Micelles are one of the most investigated nanocarriers for drug delivery. In this study, polymeric micelles based on chitosan were prepared to explore the delivery mechanism which was critical for enhancing tumor targeting but still remain elusive.
Carbohydrate Polymers 229 (2020) 115435 Contents lists available at ScienceDirect Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol Selective uptake of chitosan polymeric micelles by circulating monocytes for enhanced tumor targeting T Xiqin Yanga, Keke Liana, Yanan Tanb, Yun Zhub, Xuan Liua, Yingping Zenga, Tong Yua, Tingting Menga, Hong Yuana, Fuqiang Hua,⁎ a b College of Pharmaceutical Science, Zhejiang University, 866 Yuhangtang Road, Hangzhou 310058, People’s Republic of China Ocean College, Zhejiang University, Zheda Road, Zhoushan 316021, People’s Republic of China A R T I C LE I N FO A B S T R A C T Keywords: Chitosan polymeric micelles Delivery mechanism Circulating monocytes Tumor targeting Micelles are one of the most investigated nanocarriers for drug delivery In this study, polymeric micelles based on chitosan were prepared to explore the delivery mechanism which was critical for enhancing tumor targeting but still remain elusive The chitosan polymer COSA was synthesized and the polymeric micelles showed good self-assembly ability, good dispersion stability and low toxicity After being intravenously administered, the micelles were selectively taken up by circulating monocytes in a receptor-mediated way (almost 94% uptake in Ly-6Chi monocytes, below 7% in all other circulating cells) and reach the tumor with the subsequent travel of these cells In addition, the micelles in macrophages (differentiated from circulating monocytes) can be exocytosed and subsequently taken up by cancer cells The delivery mechanism of COSA micelles is directional for the novel strategies to enhance tumor targeting and the micelles are promising candidates for diseases in which monocytes are directly implicated Introduction In the past decades, nanotechnology is a promising approach for drug delivery in cancer therapy Unfortunately, the limited therapeutic efficacy is a trend found across many nanoparticle formulations (Bertrand, Wu, Xu, Kamaly, & Farokhzad, 2014) To improve cancer treatment, worldwide attention is captured on engineering a myriad of nanocarriers to settle some of great problems in cancer therapy (Bhushan, 2015; Kaounides, Yu, & Harper, 2007) One such examples is micelles Micelles have the advantage of a stealth shell-hydrophobic core structure, which is capable of encapsulating a variety of waterinsoluble drugs without altering their chemical structures (Gref et al., 1994; Houdaihed, Evans, & Allen, 2017) Therefore, micelles have been explored as one of the main nanocarriers for cancer nanomedicine aimed at delivering drugs to tumors (Cabral et al., 2011; Eetezadi, Ekdawi, & Allen, 2015; Elvin, Haifa, & Mauro, 2015) Chitosan (CO), as a kind of natural carbohydrate polysaccharides, has attracted much attention as an excipient for the preparation of micelles due to the desirable properties like bioavailability, non-toxicity, biodegradability, stability, and affordability Stearic acid (SA), which are compatible with the cellular membrane, are good for promoting cellular uptake With these in mind, the chitosan polymeric micelles (COSA), which combine the advantages of CO and SA, were constructed and expected to show great potential as nanocarriers in cancer therapy Unexpectedly, after being intravenously administered, a considerable proportion of COSA micelles were delivered to the center of tumor which was frequently the hypoxic/necrotic regions and rendered inaccessible for nanoparticles delivered through the typical mechanism (blood vessels leakiness) (Choi et al., 2007; Owen et al., 2011) In addition, COSA micelles were mainly accumulated in macrophages For this reason, we chose to reveal the COSA micelles delivery mechanism which was critical for overcoming the delivery obstacles but still remain elusive At present, micelles are assumed to be delivered via several targeting mechanisms, particularly extravasation It is clear that blood cells including monocytes, macrophages and dendritic cells express glycoprotein receptors such as mannose receptors, Dectin receptors, Toll-like receptor and (Liu & Zeng, 