Conjugated Polymer Brush Based on Poly(L-lysine) with Efficient

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Conjugated Polymer Brush Based on Poly(L-lysine) with Efficient Ovalbumin Delivery for Dendritic Cell Vaccine Chao Wang, Pengfei Sun, Gaina Wang, PengCheng Yuan, Rongcui Jiang, Wenjun Wang, Wei Huang, and Quli Fan ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.8b00496 • Publication Date (Web): 02 Nov 2018 Downloaded from http://pubs.acs.org on November 3, 2018

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ACS Applied Bio Materials

Conjugated Polymer Brush Based on Poly(L-lysine) with Efficient Ovalbumin Delivery for Dendritic Cell Vaccine Chao Wang, †, ※ Pengfei Sun, †, ※ Gaina Wang, † Pengcheng Yuan, † Rongcui Jiang, † Wenjun Wang, ‡ Wei Huang§ and Quli Fan*, †

†

Key Laboratory for Organic Electronics and Information Displays &Jiangsu Key

Laboratory for Biosensors, Institute of Advanced Materials (IAM), Jiangsu National Synergetic Innovation Center for Advanced Materials (SICAM), Nanjing University of Posts &

Telecommunications,

9

Wenyuan

Road,

Nanjing

210023,

China.

E-mail:

[email protected]; Fax: +86 25 8586 6533; Tel: +86 25 8586 6396 ‡ Key Lab of Optical Communication Science and Technology of Shandong Province & School of Physics Science and Information Engineering, Liaocheng University, Liaocheng 252059, China. § Shaanxi Institute of Flexible Electronics (SIFE), Northwestern Polytechnical University (NPU), 127 West Youyi Road, Xi'an 710072, Shaanxi, China

ACS Paragon Plus Environment

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KEYWORDS: Poly(L-lysine), water-soluble conjugated polymer brush; DC vaccine; cancer immunotherapy

ABSTRACT: Dendritic cell (DC)-based vaccines consist of antigens and antigen-presenting cells, such as DCs, that can induce antitumor immune response and extend the lives of patients. In this research, a water soluble conjugated polymer brush (WSCPB) made of poly(L-lysine) (PLL) and poly(p-phenyleneethynylene) (PPE) was applied to an antigen delivery system for the development of a DC vaccine. We synthesized the WSCPB with a lower proportion of the rigid PPE polymer backbone and a large amount of PLL side chains. The rigid backbone retained a stable optical performance within the experimental range, which enabled the visualization of the payload and cellular imaging as a reporter. Due to the unique brush-like structure, PPE-PLL exhibited not only excellent water solubility but also outstanding antigen-loading capacity. Ovalbumin (OVA), a model antigen in different researches, could be adsorbed onto PPE-PLL and then taken up by DCs. Subsequently, DC maturation and cytokine release would be induced by the antigen. In vivo, strong immune responses were induced after the injection of antigenpulsed DCs, and the level of cytokines in the serum was significantly increased. In addition, the study of the in vivo tumor-suppressor activity of these antigen-pulsed DCs revealed that the DC vaccine induced strong immune responses and thereby effectively inhibited tumor development.

INTRODUCTION


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Cancer has been one of the most serious dreaded diseases in recent years, and immunotherapy as an emerging means of cancer treatment has shown efficacy in the treatments of cancer.1-3 Immunotherapy using antigen-presenting cells is one of the main directions for tumor biotherapy, such as macrophages, B lymphocytes and dendritic cells (DCs). The application of DC vaccines has become an effective method of immunotherapy to activate the patient’s own immune function and bolster the anticancer response.4,

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DC is the mostly powerful antigen-

presenting cells and plays a key role in mammalian immune system. DCs send signals between innate and adaptive immune system by processing antigen materials and presenting the antigen to the T cells. Then DCs activate T cells and make them become various effector cells of the immune system.6-8 Activated effector helper T cells release different types of cytokines (interferon gamma (IFN-γ) and tumor necrosis factor) to stimulate the immune responses against cancer. Unfortunately, there are only a limited number of DCs in the human body, and the immune response can only be stimulated through the function of the proper antigen-presenting cells. To address these issues, activated DCs could be used in an antigen-pulsed DC vaccine for immunotherapy. It is found that DCs become activated into the mature state while DCs absorb a presentable antigen, and that mature DCs can activate naive and memory T cells. Ovalbumin (OVA), a main constituent of egg white, is a common antigen of activated DCs. However, in the endosomallysosomal pathway, free OVA enters DCs through macropinocytosis is often degraded, sequestered or exocytosed.9-11 Accordingly, an appropriate OVA delivery system is needed to ensure that the antigen enters the DCs. In recent years, various types of OVA delivery system have been developed by many research groups.12 One of them is the conjugation of ovalbumin with polymers or cell-penetrating peptides through chemical bonds (such as disulfide bonds and


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amide bonds), whereby the OVA is rapidly released in a specific trigger environment.13, 14 As the most commonly reported, another OVA delivery system is self-assembling nanoparticles, such as liposomes, micelles and nanogels. This method has various advantageous properties, such as the controlled size and easy processibility.15 In addition, cationic polymers, such as polyetherimide and poly(dimethylaminoethyl methacrylate), can be used to deliver anionic OVA through a strong electrostatic interaction to promote DC activation and maturation. Moreover, compared with negatively charged and neutral polymers, cationic polymers could induce the antigenspecific responses of cytotoxic T lymphocyte (CTL) and amplify immunity.16 However, these OVA delivery systems have some inherent drawbacks. For instance, the function of OVA could be affected after the chemical reactions, and the encapsulation in nanoparticles also has a relatively poor immunogenic capacity.15 Additionally, a high concentration of carriers is needed in practice due to the low loading capacity (LC), i.e., less than 10%, which limited their therapeutic effects. Since these delivery systems have no imaging ability, the real-time tracking cannot be realized. Thus, it is significative to invent an OVA delivery system with high protein LC and controlled release of OVA.17, 18 Water-soluble conjugated polymer brush (WSCPB) is a kind of polymer with a conjugated backbone and multi water-soluble side chains.19-21 WSCPBs not only exhibit strong lightharvesting property and excellent photostability but also show greater fluorescence quantum efficiency and they reduce the aggregation of conjugated backbones with the help of hydrophilic side chains.22-27 These advantages have led to the application of WSCPBs in biosensors, cell imaging, phototherapy and anionic type drugs delivering.28-32 At present, WSCPBs functionalized with cationic polymers, such as cationic polyfluorene brush (PFNBr)33, have been


