages play a vital part in innate immunity and adaptive immunity through collaborating with other types of immune cells. Upon stimulation, macrophages can be converted into two phenotypes, M1 phenotype secreting proinflammatory cytokines to promotes type I immune response against infections and tumor cells, and M2 phenotype with anti-inflammatory functions that facilitate tumor growth.2−4 In tumor environments, the interaction between cancer cells and immune cells crucially influences cancer development and diversification. Tumor-associated macrophages correlate with the suppression of anticancer immune responses, neovascularization, and metastasis in many tumor types.3,5,6 As an important strategy in cancer immunotherapy, specific alterations in the tumor microenvironment by modulating macrophages to activated M1 phenotype show promising results.3,5,7,8 To promote strong immune responses by oligodeoxynucleotides containing CpG motifs (CpG ODN) is regarded as a promising strategy to overcome cancer-associated immunosup© 2017 American Chemical Society

Received: September 8, 2017 Accepted: November 21, 2017 Published: November 21, 2017 42566

DOI: 10.1021/acsami.7b13594 ACS Appl. Mater. Interfaces 2017, 9, 42566−42576

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ACS Applied Materials & Interfaces

Scheme 1. Structure of the Dual-Targeting Delivery System and Delivery of CpG ODN for Inducing Immunological Stimulation in Macrophage Cells

Table 1. Morphology and ODN-Loading Capability of Delivery Systems system nontargeting PS/ODN nontargeting CMC/PS/ODN monotargeting HA/PS/ODN monotargeting MCMC/PS/ODN dual-targeting MCMC/HA/PS/ODN

particle size (nm) 145 170 190 165 210

± ± ± ± ±

polydispersity index (PDI)

ζ (mV)

encapsulation efficiency of ODN (%)

0.12 0.10 0.02 0.16 0.08

26.5 ± 1.2 13.5 ± 0.3 12.6 ± 0.4 15.5 ± 0.7 9.3 ± 0.5

98.5 98.7 98.1 99.0 99.5

9 2 2 5 5

on cancer cells was also investigated, and the increased expressions of NF-κB, PIK3R3, and p-Akt in MCF-7 cells were detected. Nevertheless, the upregulation of Fas and Fas ligand (FasL) induces Fas/FasL-mediated tumor cell apoptosis.

As it is well known, the cell uptake of CpG ODN is very limited in the absence of a delivery vector. To efficiently deliver CpG ODN into targeted cells and prevent the degradation of ODN by a vector is of critical importance. Recently, intense research interest has been paid to nanovectors for CpG ODN delivery.20,21 Reported delivery vectors are based on lipids,22−24 biomacromolecules,25−30 exosomes,31 synthetic polymers,32−37 inorganics, such as mesoporous silica,38 metals, such as gold nanoclusters,39,40 metal−organic framework,41 and hybrid materials.42−47 Despite the advances in designing diverse carriers, the investigations on the influences of ODN delivery on tumor cells are very rare.48 The purpose of this investigation is to develop a dual macrophage targeting delivery vector for CpG ODN delivery. In the delivery vector, mannosylated carboxymethyl chitosan (MCMC) and hyaluronan (HA), which can realize macrophage targeting because of the high expression of mannose receptors49,50 and CD44 receptors51 in macrophages, are decorated on the surface of the nanosized delivery system. Positively charged protamine sulfate (PS), which forms complexes with negatively ODN, can easily cross cell membranes and promote nuclear translocation.50,52 The delivery vector has favorable biodegradability and biocompatibility. Because of the existence of two types of ligands in the surface, dual-targeting MCMC/HA/PS/ODN nanoparticles exhibit significantly stronger immune stimulation activity than the monotargeting system containing either MCMC or HA. More importantly, the influence of the targeting delivery system