2013; Macri, Dumont, Johnston, & Mintern, 2016) While chitosan, as a cationic polysaccharide, can bind with the glycoprotein receptors expressed in blood cells, resulting ⁎ Corresponding author E-mail addresses: yangxq@zju.edu.cn (X Yang), coco_lian@yeah.net (K Lian), tanyanan@zju.edu.cn (Y Tan), zhuyun@zju.edu.cn (Y Zhu), pupliuxuan@zju.edu.cn (X Liu), zengyp@zju.edu.cn (Y Zeng), 21819010@zju.edu.cn (T Yu), mengtt@zju.edu.cn (T Meng), yuanhong70@zju.edu.cn (H Yuan), hufq@zju.edu.cn (F Hu) https://doi.org/10.1016/j.carbpol.2019.115435 Received September 2019; Received in revised form 26 September 2019; Accepted October 2019 Available online 04 October 2019 0144-8617/ © 2019 The Authors Published by Elsevier Ltd This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/) Carbohydrate Polymers 229 (2020) 115435 X Yang, et al 4.0 cm−1 Dynamic light scattering (DLS) was used to determine the particle size and zeta potential The transmission electron microscopy (TEM, JEM-1230, JEOL) was used to observe the morphology of micelles Pyrene was used as a probe, and fluorescence spectroscopy was used to determine the critical micelle concentrations (CMC) of COSA The substitution degree of amino groups was also measured by the TNBS method as previously described (Hu, Zhang, You, Yuan, & Du, 2012) in the endocytic of chitosan based nanocarriers by these cells (Chen, 2015; Seferian & Martinez, 2000) Besides, following recruitment to tissues, circulating monocytes can differentiate into macrophages within the tissues (Frederic et al., 2010; Jakubzick, Randolph, & Henson, 2017; Warren & Vogel, 1985) Inspired by these facts, we hypothesized that monocytes in blood took up COSA micelles and deposited them in the tumor In this study, the characterizations of COSA micelles were investigated The distribution of COSA micelles in tumor was analyzed and the amount of tumor macrophages that internalized micelles was quantified To reveal the delivery mechanism that directed COSA micelles accumulation in the tumor, the interaction between COSA micelles and blood cells was explored Particularly, the process by which COSA (accumulated in monocytes-derived macrophages) reached cancer cells were further investigated 2.4 Dispersion stability of COSA The stability of polymeric micelles in serum or in different temperatures was investigated by determining the particle size of micelles (Lu, Owen, & Shoichet, 2011; Tan et al., 2019) Briefly, COSA micelles in deionized water at the concentration of 0.5 mg mL−1 were prepared To investigate the stability of COSA micelles in serum, the prepared micelles aqueous solution was supplemented with 10% fetal bovine serum (FBS, v/v), and then the particle size of micelles was determined by DLS at predetermined time points To investigate the stability of COSA micelles in different temperatures, the prepared micelles aqueous solution were stored at °C, 25 °C and 37 °C, and then the particle size of micelles in different temperatures was determined by DLS at predetermined time points In addition, to investigate the stability of drug-loaded micelles, Doxorubicin base (DOX) was used as the model drug to test the in vitro drug release from micelles in phosphate buffered saline (PBS, pH 7.4) To obtain DOX-loaded micelles, mg/mL of DOX/DMSO was added dropwise into a mg/mL COSA aqueous solution and stirred for h Then, the mixture solution was dialyzed in DI water overnight and then centrifuged at 8000 rpm for 10 The supernatant was collected as the COSA/DOX micelles To investigate the in vitro release of DOX from COSA/DOX micelles, the COSA/DOX micelles was dialyzed against PBS in an incubator shaker with horizontal shaking (75 rpm) at 37 °C COSA/DOX aqueous solution (1.0 mL) was dialyzed in 20.0 mL of PBS (MWCO: 3.5 kDa) At predetermined time points, all of the medium outside of the dialysis bag was acquired and replaced with fresh PBS The DOX concentration of all the samples was determined with a fluorescence spectrophotometer, and the assays were repeated three times Materials and methods 2.1 Reagents 95% deacetylated chitosan (CO, Mw = 450 kDa, Yuhuan, China) was degraded with enzymes to acquire low molecular weight CO (Mw = 19.9 kDa) Stearic acid (SA) and D-mannose were supplied by Shanghai Chemical Reagent Co, Ltd 1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide (EDC) were purchased from