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developed by our group as the highly efficient siRNA delivery systems for cancer treatments.26, 34

In this paper, we report the application of a developed WSCPB (PPE-PLL) with high OVA loading capacity. This WSCPB is used as an OVA delivery system in the study of DC vaccines. The conjugated polymer, poly(p-phenyleneethynylene) (PPE), was employed as the backbone by virtue of its excellent light-harvesting ability and photostability, which enable it to serve as a signal reporter for delivery visualization. In addition, we utilized the cationic polymer poly(Llysine) (PLL) to act as side chains, not only because natural lysine biomolecules have characteristics such as biodegradability, biocompatibility and nontoxicity but also because they exhibit a strong selective interaction with negatively charged drugs, genes and proteins.35-38 The polymer brush PPE-PLL can take advantage of its unique structure with a high density of positive charges to display an excellent OVA-binding capability. Consequently, the polymer brush PPE-PLL allows the tracking of OVA into cells without adding fluorescent dyes, and it is suitable for the delivery of OVA with low cytotoxicity. Then the fluorescence resonance energy transfer (FRET) between PPE-PLL and fluorescein isothiocyanate (FITC) was also used for studying the release process of OVA from PPE-PLL. In this work, PPE-PLL was used as the carrier of OVA and showed a higher cellular uptake in DCs, while the antigen-pulsed DCs elicited good immune response in vitro. The production of TNF-α and interleukin-12 (IL-12p70) were enhanced by activated DCs. Importantly, the DC vaccine improved the level of immunity and inhibited the growth of cancerous tumor in vivo, and the survival rate of mice was greatly improved in our experiments. Accordingly, this OVA delivery system basing on cationic conjugated polymer brush has a bright prospect in the field of DC vaccines.8, 39 EXPERIMENTAL SECTION


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Materials. Materials: After purchasing from commercial suppliers, all the chemicals were used as received. 2,5-bis[3-(BOC-amino)-1-oxapropyl)-1,4-diiodobenzene] (M1) and 2,5-bis[3-(BOCamino)-1-oxapropyl)-1,4-diethynylbenzene] (M2) were obtained according to our group’s work before.40,

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N-[4-[(4S)-2,5-dioxooxazolidin-4-yl]butyl]-2,2,2-trifluoro-acetamide (98%) was

purchased from Nanjing Chemlin Chemical Industry Co., Ltd. Other reagents were ordered from Shanghai J&K Scientific Co., Ltd. OVA were obtained from Sigma-Aldrich Co., Ltd. The FITCOVA was prepared by using FITC Labeling Kit which is purchased from Abbkine. DCs were purchased from Icellbioscience (China) and incubated in Roswell Park Memorial Institute 1640 (RPMI 1640, KeyGEN Bio TECH). B16F10 cells were purchased from Jiangsu KeyGEN Bio TECH Corp., Ltd. Synthesis of PPE-NH2 Monomers of PPE-NH2, M1 and M2, was shown in Scheme 1. M1 (0.79 g, 1.1mmol), M2 (0.54 g, 1.1mmol), tetrakis(triphenylphosphine)palladium (0.05 g, 0.005 mmol) and cuprous iodide (0.01 g, 0.05 mmol) were dissolved in superdry toluene (22 mL) and superdry diisopropylamine (9 mL) under argon protection. Then the mixture was heated for 3 h at 70 °C. After that bromobenzene (0.9 g, 5.7 mmol) was added and then stirred for 1 h. Next the mixture was reprecipitated in methanol, and yellow solid were obtained by filtering. Column chromatography (alumina: tetrahydrofuran) was carried out to provide PPE-NH(BOC). The 1HNMR spectrum (Figure S1) and optical properties (Figure S2) of PPE-NH(BOC) was shown in Supporting Information. PPE-NH(BOC) was dissolved in trifluoroacetic acid (TFA) and then stirred for 18 h. Finally, the TFA was rotary evaporated with methanol to afford PPE-NH2. Synthesis of PPE-PLL.


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Under argon protection product, PPE-NH2 (0.36 g), N-[4-[(4S)-2,5-dioxooxazolidin-4yl]butyl]-2,2,2-trifluoro-acetamide (2.00 g) and thiocarbamide (3.00 g) were dissolved in dimethylformamide (20 mL) and stirred for 78 h. Then the mixture was reprecipitated in diethyl ether to afford a yellow powder. After dissolving the powder in methanol and NaOH, the mixture was stirred for 8 h. The solution was dialyzed by dialysis bag placed in H2O (500 mL) with HCl (3.0 mL). Then the solution was lyophilized to afford the purified polymers (PPE-PLL, yield: 1.93 g, 79%). 1H NMR spectrum of PPE-PLL was shown in Figure 1. Characterization. 1H-NMR

spectra (400 MHz) were obtained at 20.0 °C on Bruker Ultrashield Plus 400 MHz

NMR spectrometer, the solvents are deuterated reagent and the internal standard is tetramethylsilane (TMS). The number-average molecular weights (Mn) and polydispersities (PDI) of materials were analyzed using gel permeation chromatography (GPC). The Shim-pack GPC-80X (refractive index detector) column as the eluent flowing at 1.0 mL/min with THF and polystyrene. The UV-vis absorption spectras were obtained using a Shimadzu UV-3600 UV-vis-NIR spectrophotometer and the fluorescence spectras were obtained on a Shimadzu RF-5301 PC fluorometer. Zeta potential was texted by a Brookhaven Instruments ZetaPALS at 25.0 °C. Dynamic light scattering (DLS) analyze was achieved by an ALV CGS-3 light scattering spectrometer installed with a laser of He-Ne (at λ = 632.8 nm) and an ALV ALV-7004 multi-τ digital time correlator (ALV-7004; ALV, Langen, Germany). A CONTIN analysis, from the scattering intensity, served to extract the data. All test samples were filtrated though