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RESULTS AND DISCUSSION Self-Assembled Delivery Vectors. To effectively deliver CpG ODN to targeted macrophages and overcome cancerassociated immunosuppression, two types of targeting components were introduced onto the surface of PS/ODN complexes to form dual-targeting nanoparticles (Scheme 1). In the delivery vector, both hyaluronan (HA) and mannosylated carboxymethyl chitosan (MCMC) have macrophage-targeting ability and can enhance the macrophage delivery efficiency via CD44- and mannose-mediated cell uptakes. Protamine sulfate (PS) can not only encapsulate ODN through electrostatic interaction but also enhance cell uptake due to its membranetranslocating ability. Self-assembled MCMC/HA/PS/ODN nanoparticles were prepared in an aqueous medium. First, the electrostatic interaction between PS with positive charges and ODN with negative charges leads to the formation of PS/ODN nanoparticles. PS/ODN nanoparticles exhibit a well dispersed spherical shape (Figure S1) and a positive surface charge because of the excess PS in the complexes (Table 1). After that, upon addition of targeting components to positively charged PS/ODN complexes, dual-targeting MCMC/HA/PS/ODN nanoparticles were formed driven by the electrostatic

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Figure 1. Size distributions of ODN delivery systems.

Positively charged PS with arginine-rich sequences can interact with negatively charged ODN to form condensed nanoparticles. Herein, we evaluated the effect of PS amount on the ODN-loading capacity (Table S1). The encapsulation efficiency of ODN increases with increasing PS amount, and reaches 98% when the PS amount is up to 30 μg. So, in this study, we fixed the PS amount at 30 μg to fabricate the ODNloaded nanoparticles unless otherwise mentioned. All of the nanoparticles with PS amount of 30 μg can encapsulate ODN efficiently, with encapsulation efficiencies higher than 98% (Table 1). To obtain the optimal targeting effect, we prepared monotargeting nanoparticles with various HA and MCMC amounts ranging from 5 to 20 μg. For HA/PS/ODN (Table S2) or MCMC/PS/ODN (Table S3) nanoparticles, the size of nanoparticles increases and the ζ-potential decreases with increasing amount of HA or MCMC. All of the nanoparticles can encapsulate ODN efficiently with encapsulation efficiencies higher than 95%. According to flow cytometry (FCM) results (Figure S2), the highest cell uptake of Cy3-labeled ODN in J774A.1 cells is achieved at the HA or MCMC amount of 15 μg for monotargeting delivery systems (HA/PS/ODN and

interaction between them, and the macrophage-targeting components (MCMC and HA) form the surface layer. Besides electrostatic interactions, hydrophobic interactions among these components also contribute to the formation of nanoparticles.53−56 For comparison, nontargeting nanoparticles CMC/PS/ODN with carboxymethyl chitosan (CMC) and monotargeting nanoparticles (HA/PS/ODN and MCMC/PS/ODN) were also prepared. As shown in Table 1 and Figure 1, as compared with nontargeting PS/ODN nanoparticles, the particle size increases after decoration of functional components, and the dual-targeting MCMC/HA/PS/ODN nanoparticles have a larger size (210 nm) as compared with monotargeting ones. Besides, the PDI values of all of the ODN-loaded nanoparticles are lower than 0.2, implying the size distribution is relatively narrow. The particle sizes measured by dynamic light scattering are in agreement with the transmission electron microscopy (TEM) observation (Figure S1). As expected, the nanoparticles decorated by negatively charged polymers possess decreased ζpotentials and the dual-targeting delivery system exhibits the lowest ζ-potential of 9.3 mV. 42568