Shanghai Medpep Co, Ltd Fluorescein isothiocyanate (FITC), 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT), β-glucan, Lipopolysaccharide (LPS) and 2,4,6-trinitrobenzenesulfonic acid (TNBS) were obtained from Sigma-Aldrich Inc 1, 1′-dioctadecyl-3, 3, 3′, 3′-tetramethyl indotricarbocyanine iodide (DiR) was obtained from Life Technologies (Carlsbad, CA, USA) PE-labeled anti-Gr-1, PE/Cy7-labeled anti-F4/80, APC-labeled anti-Ly6C, anti-αvβ3 and Percp/Cy5.5-labeled anti-CD11b were purchased from Biolegend (San Diego, CA) Other chemicals used were of chromatographic grade or analytical grade 2.2 Cell culture and animals 4T1 and RAW264.7 cells were purchased from the Cell Bank of Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China) and cultured in DMEM supplemented with 10% fetal bovine serum (FBS, v/v), 10000 U mL−1 streptomycin and 10000 U mL−1 penicillin at 37 C in a humidified incubator with 5% CO2 6–8 week-old female BALB/c mice were purchased from the Shanghai Silaike Laboratory Animal Limited Liability Company All animal experiments were carried out in compliance with the Zhejiang University Animal Study Committee’s requirements for the care and use of laboratory animals in research 2.5 The distribution of COSA in tumor To prepare the tumor-bearing mice models, × 105 4T1 cells was implanted to the right mammary gland of 6–8 week-old female BALB/c mice To detect the in vivo distribution of COSA micelles, near infrared dye DiR was encapsulated in COSA micelles according to the preceding protocol DiR loaded COSA micelles were intravenously injected into the tail vein of tumor bearing BALB/c mice The mice were imaged at predetermined time points by a Maestro in vivo Imaging System (CRI Inc., Woburn, MA) After 24 h, the mice were sacrificed, followed by collection of heart, liver, spleen, lung, kidney and tumor The fluorescence images of these tissues were obtained by using a Maestro in vivo Imaging System (CRI Inc., Woburn, MA) To investigate the distribution of COSA in tumor, FITC labeled COSA was prepared as previously described (Zhu et al., 2018) Briefly, 2.0 mg/mL FITC (C21H11NO5S) ethanol solution was added dropwise into 1.0 mg/mL COSA aqueous solution (COSA: FITC = 1:1, mol: mol), then kept stirring overnight and dialyzed (MWCO = 000 Da) against pure water After 24 h of injection of FITC-COSA micelles, the tumors were collected Then the tumors were sectioned and stained with antibodies to examine by confocal laser scanning microscopy 2.3 COSA polymer synthesis and characterization The COSA polymer was synthesized in the presence of EDC Briefly, 20 mL of ethanol was used to dissolve stearic acid (SA) and EDC, and the mixed solution was stirred for h at 60 °C Then, 20 mL of deionized water (DI water) was used to dissolve 0.3 g CO, and the solution was incubated at 60 °C for 20 Then, the mixed solution was added into the CO solution After stirring for another 14 h, the reaction solution was collected and dialyzed against DI water for days Finally, the products were collected by lyophilization after three purifications with ethanol H NMR spectroscopy was used to elucidate the structure of COSA Briefly, mg COSA was dissolved in 0.5 mL D2O The samples were measured by 1H NMR spectrometer (AC-80, Bruker Biospin, Germany) In addition, fourier-transform infrared spectroscopy (FTIR) was used to confirm the structure of COSA The samples were sliced by KBr tableting and examined using a Bruker Tensor 27 model infrared spectrometer with a scan range of 400–4000 cm–1 and a resolution of 2.6 Quantification of macrophages that internalized COSA micelles To quantify the accumulation of FITC-COSA in the tumor cell subsets, tumors were isolated at h, 18 h and 24 h after intravenous Carbohydrate Polymers 229 (2020) 115435 X Yang, et al injection of FITC-COSA The samples were grinded and filtered using a cell strainer (Qin et al., 2018) The obtained single cell suspension was centrifuged at 350 g for 10 at °C The cell pellets were washed using PBS buffer Before antibody labeling, all the cells were pre-incubated with anti-CD16/CD32 mAb Then cells were labelled with the antibodies (anti-αvβ, PE/Cy7-labeled anti-F4/80 and Percp/Cy5.5-labeled anti-CD11b) and analyzed by flow cytometry COSA/Fe2O3 Cells were exposed to COSA/Fe2O3 for different duration and collected, washed twice with PBS