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Millipore filters (0.45 μm) before each measurement and tested in triplicate. The scattering angle was 90° and the temperature was 25.0±0.5 °C. Transmission electron microscopy (TEM) images were collected on a HT7700 transmission electron microscope. The TEM specimens were made on a carbon-coated copper grid via placing a drop of PPE-PLL (0.5 mg mL-1). Binding Capacity. To evaluate the binding capacity of PPE-PLL with OVA, the PPE-PLL@OVA complex was obtained after loading OVA onto PPE-PLL by mixing different mass ratios of PPE-PLL and OVA in PBS, then stirring at room temperature for 10 min. To track OVA during this process, OVA was modified by FITC. After that, the mixtures (0.5 mL) were centrifuged at 8,000 rpm for 30 min using ultrafiltration (Micron YM-100, Merck-Millipore, Germany) to separate the free FITC-OVA. The solution of free FITC-OVA was diluted three times with PBS, and the fluorescence spectrum was recorded on a fluorescence spectrometer with excitation at 488 nm, and the intensity compared with that of a standard concentration curve of FITC-OVA. The PPE-PLL@OVA complexes with different mass ratios of PPE-PLL and OVA (1:0, 0:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6) were separated by 0.6% agarose gel in TAE buffer at 170 V for 20 min. Coomassie blue was used to stain the complexes for visualization. The gels were photographed after elution with an eluent solution (water: methanol: acetic acid = 45: 45: 10). OVA Release Experiment. The release characteristics of OVA from PPE-PLL at different pH values were investigated with UV spectroscopy, photoluminescence (PL) spectroscopy and DLS analysis. First, 5.0 mL of the PPE-PLL@OVA solution was dialyzed in a dialysis bag (35 kDa), then placed them in 20 mL of PBS with pH 6.0 and 7.4 separately. Taking the solubility of the complexes into account,


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the mass ratio of PPE-PLL to OVA was set as 1:2. After 1, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 and 24 h, the buffer was removed, and 20 mL PBS was added, then the removed buffer was used for determining the UV absorption at 280 nm. The experiments were repeated in triplicate, the mean value of each sample was calculated. The release characteristic of OVA from the PPE-PLL was evaluated by comparing with a standard concentration curve of OVA. The FRET phenomenon will occur when PPE-PLL and FITC-OVA are combined. Thus, while the OVA is gradually released from the complexes, the FRET efficiency will decline. Initially, the mass ratio of PPE-PLL to OVA was set as 1:2 when a final OVA concentration was 5.0 μg mL-1 at pH values of 6.0 and 7.4 at room temperature. After 1, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 and 24 h, the fluorescence spectra were collected with an excitation light at 405 nm. The experiment was repeated in triplicate, and the mean value of each sample was calculated. The peaks of 425 nm and 525 nm were compared, and the amounts of released OVA at different time points were evaluated. The release process of OVA was also studied by DLS analysis. The mass ratio of PPE-PLL to OVA was set at 1:2 under the same conditions as above. Then, the particle size of these complexes were determined at 0, 1, 2, 4, 6, 8, 10 and 12 h. The released OVA during each time interval was evaluated by comparing the peaks of OVA and PPE-PLL (the peak of OVA was approximately 30 nm, and the peak of PPE-PLL was over 150 nm). Cell Viability Assay. Cell viability was analyzed using mmttetrazolium (MTT) assay (Sigma-Aldrich Co., USA). DCs were incubated in RPMI 1640 and various concentrations of PPE-PLL or PPE-PLL@OVA. In each well, MTT (0.5 mg mL-1) diluted in fresh culture medium was added after removing the


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culture medium, then the cells were incubated for 4 h. After the formazan was dissolved by DMSO, the absorbances were analyzed at 560 nm by a Bio-Rad Laboratories microplate reader. Cellular Uptake of PPE-PLL@OVA and Imaging. PPE-PLL, free OVA and PPE-PLL@FITC-OVA, at a final OVA concentration of 5.0 μg mL-1, were incubated with DCs for 0, 1, 2 and 4 h. After washing these cells with PBS, their fluorescence was collected using fluorescence-activated cell sorting (FACS) flow cytometry analysis (Merck-Millipore). The cellular imaging of the DCs was performed. PPE-PLL, FITCOVA and PPE-PLL@FITC-OVA were incubated with DCs for 2 h when final OVA concentration was 5.0 μg mL-1. After washed with PBS, these cells were observed with a Carl Zeiss Zeiss LSM confocal laser scanning microscope (CLSM) by using the Olympus Fluoview FV500 imaging software. The level of DCs Maturation. In order to test the maturation level of the DCs with different treatments (PPE-PLL, free OVA, PPE-PLL@OVA) for 24 h. After washed with FACS buffer, the DCs were stained with antiCD86-PE and anti-CD80-FITC antibodies (eBioscience, USA) for 20 min. After being washed again, these DCs were detected using FACS-flow cytometry. All samples were analyzed on three separate occasions. In Vitro Cytokine Detection Assay. Suspensions of the DC culture media were analyzed after culturing with PPE-PLL, free OVA and PPE-PLL@OVA (5.0 μg mL-1 OVA) for 24 h. IL-12p70 and TNF-α were analysed with enzyme-linked immunosorbent assay (ELISA) kits (KeyGEN Bio TECH, China). All samples were measured in triplicate.


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Animals and Tumor Model. The mice experiments were based on NIH guidelines (NIH Publication no. 85-23 Rev. 1985), and these experiments were approved by the Animal Ethics Committee of Simcere Pharmaceutical Group. C57BL/6 mice were purchased from Shanghai LingChang Bio TECH Corp., Ltd. The right armpit of C57BL/6 mouse subcutaneously injected with B16F10 tumors cells for antitumor study. The volume of tumor was monitored every two days, from 2 d to 60 d. After the volume of tumor exceeded 2,000 mm3, the mice were euthanized. DC Vaccine and in Vivo Cytokine Detection Assay. DCs were incubated with PPE-PLL, free OVA or PPE-PLL@OVA (5.0 μg mL-1 OVA) for 2 h. After replacing the culture medium and further culturing for 24 h, the cells were collected as PPE-PLL-pulsed, OVA-pulsed and PPE-PLL@OVA-pulsed DCs. The mice were divided into five groups randomly (6 mice per group), and the mice from each group were vaccinated subcutaneously twice at weekly intervals with 2 × 106 DCs, PPE-PLLpulsed, OVA-pulsed or PPE-PLL@OVA-pulsed DCs. Ten days later, the serum samples taken from the vaccinated mice were analyzed after the appropriate dilution. According to the instructions, Interleukin 1β (IL-1β) and TNF-α were analysed by using ELISA kits (KeyGEN Bio TECH). Antitumor Study. The mice were divided into five groups (6 mice per group) and were vaccinated with 2 × 106 DCs, PPE-PLL-pulsed, OVA-pulsed or PPE-PLL@OVA-pulsed DCs three times every 7 days. 10 days after the last injection, B16F10 tumors were generated by subcutaneous injection of 6 × 105 tumor cells in the right armpit of mice from these 5 groups. The volume of tumor was monitored every two days from 2 d to 60 d. The volume of tumor was estimated by the equation:


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V = (a × b2)/2 (a, length; b, width). At same time points, the body weights were also monitored. After the volume of tumor exceeded 2,000 mm3, the mice were euthanized. Statistical Analysis. All results were shown with average ± standard deviation. Statistical comparison was analysed statistically using one-way analysis of variance (95% confidence interval). Significant difference was set at p < 0.05.