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simultaneous decoration of MCMC and HA on the delivery system surface results in the most efficient ODN delivery, which is due to the interaction between mannose-incorporated MCMC and mannose receptors as well as HA and CD44 receptors in J774A.1 cells. The intracellular delivery of ODN mediated by MCMC/HA/PS/ODN nanoparticles was also observed by confocal laser scanning microscopy (CLSM) (Figure S3). After endocytosis and endo/lysosomal escape, ODN is intracellularly released due to the biodegradation of the delivery vector. The cellular uptake was further quantitatively studied by flow cytometry (FCM). Clearly, the simultaneous introduction of HA and MCMC to the delivery system leads to an obviously increased intracellular ODN accumulation as compared with nontargeting PS/ODN complexes and two types of monotargeting nanoparticles (Figure 3B). The FCM result is in good agreement with the confocal microscopy observation, confirming the surface decoration by HA and MCMC can achieve macrophage targeting. In tumor environments, tumorous cells and immunological cells coexist. The influence of ODN delivery on tumor cells cannot be ignored. In this study, we also studied the cell uptake of ODN delivery systems for cancerous MCF-7 cells. Being different from J774A.1 cells, the cell uptake of HA/PS/ODN nanoparticles is highest in MCF-7 cells (Figures S4). Obviously, the HA-targeting effect leads to the highest cell uptake of HA/PS/ODN nanoparticles in MCF-7 cells. Although hyaluronan can also target tumor cells,57 its tumortargeting efficiency is lower than the macrophage-targeting efficiency because cancerous MCF-7 cells have a significantly lower expression of CD44 compared with J774A.1 cells (Figure S5). Because mannose moiety does not target cancerous MCF7 cells, the presence of MCMC on the nanoparticle surface is not helpful for tumor cell uptake. The enlarged size caused by MCMC decoration is the main reason for the slightly decreased cell uptake of the nanoparticles containing MCMC as compared with their counterparts without MCMC. Activation of Macrophages by ODN Delivery Systems. In the current study, no apparent changes in cytokine levels can be detected in J774A.1 cells co-incubated with blank vectors (Table S4), indicating the immune stimulation caused by blank vectors can be ignored. As demonstrated in Figure 4, proinflammatory cytokines secreted by J774A.1 cells co-incubated with ODN-loaded vectors increase significantly. The extent of increase in the levels of interleukin (IL)-12, IL-6, tumor necrosis factor (TNF)-α, and inducible nitric oxide synthase (iNOS) depends on the delivery capability of ODN-loaded nanoparticles. Monotargeting delivery systems (MCMC/PS/ODN and HA/ PS/ODN nanoparticles) possess a higher immunostimulatory activity as compared with nontargeting systems, and dualtargeting nanoparticles exhibit the highest activity in stimulating immune responses. Herein, we also monitor the secretion of anti-inflammatory IL-10 in J774A.1 cells. After being treated by ODN delivery systems, increased IL-10 levels can be detected and the enhancement of IL-10 secretion also relies on the delivery efficiency of vectors. Nevertheless, the IL-10 concentration (∼160 pg/mL) is significantly lower as compared with proinflammatory cytokines after the treatment by the most effective dual-targeting delivery system. As is well known, the enhanced expression of proinflammatory cytokines is a major characteristic of M1 phenotype

MCMC/PS/ODN nanoparticles). A further increase in MCMC or HA amount to 20 μg leads to a slightly decreased cell uptake, which is caused by the increased size of ODNloaded nanoparticles. For the dual-targeting delivery system (MCMC/HA/PS/ODN nanoparticles), the enhancement in cell uptake increases with HA and MCMC amounts in the range of 5−15 μg. If the HA and MCMC amounts are further increased to 20 μg, the size of particles increases dramatically and the particles become unstable. So, the amounts of MCMC and HA were fixed at 15 μg to prepare the ODN delivery systems to achieve an optimized targeting ability unless otherwise mentioned. Evaluation on Cytotoxicity of ODN Delivery Systems. The biocompatibility of our delivery vectors was studied by evaluating the cytotoxicity of nanoparticles in J774A.1 macrophages. As demonstrated in Figure 2, no apparent decrease in

Figure 2. Viability of J774A.1 cells after being treated by different agents.

the cell viability (>95%) can be observed after the treatment by all of the nanoparticles with PS, CMC, MCMC, and HA components for 24 h. In contrast, Lipofectamine 2000/ODN complexes lead to decreased cell viability (∼70%). Clearly, our ODN delivery vectors have a better biocompatibility as compared with commercialized Lipofectamine 2000. Evaluation on Targeting Capability of Delivery Vectors. For the evaluation on the macrophage-targeting delivery efficiency of our ODN vectors, diverse Cy3-labeled ODN-encapsulated vectors were co-incubated with J774A.1 cells. As visualized by a confocal microscope (Figure 3A), a very low fluorescence is detected in naked ODN-treated cells because naked ODN can hardly pass the cell membranes. Enhanced red fluorescences can be detected in the J774A.1 cells treated by PS/ODN and CMC/PS/ODN nanoparticles. Clearly, the positively charged PS can effectively interact with ODN to achieve enhanced cell uptake. However, without the cell-targeting components, the delivery efficiency of the PS/ ODN complexes is still limited. Monotargeting delivery systems (MCMC/PS/ODN nanoparticles with mannose moieties and HA/PS/ODN nanoparticles with hyaluronan) result in further enhanced cell uptake of Cy3-labeled ODN because of the mannose- and hyaluronan-mediated endocytosis, respectively. The dual-targeting delivery system with both MCMC and HA leads to the highest intracellular fluorescence. Clearly, the 42569