and centrifuged at 350 × g for 10 The cells were pre-fixed with formaldehyde overnight and then dehydrated with increasing concentrations of ethanol (50, 60, 70, 80, 90, and 100%) for 15 each, and stained with 2% uranyl acetate in 70% ethanol overnight, then embedded in Epon Ultrathin sections of macrophages were cut using a sliding ultramicrotome and the thin sections were supported by copper grids and observed using a TEM system 2.7 The interaction between COSA micelles and blood cells Mouse whole blood from eyeball was drawn into tubes containing heparin sodium at h, 18 h and 24 h after intravenous injection of FITC-COSA Red blood cell lysis buffer (Biolegend, San Diego, CA) was added to tubes and vortexed for several seconds After incubated 15 at room temperature, blood samples were centrifuged at 350×g for 10 Before antibody labeling, the remaining immune cells were preincubated with anti-CD16/CD32 mAb and then stained with specific antibodies (PE-labeled anti-Gr-1, APC-labeled anti-Ly6C and Percp/ Cy5.5-labeled anti-CD11b) for flow cytometry analysis 2.11 Cancer cells uptake of COSA excreted by macrophages Three-dimensional (3D) models were used to investigate whether the excreted COSA would indeed be taken up by cancer cells To construct 3D models, 4T1 cells, RAW264.7 cells and 3T3 cells were mixed at the same number × 105 mixed cells were seeded in 96-well plates pretreated with 2% agarose to construct the cell spheres as previously described (Yang et al., 2018) After 3days, the cell spheres were divided into two groups One group was added with FITC-COSA and incubated for different duration Another group was treated with FITC-COSA for 24 h and washed with PBS and then incubated with fresh culture medium (without FITCCOSA) for different duration The group without culture medium replacement was used as control All the cell spheres were collected at determined time points and digested with enzymes to obtain single cell suspension Then the cells were pre-incubated with anti–CD16/CD32 mAb and labelled with antibodies (anti-αvβ, PE/Cy7-labeled anti-F4/ 80 and Percp/Cy5.5-labeled anti-CD11b) for flow cytometry analysis 2.8 Cellular uptake mechanisms To reveal the cellular uptake mechanisms of COSA, monocytes from peripheral blood were isolated according to the previous protocol (Macparland et al., 2017) Whole blood was collected into tubes containing heparin sodium Peripheral blood mononuclear cells (PBMC) were isolated from blood by gradient centrifugation in Ficoll-paque Plus (GE Healthcare) To purify monocytes from PBMC, the negative-selection-based monocyte isolation kit II (Miltenyi Biotec, Auburn, CA) was used, and then the purified monocytes were suspended in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin The collected monocytes were respectively pre-incubated with specific ligands (mannose, β-glucosan and lipopolysaccharide) of glycoprotein receptors (mannose receptors, Dectin receptors and Tolllike receptor and 4) After removal of inhibitor solutions, cells were washed twice with PBS and then incubated with 20 μg/mL of FITCCOSA for another 10 The cells without inhibitor treatment were used as control The cellular uptake of FITC-COSA was measured by flow cytometry 2.12 Statistical analysis All the data were reported as mean ± SD Differences between groups were tested using the two-tailed student’s t-test The differences with p < 0.05 were considered statistically significant Results and discussion 3.1 The synthesis and characteristics of COSA The polymer COSA was synthesized by reactions between chitosan (CO) amine groups and stearic acid (SA) carboxyl groups in the presence of EDC (Fig 1A) The COSA structure was confirmed based on the H NMR and FTIR spectroscopy As shown in Fig 1B, new peaks at approximately 1.00 ppm indicated the synthesis of COSA In addition, the absorption peaks in FTIR spectra at about 3091 cm−1, 1645 cm−1, 1521 cm−1 and 1288 cm−1 which resulted from amide bonds (eCONHe) indicated the amidation reaction between CO and SA (Fig 1C) The particle size and zeta potential of COSA were investigated (Fig and Table 1) The average size of COSA was determined as 85.93 ± 0.68 nm and COSA micelles showed positive zeta potential (23.17 ± 0.90 mv) which was good for cellular uptake In addition, the spherical morphology of micelles was presented in the TEM