RESULTS AND DISCUSSION Preparation and Characterization of PPE-PLL PPE-PLL was obtained through the main route depicted in Scheme 1. The fluorescent conjugated polymer PPE-NH2 was prepared from M1 and M2 as illustrated in the experimental section. Then, the brush copolymer PPE-PLL(TFA) was prepared via ring-opening polymerization of L-lysine, while trifluoroacetyl (TFA) is the protective group of the amino.42-44 PPE-PLL, the end product, was prepared by taking off the protective groups of PPE-PLL(TFA). The structure of PPE-PLL was confirmed by 1H-NMR spectroscopy, from the spectrum in Figure 1. The peaks of PLL can be clearly observed at 1.80, 3.10, 4.25 and 8.10 ppm, thus validating the successful synthesis of the PLL side chains. According to the 1H-NMR spectroscopy result, the polymerization degree of PLL was calculated to be 20 by comparing the integration of (-CH2CH2-O-) (2.75, 3.80 ppm for the backbone) and (-CH2-CH2-CH2-) (1.80 ppm for the PLL). The Mn and PDI of PPE-PLL(TFA) measured by GPC were 38,000 and 1.48, while those of PPENH(BOC) were 17,000 and 1.73, respectively (Figure S3). By applying the PLL side chains, the water solubility of PPE-PLL was up to 8.75 mg mL-1.


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Scheme 1. The synthetic route of PPE-PLL.


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Figure 1. The 1H-NMR spectrum of PPE-PLL in DMSO-D6. To study the optical properties of PPE-PLL, the UV-visible absorption and PL spectra of PEEPLL were determined. From Figure 2a, PPE-PLL exhibited a maximal absorption at 415 nm and a fluorescence emission at 485 nm under an excitation of 425 nm in aqueous solution. Additionally, no obvious changes were shown in the fluorescence spectrum (Figure 2b) within pH range of 5-8. Accordingly, the polymer can achieve superb optical properties in a physiological environment.45 However, due to the deionization of PPE-PLL reduced its solubility in a basic solution, the aggregation of PPE-PLL caused the fluorescence signal of PPE-PLL quenched. Therefore, a sharp decrease in PL appeared when the pH increased over 9. The particle sizes of PPE-PLL were determined by TEM and DLS analysis (Figure 2c and 2d). The TEM image in Figure 2c revealed that the radius of PPE-PLL was approximately 110 nm,


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while the hydrodynamic particle size of PPE-PLL according to the DLS analysis was also approximately 130 nm. Judging from the above findings, the hydrodynamic particle size of PPEPLL was slightly larger than the particle size in the TEM images, this is due to the electrostatic spatial repulsion among the PLL. In aqueous solution, the cationic PLL side chains can fully extend. The solvents during the drying process caused the surface tension, which subsequently resulted in the aggregation of PPE-PLL. Figure 2d revealed the same hydrodynamic sizes of PPE-PLL at different time points as about 130 nm, it indicated that PPE-PLL maintain good stability within 24 h.


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Figure 2. (a) UV spectrum of PPE-PLL (black); Fluorescence spectrum of PPE-PLL (red). (b) PL spectrum of PPE-PLL at different pH values. (c) TEM image of PPE-PLL. (d) Hydrodynamic sizes of PPE-PLL at different time points. Binding Capability of PPE-PLL with OVA To evaluate the binding capacity of PPE-PLL with OVA, ultrafiltration technology was used to separate free FITC-OVA from the PPE-PLL@FITC-OVA complexes, and the calculation of the LC was obtained by measuring free FITC-OVA outside the ultrafiltration membrane via fluorescence intensity. The standard curve of the FITC-OVA concentration is shown in Figure S4. From Table S1, LC of the brush polymer PPE-PLL was 88.69%, which is much higher than that of the linear polymer PPE-propanamine (8.99%) and linear polymer PLL (16.37%), while some other delivery systems of free OVA had an LC of only approximately 10%.8 The molecular structure of liner polymer PPE-propanamine and liner polymer PLL was shown in Figure S5. The high LC of PPE-PLL benefits from a special brush chemical structure with cationic side chains. These results demonstrate that this brush polymer, PPE-PLL, has better transport capacity than linear polymer PPE-propanamine, linear polymer PLL and other commonly used carrier systems.46, 47 In addition, since the PLL side chains provided electrostatic interactions to bind OVA, the cations on PPE-PLL decreased when successful combination of OVA with PPE-PLL by electrostatic attraction. As shown in Figure 3a, PPE-PLL was highly positively charged, which resulted from the amino group on every L-lysine residue. After mixing the PPE-PLL with OVA, the zeta potential decreased significantly. Along with the decrease of the mass ratio of PPE-PLL to OVA from 1:0 to 1:4, the zeta potential decreased from +54 to +6 mV. Additionally, the numerical results revealed that the zeta potential became negative (-16 mV) when the mass ratio


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of PPE-PLL to OVA changed to 1:5. In addition, the agarose gel electrophoresis shift assay (Figure S6) was used to determine the OVA-binding capacity of PPE-PLL. Coomassie blue, which binds to proteins, was used to visualize OVA. It has previously been established that the negatively charged free OVA moves to the positive electrode, while the cationic PPE-PLL moves in the opposite direction. The results indicated that along with the decrease of the PPEPLL/OVA mass ratio, the charges of PPE-PLL were neutralized bit by bit and the complex moved from the sample holes to the negative electrode with reduced mobility. When the mass ratio of PPE-PLL to OVA was decreased to 1:4, the negatively charged free OVA appeared to move to the positive electrode. We consider the potential effect of an over load PPE-PLL with less positive charge and the poor immunogenicity caused by a complex with low OVA content. Accordingly, to achieve good endocytosis ability and strong immunogenicity, the mass ratio of 1:2 was chosen for further experiments. Additionally, the size of the complex was investigated by TEM and DLS analysis. From Figure 3b and 3c, the radius of the PPE-PLL@OVA complexes were approximately 110 nm in the TEM image, while the hydrodynamic particle sizes of PPEPLL and PPE-PLL@OVA complexes were approximately 130 and 195 nm, respectively. The hydrodynamic particle size of the PPE-PLL@OVA complexes were larger than that of PPE-PLL, which was due to the combination with OVA.