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Figure 3. Confocal microscopy images (A) and flow cytometric analysis (B) on the delivery of ODN (Cy3 labeled) to J774A.1 cells. The cells were treated by (a) naked ODN, (b) nontargeting system without CMC, (c) nontargeting system with CMC (CMC/PS/ODN), (d) monotargeting system with HA (HA/PS/ODN), (e) monotargeting system with MCMC (MCMC/PS/ODN), and (f) dual-targeting system (MCMC/HA/PS/ ODN) for 4 h. Scale bar: 24 μm.

macrophages.4−6 In the current study, the dramatically enhanced secretion levels of proinflammatory cytokines indicate the strong immunostimulatory capability of the dualtargeting ODN delivery system. It should be noted that both proinflammatory cytokines and anti-inflammatory cytokines exert highly pleiotropic effects on tumor development. For example, despite the antitumor activities, TNF-α may also be responsible for tumor cell survival because it stimulates the generation of genotoxic molecules. Although IL-10 leads to suppressed adaptive immune responses, it may also suppress tumor development by inhibiting NF-κB activation.58 Upregulated proinflammatory cytokines are essential to obtain antitumor immunity. However, the inflammatory responses induced by the high level of proinflammatory cytokines may provide a breeding ground for tumor growth and metastases. In a particular local environment, the polarity of highly plastic macrophages can be switched to proinflammatory M1 polarity or anti-inflammatory M2 polarity.7 To study the relationship between the ODN delivery and the macrophage polarization, the expressions of CD80 (a M1 marker)7 and CD206 (a M2 marker)59 after being treated by the ODNloaded nanoparticles were studied by flow cytometry. As shown in Figure 5, compared with naked ODN-treated J774A.1 cells with a limited CD80 expression, the J774A.1 cells treated by all ODN-loaded nanoparticles show an apparent increase in CD80 expressions. The expression level of CD80

increases monotonically with the delivery efficiency of the vectors. After being treated by the most efficient dual-targeting MCMC/HA/PS/ODN nanoparticles, CD80 is expressed in more than 70% J774A.1 cells. In contrast to the dramatic increase in CD80 expression, the expression of CD206 increases very slightly, i.e., the CD206 expressed cells after ODN delivery are in the range of 2−5%. Western blot analysis confirms the significant enhanced level of CD80 (Figure S6). These results indicate that the ODN delivery systems can promote M1-type polarization in macrophages to overcome cancer-associated immunosuppression. Analysis of Signal Transduction Pathways in Macrophages. Based on previous studies, NF-κB and MAPK are the major signal pathways participating in the expression of a variety of cytokines after the stimulation of CpG ODN.12,13 After the cell uptake of an ODN delivery system, microbial DNA sequence containing ODN is transported from early endosomes into tubular lysosomes. During this process, ODN binds to redistributed TLR9.60 After recognition of ODN, TLR9 sequentially recruits myeloid differentiation factor 88, followed by the activation of IL-1 receptor-activated kinase/ tumor necrosis factor receptor-associated factor 6 complexes, which further mediate NF-κB and MAPK activation and then lead to the cytokine synthesis.11,61 Stimulated by CpG ODN, activated NF-κB pathway plays a vital part in diverse cytokine expressions.9−11 Before ODN delivery, NF-κB complexed with inhibitory IκB proteins 42570


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Figure 4. Cytokine secretions of J774A.1 cells after being treated by different agents for 24 h. Control cells were untreated J774A.1 cells.

NF-κB is a major factor to induce immune responses. However, inappropriate activation of NF-κB may be responsible for the undesired outcomes, such as cancer, inflammatory, and autoimmune diseases.15 For example, NF-κB is constitutively active in many types of tumor cells, promoting the gene expressions that are favorable for cell proliferation and survival.15,16 As an important antitumor strategy, inhibiting NF-κB in tumor cells can cause cell growth suppression and enhance the sensitivity to antitumor agents.16 To effectively

remains in a quiescent state. Upon activation, IκBα is phosphorylated, resulting in the dissociation of IκBα from NF-κB. Then, the free NF-κB translocated into cell nuclei initiates gene transcription.11,62 As shown in Figure 6, the treatment of naked ODN and diverse ODN delivery systems leads to the significantly enhanced phosphorylated IκBα (PIκBα) and free NF-κB p65, and enhancement in the expression levels increases with increasing delivery efficiency of the vector. 42571