images (Fig 2A) The degree of amino substitution (SD%) of COSA was measured as 7.03% (Table 1) and the polymer COSA could self-assemble into nano-scaled micelles Fig 2B showed the variation of the I1/I3 ratio against the logarithmic concentration (Log C) of COSA The inflection point corresponded to the critical micelle concentration (CMC) value which was 52.02 μg/mL The relatively low CMC values indicated the good self-assembly ability and structural stability of COSA micelles The stability of polymeric micelles in serum is critical for in vivo applications (Cao et al., 2013) Therefore, the particle size of COSA micelles in solution with 10% serum (v/v) was investigated The adsorption of blood proteins onto the micelles surface can contribute some changes in particle size of micelles and the protein-particle interactions can increase the particle size by 3–35 nm (Dobrovolskaia et al., 2009; 2.9 Cytotoxicity evaluation The cytotoxicity of COSA was evaluated by MTT assay (Cheng et al., 2017) Briefly, × 103 monocytes were seeded in 96-well plates After 12 h incubation, a series of concentrations of COSA were added to the cells and co-cultured for another 48 h 15 μL MTT solution (5 mg/mL) was added to each well and incubated for another h After removing the medium, the cells were incubated with 200 μL Dimethyl sulfoxide (DMSO) in an automated shaker Finally, the absorbance of each well at 570 nm was read by an automatic reader (BioRad, Model 680, USA) 2.10 Cellular uptake and exocytosis of COSA To determine in vitro cellular uptake, × 105 RAW264.7 cells were seeded in 6-well plates and incubated in 5% CO2 for h Different concentration of FITC-COSA micelles were added to each well and incubated with cells Then the cells were rinsed with PBS, collected and analyzed by flow cytometry Transmission electron microscope (TEM) was used to investigate the cellular uptake and exocytosis of COSA Firstly, Fe2O3 loaded micelles were prepared as previous description (Tan et al., 2017) Briefly, Fe2O3 nanoparticles solution (5 nm, mg/mL in oleic acid) was added to COSA micelles under being sonicated with a probe type ultrasonicator (JY92-II, Ningbo scientz biotechnology Co., Ltd., China) at 100 W for 30 The solution was centrifuged at 1500×g for 20 to obtain Carbohydrate Polymers 229 (2020) 115435 X Yang, et al Fig Synthesis of COSA (A) The synthetic scheme of COSA (B) The 1H NMR spectra of COSA (C)The FTIR spectra of COSA obvious fluorescent signals, which indicated the ability of COSA micelles to accumulate at the tumor site To effectively kill cancer cells, the drug delivery system should be targeted to cancer cells In order to investigate whether the COSA micelles were exactly delivered to cancer cells, the tumors were collected for immunofluorescent analysis at 24 h after intravenous administration of COSA As shown in Fig 4A, a considerable proportion of micelles were delivered to the center of tumor which is frequently the hypoxic/ necrotic regions and rendered inaccessible for nanoparticles delivered through the typical mechanism (blood vessels leakiness) In addition, macrophages (in red) showed much more overlaps with micelles (in green) compared with cancer cells As shown in Fig 4B, up to 68.10% of tumor macrophages internalized COSA at 24 h While as the targeted cells, cancer cells did not actively interact with micelles and the proportion was less than 3% which can be negligible when compared with macrophages The result indicated that macrophages were the key cells in the sequestration of intravenously administrated micelles Hak Soo et al., 2007; Monopoli et al., 2011) As shown in Fig 3, the particle size of COSA micelles in solution with serum was larger than that without serum, which resulted from the interactions between micelles and blood proteins As expected, with or without serum, there was no obvious change in COSA micelle diameters at different time points Besides, the stability of COSA in different temperatures was also tested As shown in Fig.S1, no obvious change of the micelle particle size was observed in different temperatures, which also indicated the stability of COSA micelles In addition, to investigate the stability of drug-loaded micelles, DOX was chosen as the model drug to test the in vitro drug release from COSA/DOX micelles in PBS (pH 7.4) The in vitro drug release