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Figure 3. Binding capacity of PPE-PLL. (a) Zeta potential of PPE-PLL and OVA with different PPE-PLL/OVA mass ratios. (b) TEM image of PPE-PLL@OVA. (c) Hydrodynamic sizes of PPE-PLL and PPE-PLL@OVA. (d) Zeta potential of PPE-PLL and PPE-PLL@OVA. To understand the detailed process of OVA release from PPE-PLL, the following experiments were conducted.33 First, as mentioned in the experimental section, the release of OVA from PPEPLL was analyzed by detecting the concentration of OVA for different periods with UV absorption spectroscopy at 280 nm, whose standard curve is presented in Figure S7. The release of OVA at pH values of 7.4 and 6.0 were shown in Figure 4a. At pH 6.0, the largest amount of OVA was released after 6 h, and the release time extended to 10 h while pH is 7.4. This is due to


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the relatively enhanced degree of protonation, as the electrostatic attraction between PPE-PLL and OVA grew weaker under the acidic condition. Moreover, FRET was also used to investigate the release process of FITC-OVA from PPE-PLL. From Figure S8, an efficient FRET from the PPE-PLL to FITC-OVA occurred when the PPE-PLL was combined with FITC-OVA due to the obvious overlap between the absorption of FITC-OVA and the emission of PPE-PLL. The release process of FITC-OVA at pH values of 7.4 and 6.0 were shown in Figure 4b by measuring the ratio of the intensity of PPE-PLL to the intensity of FITC. At 24 h, the ratio between the intensity of PPE-PLL to FITC was found to reach approximately 1.84 at pH 6.0, while it only reached 1.69 at pH 7.4. Additionally, the ratio at pH 6.0 grew slower than that at pH 7.4. Thus, all these results showed that OVA was more easily released under the acidic condition. We also traced the release process of OVA by analyzing the particle sizes. The particle size of PPEPLL@OVA by DLS analysis was over 150 nm, while that of OVA was approximately 30 nm. The results shown in Figure S9 reveal that at pH 6.0 the peak of OVA increased faster than it at pH 7.4. Together, these results suggested that the electrostatic attraction between PPE-PLL and OVA is weakened in acidic conditions.


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Figure 4. Analysis of OVA release from the complexes at different pH values. (a) Via UV-vis absorption spectrum; (b) via fluorescence emission spectrum. Cellular Uptake and Cellular Viability To investigate the potential application of the PPE-PLL@OVA nanoparticles, the MTT assay was used to analyze their cytotoxicity. The MTT assay showed that the PPE-PLL and PPEPLL@OVA exerted lower cell toxicity on DCs, which is due to the biodegradable, biocompatible and nontoxic nature of the side chains of PPE-PLL. The results shown in Figure 5 indicated that treatment of the DCs with either PPE-PLL or PPE-PLL@OVA did not significantly decrease DC viability in a certain concentration range (10-50 μg mL-1), and suggested that this system has good biocompatibility when incubated with DCs and can be further used in a DC vaccine.

Figure 5. Cell viability under different concentrations (0-50 μg mL-1) of (a) PPE-PLL and (b) PPE-PLL@OVA for 24 h. Previous studies have shown that negatively charged free OVA inhibits cellular uptake, while the positively charged PPE-PLL@OVA effectively enhances cellular uptake and intracellular


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levels of OVA.48 To investigate the process of cellular uptake of PPE-PLL@OVA, OVA was prelabeled with FITC and the FITC-OVA was then used to prepare the complexes, which were then incubated with DCs. Flow cytometry analysis has been used to monitor the entry of FITCOVA from PPE-PLL or in its free form. DCs were separately treated with PPE-PLL, OVA or PPE-PLL@OVA for 2 h. The cultured time was determined by flow cytometry analysis (Figure S10). Compared to the OVA group, the PPE-PLL@OVA group exhibited clearly enhancement of fluorescence intensity, as shown in Figure 6a. The result confirmed that the uptake of OVA in the DCs was enhanced when OVA was complexed with PPE-PLL. Confocal fluorescence microscope images of the uptake of PPE-PLL and FITC-OVA by DCs were also shown in Figure 6b. Specifically, the first channel was assigned to the FITC-OVA at 488 nm laser excitation (in green), while the second channel was assigned to the PPE-PLL at 405 nm laser excitation (in blue). When DCs were incubated with PPE-PLL@OVA for 2 h, the green signal of FITC-OVA and the blue signal of PPE-PLL both exhibited strong fluorescence intensity. Compared to the FITC-OVA group with weak green fluorescence in the first channel, the PPEPLL@OVA-treated DCs exhibited significant enhancement of the fluorescence intensity of FITC-OVA. Meanwhile, the DCs incubated with PPE-PLL also exhibited a clear blue signal in the second channel. These results indicated that compared to free OVA, PPE-PLL@OVA effectively improved the uptake of OVA in DCs.


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Figure 6. Intracellular delivery of PPE-PLL@OVA into DCs. (a) Flow cytometry analysis of FITC labeled OVA in DCs treated with PPE-PLL, OVA or PPE-PLL@OVA. (b) CLSM images of PPE-PLL@OVA-pulsed DCs. The blue signals were from PPE-PLL; the green signals were from FITC-OVA. DC maturation and cytokine release Previous reports have shown that immature DCs possess the potent ability of antigen endocytosis, and mature DCs mainly function in the antigen-presenting process. The DCs


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transform from an immature to a mature state after capturing the antigens or pathogens. During this conversion process, the antigen would be degraded into peptides after being imbibed by DCs. Then in draining lymph node, major histocompatibility complex (MHC) presents the signal onto the naive T cells, eventually inducing the immune response.8, 49 Mature DCs show higher levels of MHC class II and costimulatory molecules (CD40, CD80 or CD86) which induce T cell activation.50 The degree of DC maturation can be tested by monitoring CD80+ and CD86+ on their membrane, because CD80+ and CD86+ are the important indices to evaluate the immune responses in immunotherapy based on DC. To study the maturation level of DCs in different treatments, the levels of CD86+ and CD80+ were analyzed using flow cytometry analysis.51, 52 From Figure 7, after treatment with PPE-PLL@OVA, the percentage of matured cells outstandingly increased from 14.9 to 64.9%, while the DC maturation level only increased to 52.5% after treating with the same dose of free OVA, and DCs treated with PPE-PLL only increased to 22.1%. These results confirmed that the enhanced DC maturation level of PPEPLL@OVA was due to the easy uptake by DCs, which subsequently triggered a stronger immune response. Proinflammatory cytokines (IL-12p70, TNF-α) lead to the further activation, maturation and immune stimulation activity of DCs.53 IL-12p70, a member of the interleukin family, is naturally secreted by DCs, and it plays a key role in the immune process through to activate natural killer cells and T lymphocytes. In addition, TNF-α is another one cytokines, involved in systemic inflammation, that makes up the phase reaction cell signaling protein. In our study, the secretion of TNF-α and IL-12p70 into the supernatants of DCs were detected through an ELISA kit. From Figure 8, the levels of TNF-α and IL-12p70 were outstanding increased after incubation with PPE-PLL@OVA compared to other groups. These results indicated that PPE-PLL can


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effectively deliver OVA into DCs, leading to DC maturation and cytokine release. Therefore, DC vaccines with the ability to promote the release of immune factors can be applied in DC-based immunotherapy.54

Figure 7. Quantification of CD86+ and CD80+ expression in vitro.