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Figure 5. Flow cytometric analysis on phycoetythrin-labeled CD80 and fluorescein isothiocyanate-labeled CD206 in J774A.1 cells (a) without treatment, (b) treated by naked ODN, (c) treated by nontargeting system (PS/ODN), (d) treated by monotargeting system with HA, (e) treated by monotargeting system with MCMC, and (f) treated by dual-targeting system for 24 h.

evolved in the ERK and p38 pathways in J774A.1 cells stimulated with ODN delivery systems were detected (Figure 6). The treatment of ODN delivery systems results in enhanced phosphorylated ERK (P-ERK), as well as increased phosphorylated p38 (P-p38). Similarly to P-IκBα and NF-κB, the extent of enhancement of P-p38 and P-ERK is dependent on the delivery efficiency of the ODN delivery systems. Different MAPK pathways may have distinct effects on cytokine production. According to previous reports, p38 activation contributes to the proinflammatory cytokine expressions. However, ERK activation negatively regulates the

stimulate antitumor immune responses with minimized side effects, we designed the dual macrophage targeting nanovector to deliver ODN into macrophage specifically. The cell uptake study indicates the ODN accumulation in macrophage cells is much higher than that in the tumor cells. In addition to the NF-κB pathway, CpG ODN stimulation can also activate MAPK signaling pathways.9 Among the MAPK pathways, MAPK/extracellular regulated protein kinase (ERK), p38 MAPK, and MAPK/c-Jun N-terminal kinase pathways were reported to be involved in CpG ODN-induced activation in macrophages.9,11 Herein, the expression levels of the proteins 42572


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Figure 6. Western blot analysis on proteins involved in NF-κB and MAPK signal pathways in J774A.1 cells (a) without treatment, (b) treated by naked ODN, (c) treated by nontargeting system (PS/ ODN), (d) treated by monotargeting system with HA, (e) treated by monotargeting system with MCMC, and (f) treated by dual-targeting system for 24 h.

Figure 7. Western blot analysis of related protein expressions in MCF7 cells (a) without treatment, (b) treated by naked ODN, and (c) treated by dual-targeting system for 24 h.

totic function.64 More importantly, NF-κB activation mediated by CpG ODN is responsible for the upregulated Fas and Fas ligand (FasL).65 As a result, NF-κB may promote Fas/FasLmediated apoptosis in cancer cells. In our investigation, Fas is significantly upregulated and FasL is weakly increased after the ODN treatment. As is well known, Fas/FasL signaling pathway plays a vital part in cell apoptosis and immune response.66 The significantly enhanced Fas expression on the surface of MCF-7 cells treated by our ODN delivery system is beneficial for the elimination of tumor cells by immunological cells like cytotoxic T cells and natural killer cells through FasL-induced apoptosis. Most commonly, FasL-expressed activated immune cells can kill Fas-expressed cancer cells. Nevertheless, FasL-expressed tumor cells can also induce apoptosis of Fas-expressed immune cells.66,67 According to previous studies, the expressions of both Fas and FasL on tumor cells may induce tumor cell death through Fas/FasL-mediated apoptosis.67 In our study, the simultaneously expressed Fas and FasL in MCF-7 after ODN treatment would induce cell apoptosis. In this investigation, the obviously enhanced expression of caspases in tumor cells after the ODN treatment is most probably caused by the Fas/FasLmediated apoptosis.66 Undoubtedly, the effect of CpG ODN on cancerous cells is pleiotropic. Upon ODN stimulation, the upregulated proteins may have contradictory effects on cell growth and proliferation. As an overall result, our ODN delivery systems do not show apparent effects on the viability of MCF-7 cells (Figure S7). Our observations provide a more inclusive view on CpG ODN in tumor treatments.