curves in Fig S2 showed that less than 25% of DOX was released from COSA/DOX micelles after 72 h, illustrating that the drug-loaded micelles were very stable under physiological conditions All these results suggested the good dispersion stability of COSA which was desirable for in vivo applications as nanocarriers 3.2 Distribution of COSA in tumor 3.3 Selective uptake of COSA by circulating monocytes To effectively inhibit tumor growth, drug delivery system should be targeted to tumors The in vivo distribution of COSA micelles was macroscopically investigated As shown in Fig S3, the tumor showed The hypoxic and necrotic regions of tumor are accessible for monocytes (Anselmo et al., 2015; Murdoch, Giannoudis, & Lewis, 2004) Fig Characterizations of COSA (A)The size distribution and transmission electron microscopy (TEM) image of COSA The image represented one of three experiments with similar results (B)The critical micelle concentration (CMC) of COSA Carbohydrate Polymers 229 (2020) 115435 X Yang, et al Table Characterizations of COSA Micelles Diameter (nm) PDI Zeta potential (mv) CMC (μg/mL) SD% COSA 85.93 ± 0.68 0.13 ± 0.04 23.17 ± 0.90 52.02 ± 5.67 7.03 ± 0.84 Data represent the mean ± standard deviation (n = 3) Jakubzick et al., 2017; Warren & Vogel, 1985) It was found that a considerable proportion of COSA micelles were delivered to the center of tumor and the micelles were mainly accumulated in macrophages Inspired by these facts, we hypothesized that circulating monocytes took up COSA micelles and deposited them in tumor Intrigued by the hypothesis, we used highdimensional 14-parameter (12-colour) FACS analysis to identify the subset(s) of immune cells that took up COSA in blood Interestingly, of all the myeloid cells, only a single monocyte subset—Ly-6Chi monocytes—displayed substantial COSA uptake As shown in Fig 5, up to 94.30% of CD11b—Ly-6Chi monocytes took up COSA micelles at 18 h after intravenous injection In contrast, neutrophils, which expressed higher levels of surface CD11b and Gr-1, took up negligible amounts of COSA (less than 2% of neutrophils took up the micelles at 18 h) Similarly, the cellular uptake of COSA by other circulating white blood cells was also negligible in comparison with Ly-6Chi monocytes (Fig S4) The result demonstrated that COSA micelles can be internalized by circulating cells in blood and showed high selectivity for Ly-6Chi monocytes It was confirmed that COSA micelles were selectively taken up by circulating monocytes, which would raise the question: what was the mechanism of cellular uptake by circulating monocytes? To answer the question, we investigated the mechanism of cellular uptake and found Fig Particle size of COSA micelles with/without serum at different time points Data was expressed as mean ± standard deviation (n = 3) but inaccessible for nanoparticles delivered through blood vessels leakiness (Choi et al., 2007; Owen et al., 2011) Tumors can generate molecular gradients that attract circulating monocytes which can differentiate into macrophages within the tissues (Frederic et al., 2010; Fig Macrophages internalized COSA micelles (A) Immunofluorescent staining of tumor COSA was labeled by FITC (green), cell nucleus were labeled by DAPI (blue), macrophages were labeled by anti-F4/80 antibodies (red) and cancer cells were labeled by anti-αvβ3 antibodies (red) The images represented one of three experiments with similar results (B) Flow cytometry plots showing FITC-COSA selective accumulation in tumor macrophages Flow cytometry plot data were representative of n = mice per group (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) Carbohydrate Polymers 229 (2020) 115435 X Yang, et al Fig Selective uptake of FITC-COSA by circulating monocytes Flow cytometry plots showing selective uptake of FITC-COSA into Ly-6Chi monocytes Blood was harvested at h, 18 h and 24 h after intravenous injection of FITC-COSA and stained with specific antibodies for flow cytometry analysis Flow cytometry plot data were representative of n = mice per group Fig The cellular uptake of COSA by monocytes (A) The cellular uptake of COSA by monocytes “F” represents FITC-COSA, “M” represents mannose, “L” represents lipopolysaccharide (LPS) and “β” represents β-glucan Data was expressed as mean ± standard deviation (n = 3) (*p < 0.05, **p < 0.01) (B) In