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Figure 8. Immunoregulatory activities of PPE-PLL@OVA in vitro. Secretion of (a) TNF-α and (b) IL-12p70 measured by ELISA assay. To further explore the therapeutic potential of PPE-PLL@OVA, in vivo immunostimulatory activity was investigated. C57BL/6 mice were vaccinated with DCs, PPE-PLL-pulsed, OVApulsed or PPE-PLL@OVA-pulsed DCs twice a week. After last intradermal injection, the serum level of the cytokines was determined. From Figure 9, the serum level of TNF-α and IFN-γ did not obvious increase when the mice were injected PPE-PLL-pulsed DCs or OVA-pulsed DCs. In contrast, after vaccinating with PPE-PLL@OVA-pulsed DCs, the serum level of TNF-α and IFN-γ was clearly increased. These results suggested that the PPE-PLL@OVA-pulsed DC vaccine had successfully induced cellular immunity. In addition, we made the lymph nodes from the C57BL/6 mice which vaccinated with normal saline, DCs, PPE-PLL-pulsed, OVA-pulsed or PPE-PLL@OVA-pulsed DCs into single cell suspensions, then studied the maturation level of these group by monitoring the quantification of CD80+ and CD86+. The results are in the following Table S2. As the results, the quantification of CD80+ and CD86+ expression in PPEPLL@OVA-pulsed DCs group was clearly increased than other groups. This result has helped prove that PPE-PLL@OVA-pulsed DC vaccine can contribute to higher tumor suppressor activity.


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Figure 9. In vivo immune responses by vaccinating with DC vaccines. (a) Level of TNF-α. (b) Level of IFN-γ. (n = 6/group) Antitumor efficacy According to the established effect of the PPE-PLL@OVA-pulsed DC vaccine on in vivo immunostimulatory activity, our group further studied the efficacy of the vaccine for cancer immunotherapy by using B16F10 cells as tumor cell models. C57BL/6 mice were vaccinated with normal saline (NS), DCs, PPE-PLL-pulsed, OVA-pulsed or PPE-PLL@OVA-pulsed DCs once every 7 d at two-week intervals.4,

5

As shown in Figure 10a, we found that the PPE-

PLL@OVA-pulsed DC treatment led to greatly enhanced tumor suppressor activity in the B16F10 tumor model within 21 days. In contrast, the treatment with NS, DCs, PPE-PLL-pulsed DCs or OVA-pulsed DCs did not produce the same effect. In the experimental results, the PPEPLL-pulsed DC group induced minor tumor suppressor activity. The reason for this could be that the cation on the surface rendered PPE-PLL more accessible to the plasma membrane compared to OVA. Thus, the positive charged substances more easily induced antigen-specific CTL responses compared with the negative charged and neutral substances. Additionally, we also monitored the body weight of mice after different vaccine treatments. However, there is no significant change between the mice group treated with PPE-PLL@OVA-


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pulsed DCs and the other groups (Figure S11). Finally, the survival analysis of tumor-bearing mice after treatment with the DC vaccine revealed that only the mice treated with PPEPLL@OVA-pulsed DCs showed a 100% survival rate after 60 days of tumor inoculation. These results clearly demonstrated the induction of the antitumor immune responses by this vaccine, and these results also indicated the great therapeutic potential of this DC vaccine.

Figure 10. Antitumor efficacy of DC vaccines with different pulsed in B16F10-bearing C57BL/6 mice. (a) Tumor volume and (b) survival rate after tumor inoculation. Conclusions In summary, we developed a WSCPB with good photostability, strong antigen-loading capacity and excellent water solubility for use as an OVA delivery system. Compared with free OVA, the PPE-PLL@OVA complexes not only showed a higher cellular uptake by DCs but also induced DC maturation and cytokines release. As a DC vaccine for cancer immunotherapy, the PPE-PLL@OVA-pulsed DCs effectively induced a strong immune response in vivo. In addition, this DC vaccine showed a high antitumor activity in the B16F10 tumor model. Although this DC vaccine cannot currently be directly applied to human patients, the successful tumor-suppressor activity in the animal models indicated its potential application in DC-based cancer immunotherapy and other cancer treatments.


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ASSOCIATED CONTENT Supporting Information. 1H-NMR

spectra of fabricated PPE-NH(BOC). The optical properties of PPE-NH(BOC). The

GPC of PPE-NH(BOC) and PPE-PLL(TFA). Spectra overlapping between PPE-PLL and FITCOVA. Loading efficiency of PPE-propanamine, PLL and PPE-PLL. The agarose gel electrophoresis shift assay of PPE-PLL. Details of the standard curve of FITC-OVA and OVA concentration. Details of DLS and flow cytometric analysis. Body weight changes after various treatments. AUTHOR INFORMATION Corresponding Author * E-mail: [email protected] ORCID Quli Fan: 0000-0002-9387-0165 Pengfei Sun: 0000-0001-7412-5154

Author Contributions

※These authors contributed equally.


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Notes The authors declare no competing financial interest. ACKNOWLEDGMENT This work was financially supported by the National Natural Science Foundation of China (No. 21674048, 21574064, 21604042, and 51503103), Synergetic Innovation Center for Organic Electronics and Information Displays, 333 project of Jiangsu province (No. BRA2016379), Jiangsu National Synergetic Innovation Center for Advanced Materials, the Natural Science Foundation of Jiangsu Province of China (No. BK20150843), Primary Research & Development Plan of Jiangsu Province (BE2016770) and NUPTSF (No. NY214182 and NY211003).