CpG ODN-mediated Th1-type response by suppressing IL-12 secretion and inducing IL-10 secretion in RAW264.7 cells.63 In the current study, the upregulation of proteins involved in p38 and ERK signal pathways (P-p38 and P-ERK, respectively) is observed. The cellular signal pathway analysis after the treatment of ODN delivery systems is consistent with the enzyme-linked immunosorbent assay (ELISA) test on cytokine productions, indicating that the upregulation of Th1 cytokines is dominant, whereas the enhanced production of a Th2 cytokine (IL-10) can also be detected, although the enhancement is limited. Influences of ODN Treatment on Tumor Cells. In the current study, we chose CD44 overexpressed MCF-7 cells as the typical tumorous cells to analyze the related protein expressions after ODN delivery by the dual macrophage targeting delivery system. The western blot analysis (Figure 7) shows the upregulated proteins including NF-κB, PRK3R3, pAkt, Fas, FasL, caspase 8, caspase 3, and caspase 7 after the treatment by naked ODN and the dual-targeting ODN delivery system. Compared with naked ODN with a limited regulation effect due to its poor cell internalization, MCMC/HA/PS/ ODN nanoparticles can exert the regulation more effectively. Clearly, the upregulation of NF-κB protein in tumorous cells is caused by the activated NF-κB signaling. The increased PIK3R3 and phosphorylated Akt (p-Akt) levels in treated MCF-7 tumor cells indicate the activation of PI3K/Akt signaling pathway. Based on previous studies, PIK3R3, a PI3K regulatory subunit, is favorable for tumor migration and invasion.18,19 NF-κB and PI3K/Akt are well-known signaling pathways that promote tumor proliferation and metastasis.18 In this respect, ODN have unfavorable effects. Even though NF-κB is identified as a critical promoter of cancer development, experimental data have also shown that NF-κB appears to have potential antioncogenic and proapop-

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CONCLUSIONS The decoration of two types of targeting components, MCMC with mannose ligands and HA, on the surface of the delivery vector of ODN can realize efficient macrophage targeting and significantly enhance the immune stimulation. By dramatically upregulating proinflammatory cytokines, the dual macrophage 42573


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ACS Applied Materials & Interfaces targeting delivery system can successfully shift the phenotype of J774A.1 macrophages to activated M1 polarity. ODN delivery does not exhibit apparent effects on the survival and growth of cancerous cells due to the contradictory effects of the activated NF-κB and PI3K/Akt signalings and upregulation of Fas/FasLmediated apoptosis.

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MATERIALS AND METHODS

Materials. Protamine sulfate, sodium hyaluronate with Mw = 5.0 × 104 g/mol, carboxymethyl chitosan with Mw = 9.1 × 104 g/mol, Lipofectamine 2000, and CpG ODN 1826 with and without Cy3 labeling were commercially available and used as received. The suppliers are provided in the Supporting Information (SI). Mannosylated carboxymethyl chitosan was synthesized by reacting carboxymethyl chitosan with α-D-mannopyranosylphenyl isothiocyanate (Scheme S1) as detailed in the SI.43,49 The molecular weight (Mw) of MCMC was 7.4 × 104 g/mol as determined by size-exclusion chromatography. Preparation and Characterizations of ODN Delivery Systems. The preparation of dual-targeting MCMC/HA/PS/ODN nanoparticles was as follows. PS/ODN complexes were formed by adding PS solution (15 μL) containing 30 μg PS into ODN solution (2 μL) containing 1 μg ODN slowly, followed by gentle stirring for about 10 min. The solution containing PS/ODN complexes was then diluted to obtain solution I (50 μL). Certain amounts of MCMC solution (2 mg/mL) and HA solution (2 mg/mL) were mixed and diluted to obtain solution II (50 μL) containing 15 μg of MCMC and 15 μg of HA unless otherwise specified. Then, MCMC/HA/PS/ODN nanoparticles (in 100 μL of aqueous medium) were formed by adding solution II into solution I followed by gentle stirring for about 10 min. For comparison, nontargeting ODN-loaded nanoparticles and monotargeting ODN-loaded nanoparticles were also fabricated. The preparation details of these nanoparticles and Lipofectamine 2000/ ODN complexes are provided in the SI. The characterizations of the ODN-loaded nanoparticles are detailed in the SI. Study on Immune Stimulation. The secretion of cytokines in J774A.1 cells treated by the ODN delivery systems was assessed by ELISA. The experimental details are provided in the SI. The expressions of CD80 and CD206 in J774A.1 cells after being treated by different agents were evaluated by flow cytometry (FCM). The experimental details are provided in the SI. The expressions of CD80, CD206, and the proteins involved in NFκB and MAPK pathways in J774A.1 cells, as well as the apoptosisrelated proteins and the proteins involved in PI3K/Akt and Fas/FasL pathways in cancerous MCF-7 cells, were studied by western blot assay. The experimental details are provided in the SI. The suppliers of antibodies used are listed in Table S5.