vitro cytotoxicity against monocytes after treatment with COSA for 48 h Data was expressed as mean ± standard deviation (n = 6) selectively taken up by circulating monocytes and reach the tumor with the subsequent travel of these cells The delivery mechanism was independent of the blood vessels leakiness, and could afford new strategies to improve tumor targeting by increasing monocyte homing to tumors In addition, monocytes can easily enter and travel throughout tumors Thus, the hypoxic and necrotic regions of tumor, which were rendered inaccessible for nanoparticles delivered through blood vessels leakiness, can now be reached with this delivery mechanism Moreover, we quantified the monocytes in tumor and found that monocytes continued to home to tumor over time (Fig 7B) To investigate whether this mechanism (targeting tumors via monocyte) has a substantial effect on the total amount of COSA accumulating in tumor, we assessed the effect by computing the proportion of COSA in tumor contained within monocytes and comparing this with the total amount of COSA in the tumor (which may arrive as a consequence of other targeting mechanisms such as extravasation (Smith et al., 2013)) As shown in Fig 7C, ∼41% of COSA in tumor on day were due to monocyte delivery and the proportion was increased to nearly 50% on day 2, which suggested that this delivery mechanism can account for a considerable proportion of COSA delivered to tumors that the cellular uptake of COSA was obviously inhibited by mannose, especially in the group pretreated with mannose and β-glucan simultaneously (Fig 6A) The result indicated that COSA micelles can be internalized mainly by mannose receptor-mediated mechanism and secondly by Dectin receptor-mediated mechanism Beside, we investigated the cytotoxicity of COSA against monocytes with MTT assay The result revealed that COSA micelles showed low toxicity to monocytes (Fig 6B), which can ensure the intrinsic homing property of monocytes 3.4 The delivery of COSA to tumor by circulating monocytes After confirming that COSA can be selectively took up by circulating monocytes in a receptor-mediated way, we then investigated whether monocyte would indeed deposited COSA in tumor When COSA micelles were intravenously injected, APC-labeled anti-mouse Ly-6C Abs (a specific marker of monocytes) was subcutaneously injected around the tumor to stain monocytes homing to tumor from the blood (Chu, Dong, Zhao, Gu, & Wang, 2017) As shown in Fig 7A, monocytes (in red) showed lots of overlaps with micelles (in green), which indicated that monocytes can home to tumor after taking up COSA in blood It can be concluded that the intravenously administered micelles were Carbohydrate Polymers 229 (2020) 115435 X Yang, et al Fig COSA-laden monocytes enter the tumor (A) Immunofluorescent staining of tumors COSA was labeled by FITC (green), cell nucleus were labeled by DAPI (blue) and monocytes were labeled by anti-Ly6C antibodies (red) (B) Monocytes accumulating in tumors (C) Relative amounts of COSA that were ferried in the tumor via monocytes (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) we then used 3D cell models to further investigate whether cancer cells would took up COSA exocytosed by macrophages One group of cell spheres was pretreated with COSA for 24 h and then the exposure solution was replaced with fresh culture medium (without COSA) The group of cell spheres, without culture medium replacement, was used as control As shown in Fig 8C, the total amount of macrophages taking up COSA in control group (the first row) was increased within 24 h and after that it was decreased While in another group (the second row), the amount of macrophages taking up COSA was decreased over time, which resulted from the continued exocytosis In contrast, the situation was different for cancer cells (Fig 8D) In control group (the first row), the total amount of cancer cells that took up COSA showed continued increase during 48 h, although much lower than that of macrophages Similarly, the amount in another group (the second row) also increased over time The uptake of COSA by cancer cells in control group resulted from remaining COSA in culture medium or the exocytosed COSA by macrophages Once replacing the exposure solution with fresh culture medium without COSA, the increased uptake by cancer