ABBREVIATIONS DC, dendritic cell; OVA, Ovalbumin; WSCPB, water-soluble conjugated polymer brush; IFN-γ, interferon gamma; TNF, tumor necrosis factor; CTL, cytotoxic T lymphocyte; PPE, poly(pphenyleneethynylene); PLL, poly(L-lysine); FITC, fluorescein isothiocyanate. REFERENCES (1) Chen, Q.; Xu, L.; Liang, C.; Wang, C.; Peng, R.; Liu, Z. Photothermal Therapy with Immune-adjuvant Nanoparticles Together with Checkpoint Blockade for Effective Cancer Immunotherapy. Nat. Commun. 2016, 7, 13193. (2) Wang, Z.; Liu, W.; Shi, J.; Chen, N.; Fan, C. Nanoscale Delivery Systems for Cancer Immunotherapy. Mater. Horiz. 2018, 5, (3), 344-362.


29


Page 30 of 37

(3) Yang, G.; Xu, L.; Chao, Y.; Xu, J.; Sun, X.; Wu, Y.; Peng, R.; Liu, Z. Hollow MnO(2) as a Tumor-microenvironment-responsive Biodegradable Nano-platform for Combination Therapy Favoring Antitumor Immune responses. Nat. Commun. 2017, 8, 902. (4) Zhang, Y.; Cui, Z. F.; Kong, H. T.; Xia, K.; Pan, L.; Li, J.; Sun, Y. H.; Shi, J. Y.; Wang, L. H.; Zhu, Y.; Fan, C. H. One-Shot Immunomodulatory Nanodiamond Agents for Cancer Immunotherapy. Adv. Mater. 2016, 28, (14), 2699-2708. (5) Coumes, F.; Huang, C.-Y.; Huang, C.-H.; Coudane, J.; Domurado, D.; Li, S.; Darcos, V.; Huang, M.-H. Design and Development of Immunomodulatory Antigen Delivery Systems Based on Peptide/PEG–PLA Conjugate for Tuning Immunity. Biomacromolecules. 2015, 16, (11), 3666-3673. (6) Liu, Y.-J. Dendritic Cell Subsets and Lineages, and their Functions in Innate and Adaptive Immunity. Cell. 2001, 106, 259-262. (7) Steinman, R. M. Dendritic Cells Versatile Controllers of the Immune System. Nat. Med. 2007, 13, 1155-1159. (8) Xiang, J.; Xu, L. G.; Gong, H.; Zhu, W. W.; Wang, C.; Xu, J.; Feng, L. Z.; Cheng, L.; Peng, R.; Liu, Z. Antigen-Loaded Upconversion Nanoparticles for Dendritic Cell Stimulation, Tracking, and Vaccination in Dendritic Cell-Based Immunotherapy. ACS Nano. 2015, 9, (6), 6401-6411. (9) Moore, M. W.; Carbone, F. R.; Bevan, M. J. Introduction of Soluble Protein into the Class I pathway of Antigen Processing and Presentation. Cell. 1988, 54, 777-785. (10) Wattiaux, R.; Laurent, N.; Wattiaux-De Coninck, S.; Jadot, M. Endosomes, lysosomes: their Implication in Gene Transfer. Drug Deliver Rev. 2000, 41, 201-208. (11) Mukherjee, S.; Ghosh, R. N.; Maxfield, F. R. Endocytosis. Physiol. ReV. 1997, 77, 759-803.


30

Page 31 of 37 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


(12) Fontana, F.; Liu, D. F.; Hirvonen, J.; Santos, H. A. Delivery of Therapeutics with Nanoparticles: What's New in Cancer Immunotherapy? Wiley Interdiscip. Rev.: Nanomed. Nanobiotechnol. 2017, 9, (1), e1421. (13) Ruiz-de-Angulo, A.; Zabaleta, A.; Gómez-Vallejo, V.; Llop, J.; Mareque-Rivas, J. C. Microdosed Lipid-Coated 67Ga-Magnetite Enhances Antigen-Specific Immunity by Image Tracked Delivery of Antigen and CpG to Lymph Nodes. ACS Nano. 2016, 10, (1), 1602-1618. (14) Flanary, S.; Hoffman, A. S.; Stayton, P. S. Antigen Delivery with Poly(Propylacrylic Acid) Conjugation Enhances MHC-1 Presentation and T-Cell Activation. Bioconjugate Chem. 2009, 20, (2), 241-248. (15) Kojima, C.; Kameyama, R.; Yamada, M.; Ichikawa, M.; Waku, T.; Handa, A.; Tanaka, N. Ovalbumin Delivery by Guanidine-Terminated Dendrimers Bearing an Amyloid-Promoting Peptide via Nanoparticle Formulation. Bioconjugate Chem. 2015, 26, (8), 1804-1810. (16) Nakanishi, T.; Kunisawa, J.; Hayashi, A.; Tsutsumi, Y.; Kubo, K.; Nakagawa, S.; Nakanishi, M.; Tanaka, K.; Mayumi, T. Positively Charged Liposome Functions as an Efficient Immunoadjuvant in Inducing Cell-mediated Immune Response to Soluble Proteins. J. Controlled Release. 1999, 61, 233-240. (17) Chen, W.; Zhou, S.; Ge, L.; Wu, W.; Jiang, X. Translatable High Drug Loading Drug Delivery Systems Based on Biocompatible Polymer Nanocarriers. Biomacromolecules. 2018, 19, (6), 1732-1745. (18) Chen, W.; Su, L.; Zhang, P.; Li, C.; Zhang, D.; Wu, W.; Jiang, X. Thermo and pH DualResponsive Drug-linked Pseudo-polypeptide Micelles with a Comb-shaped Polymer as a Micellar Exterior. Polym. Chem. 2017, 8, (44), 6886-6894.