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ent MCMC/HA amounts; CLSM observation and flow cytometry on MCF-7 cells co-incubated with ODN delivery systems; CD44 expressions in J774A.1 cells and MCF-7 cells; CD80 and CD206 in J774A.1 cells determined by western blotting; viability of MCF-7 cells treated by ODN delivery systems (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Si-Xue Cheng: 0000-0001-9611-4421 Author Contributions

All of the authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by National Natural Science Foundation of China (51533006). ABBREVIATIONS ODN, oligodeoxynucleotides; HA, hyaluronan; MCMC, mannosylated carboxymethyl chitosan; CMC, carboxymethyl chitosan; PS, protamine sulfate; SEC, size-exclusion chromatography; FCM, flow cytometry; CLSM, confocal laser scanning microscopy; ELISA, enzyme-linked immunosorbent assay; TLR9, toll-like receptor 9; NF-κB, nuclear transcription factor-κB; MAPK, mitogen-activated protein kinase; IRAK/ TRAF6, IL-1 receptor-activated kinase/tumor necrosis factor receptor-associated factor 6; MyD88, myeloid differentiation factor 88; IκB, inhibitor of NF-κB; ERK, extracellular regulated protein kinases; PI3K, phosphoinositide 3-kinase; Akt, protein kinase B

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REFERENCES

(1) Mellman, I.; Coukos, G.; Dranoff, G. Cancer Immunotherapy Comes of Age. Nature 2011, 480, 480−489. (2) Wynn, T. A.; Chawla, A.; Pollard, J. W. Macrophage Biology in Development, Homeostasis and Disease. Nature 2013, 496, 445−455. (3) Ostuni, R.; Kratochvill, F.; Murray, P. J.; Natoli, G. Macrophages and Cancer: From Mechanisms to Therapeutic Implications. Trends Immunol. 2015, 36, 229−239. (4) Yuan, A.; Hsiao, Y. J.; Chen, H. Y.; Chen, H. W.; Ho, C. C.; Chen, Y. Y.; Liu, Y. C.; Hong, T. H.; Yu, S. L.; Chen, J. J.; Yang, P. C. Opposite Effects of M1 and M2 Macrophage Subtypes on Lung Cancer Progression. Sci. Rep. 2015, 5, No. 14273. (5) Komohara, Y.; Fujiwara, Y.; Ohnishi, K.; Takeya, M. TumorAssociated Macrophages: Potential Therapeutic Targets for AntiCancer Therapy. Adv. Drug Delivery Rev. 2016, 99, 180−185. (6) Singh, Y.; Pawar, V. K.; Meher, J. G.; Raval, K.; Kumar, A.; Shrivastava, R.; Bhadauria, S.; Chourasia, M. K. Targeting Tumor Associated Macrophages (TAMs) via Nanocarriers. J. Controlled Release 2017, 254, 92−106. (7) 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, No. 16632. (8) Tariq, M.; Zhang, J.; Liang, G.; Ding, L.; He, Q.; Yang, B. Macrophage Polarization: Anti-Cancer Strategies to Target TumorAssociated Macrophage in Breast Cancer. J. Cell. Biochem. 2017, 118, 2484−2501.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.7b13594. Experimental; study on assembly efficiency; observation on intracellular delivery of ODN; determination of CD44 on J774A.1 macrophages and MCF-7 cells; ODN encapsulation efficiency of PS/ODN nanoparticles with different amounts of PS; size, ζ-potential, and ODN encapsulation efficiency of HA/PS/ODN nanoparticles with different amounts of HA and MCMC/PS/ODN nanoparticles with different amounts of MCMC; synthesis of MCMC; TEM images of the nanoparticles; flow cytometric analysis on cell uptake of Cy3-labeled ODN in J774A.1 cells treated by monotargeting systems with different HA amounts and different MCMC amounts as well as MCMC/HA/PS/ODN nanoparticles with differ42574


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