cells was only due to the exocytosed COSA It can be concluded that macrophages can exocytose the internalized COSA and the excreted COSA would be taken up by cancer cells This interaction between macrophages and cancer cells showed great significance and could demonstrate novel ways to influence cancer cells for cancer therapy 3.5 The interaction between macrophages and cancer cells COSA micelles were selectively taken up by circulating monocytes Subsequently, the COSA-loaded monocytes were recruited to tumors and became the source of tumor-infiltrating macrophages We then asked how COSA micelles which located in macrophages were delivered to cancer cells to realize therapeutic efficacy Based on previous study, macrophages were able to exocytose the internalized nanoparticles (Jiang et al., 2017; Oh & Park, 2014) Accordingly, we hypothesized that the internalized COSA could be excreted by macrophages First, flow cytometry was used to investigate the COSA endocytosis of macrophages exposed to 5, 10, 20 and 40 μg/mL COSA As shown in Fig S5, the cellular uptake became saturated at the COSA concentration of 20 μg/mL Then, we further determined the cellular uptake of COSA over time in 20 μg/mL COSA exposure It was surprising that the amount of cells taking up COSA obviously decreased at 36 h and 48 h in comparison with that at 24 h (Fig S6A) These data, although limited, partly suggested the occurrence of COSA exocytosis In addition, transmission electron microscope (TEM) images revealed the endocytosis process of COSA, which began with the interaction between COSA and cell membrane (Fig 8A) However, when we replaced the exposure solution with fresh culture medium (after h exposure), the amount of cells that took up COSA significantly decreased with prolonged incubation time (Fig S6B) and the fluorescence intensity of the culture medium increased over time (Fig S7), which suggested the occurrence of COSA exocytosis by macrophages Moreover, the TEM images also showed that the cells exocytosed internalized contents via vesicle-related secretion (Fig 8B) Having shown that macrophages would excrete internalized COSA, Conclusions In summary, the chitosan polymer COSA was synthesized and the polymeric micelles showed good self-assembly ability, good dispersion stability and low toxicity After being intravenously administrated, the Carbohydrate Polymers 229 (2020) 115435 X Yang, et al Fig Cancer cells uptake of COSA secreted by macrophages (A) Typical TEM images showing endocytosis (B) Typical TEM images showing cellular exocytosis The images represented one of three experiments with similar results (C) Cellular uptake of COSA by macrophages at different time points (D) Cellular uptake of COSA by cancer cells at different time points Gating strategy: cells taking up COSA were identified first from all the cells based on FITC staining Macrophages were then identified from FITC+ cells by F4/80+ gating and cancer cells were identified from FITC+ cells by αvβ3+ gating Flow cytometry plot data represented one of three experiments with similar results to tumors In addition, the internalized COSA can be exocytosed by macrophages and then taken up by cancer cells This interaction between macrophages and cancer cells would demonstrate novel ways to influence cancer cells for cancer therapy Overall, the delivery mechanism identified in this work is directional for enhancing tumor COSA micelles were selectively taken up by nearly 94% of circulating monocytes (Ly-6Chi monocytes) in a receptor-mediated way The subsequent travel of these cells resulted in a considerable proportion of COSA accumulation in tumor This delivery mechanism can afford new strategies to improve tumor targeting by increasing monocytes homing Carbohydrate Polymers 229 (2020) 115435 X Yang, et al targeting and the COSA micelles exhibited 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COSA by circulating monocytes To effectively inhibit tumor. .. (2020) 115435 X Yang, et al Fig Selective uptake of FITC-COSA by circulating monocytes Flow cytometry plots showing selective uptake of FITC-COSA into Ly-6Chi monocytes Blood was harvested at... directional for enhancing tumor COSA micelles were selectively taken up by nearly 94% of circulating monocytes (Ly-6Chi monocytes) in a receptor-mediated way The subsequent travel of these cells