31


Page 32 of 37

(19) Jiang, Y.; Pu, K. Multimodal Biophotonics of Semiconducting Polymer Nanoparticles. Acc. Chem. Res. 2018, 51, (8), 1840-1849. (20) Miao, Q.; Pu, K. Organic Semiconducting Agents for Deep-tissue Molecular Imaging: Second Near-Infrared Fluorescence, Self-Iuminescence, and Photoacoustics. Adv. Mater. 2018, 30, 1801778. (21) Miao, Q.; Xie, C.; Zhen, X.; Lyu, Y.; Duan, H.; Liu, X.; Jokerst, J. V.; Pu, K. Molecular Afterglow Imaging with Bright, Biodegradable Polymer Nanoparticles. Nat. Biotechnol. 2017, 35, 1102-1110. (22) Li, J.; Rao, J.; Pu, K. Recent Progress on Semiconducting Polymer Nanoparticles for Molecular Imaging and Cancer Phototherapy. Biomaterials. 2018, 155, 217-235. (23) Zhang, Z. Y.; Lu, X. M.; Fan, Q. L.; Hu, W. B.; Huang, W. Conjugated Polyelectrolyte Brushes with Extremely High Charge Density for Improved Energy Transfer and Fluorescence Quenching Applications. Polym. Chem. 2011, 2, (2), 2369-2377. (24) Zhang, Z. Y.; Fan, Q. L.; Sun, P. F.; Liu, L. L.; Lu, X. M.; Li, B.; Quan, Y. W.; Huang, W. Highly Selective Anionic Counterion-based Fluorescent Sensor for Hg2+ by Grafted Conjugated Polyelectrolytes. Macromol. Rapid Commun. 2010, 31, 2160-2165. (25) Yuan, Y.; Liu, B. Self-Assembled Nanoparticles Based on PEGylated Conjugated Polyelectrolyte and Drug Molecules for Image-Guided Drug Delivery and Photodynamic Therapy. ACS Appl. Mater. Interfaces. 2014, 6, 14903-14910. (26) Pu, K.; Mei, J.; Jokerst Jesse, V.; Hong, G.; Antaris Alexander, L.; Chattopadhyay, N.; Shuhendler Adam, J.; Kurosawa, T.; Zhou, Y.; Gambhir Sanjiv, S.; Bao, Z.; Rao, J. Diketopyrrolopyrrole‐Based Semiconducting Polymer Nanoparticles for In Vivo Photoacoustic Imaging. Adv. Mater. 2015, 27, (35), 5184-5190.


32

Page 33 of 37 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


(27) Zhang, Y.; Zhang, Z.; Liu, C.; Chen, W.; Li, C.; Wu, W.; Jiang, X. Synthesis and Biological Properties

of

Water-soluble

Polyphenylthiophene

Brushes

with

Poly(ethylene

glycol)/polyzwitterion Side Chains. Polym. Chem. 2017, 8, (10), 1672-1679. (28) Sun, P. F.; Wang, G. N.; Hou, H. Z.; Yuan, P. C.; Deng, W. X.; Wang, C.; Lu, X. M.; Fan, Q. L.; Huang, W. A Water-soluble Phosphorescent Conjugated Polymer Brush for Tumortargeted Photodynamic Therapy. Polym. Chem. 2017, 8, (38), 5836-5844. (29) Zhao, H.; Hu, W. B.; Ma, H. H.; Jiang, R. C.; Tang, Y. F.; Ji, Y.; Lu, X. M.; Hou, B.; Deng, W. X.; Huang, W.; Fan, Q. L. Photo-Induced Charge-Variable Conjugated Polyelectrolyte Brushes Encapsulating Upconversion Nanoparticles for Promoted siRNA Release and Collaborative Photodynamic Therapy under NIR Light Irradiation. Adv. Funct. Mater. 2017, 1702592. (30) Zhen, X.; Xie, C.; Pu, K. Temperature‐Correlated Afterglow of a Semiconducting Polymer Nanococktail for Imaging ‐ Guided Photothermal Therapy. Angew. Chem., Int. Ed. 2018, 57, (15), 3938-3942. (31) Xie, C.; Zhen, X.; Miao, Q.; Lyu, Y.; Pu, K. Self-Assembled Semiconducting Polymer Nanoparticles for Ultrasensitive Near-Infrared Afterglow Imaging of Metastatic Tumors. Adv. Mater. 2018, 42, (10), 1801331. (32) Jiang, Y.; Cui, D.; Fang, Y.; Zhen, X.; Upputuri, P. K.; Pramanik, M.; Ding, D.; Pu, K. Amphiphilic

Semiconducting

Polymer

as

Multifunctional

Nanocarrier

for

Fluorescence/Photoacoustic Imaging Guided Chemo-Photothermal Therapy. Biomaterials. 2017, 145, 168-177.


33


Page 34 of 37

(33) Jiang, R. C.; Lu, X. M.; Yang, M. H.; Deng, W. X.; Fan, Q. L.; Huang, W. Monodispersed Brush-Like Conjugated Polyelectrolyte Nanoparticles with Efficient and Visualized SiRNA Delivery for Gene Silencing. Biomacromolecules. 2013, 14, (10), 3643-3652. (34) Pu, K.; Shuhendler, A. J.; Jokerst, J. V.; Mei, J.; Gambhir, S. S.; Bao, Z.; Rao, J. Semiconducting Polymer Nanoparticles as Photoacoustic Molecular Imaging Probes in Living Mice. Nat. Nanotechnol. 2014, 9, 233-239. (35) Zhan, W.; Qu, Y.; Wei, T.; Hu, C.; Pan, Y.; Yu, Q.; Chen, H. Sweet Switch: SugarResponsive Bioactive Surfaces Based on Dynamic Covalent Bonding. ACS Appl. Mater. Interfaces. 2018, 10, (13), 10647-10655. (36) Cheol-Hee, A.; Su, Y. C.; You, H. B.; Sung, W. K. Synthesis of Biodegradable Multi-Block Copolymers of Poly(l-lysine) and Poly(ethylene glycol) as a Non-viral Gene Carrier. J. Control. Release. 2004, 97, 567-574. (37) Hynes, S. R.; McGregor, L. M.; Rauch, M. F.; Lavik, E. B. Photopolymerized Poly(ethylene glycol)/poly(L-lysine) Hydrogels for the Delivery of Deural Progenitor Cells. J. Biomater. Sci. Polym. Ed. 2007, 18, 1017-1030. (38) Kim, S. Y.; Phuengkham, H.; Noh, Y. W.; Lee, H. G.; Um, S. H.; Lim, Y. T. Immune Complexes Mimicking Synthetic Vaccine Nanoparticles for Enhanced Migration and CrossPresentation of Dendritic Cells. Adv. Funct. Mater. 2016, 26, (44), 8072-8082. (39) Dömling, A.; Holak, T. A. Programmed Death-1: Therapeutic Success after More than 100 Years of Cancer Immunotherapy. Angew. Chem., Int. Ed. 2014, 53, (9), 2286-2288. (40) Fan, Q. L.; Zhou, Y.; Lu, X. M.; Hou, X. Y.; Huang, W. Water-Soluble Cationic Poly(pphenyleneethynylene)s (PPEs): Effects of Acidity and Ionic Strength on Optical Behavior. Macromolecules. 2005, 38, (7), 2927-2936.


34

Page 35 of 37 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


(41) Zhao, Y. L.; Liu, L. H.; Zhang, W. Y.; Sue, C.-H.; Li, Q. W.; Miljanić, O. Š.; Yaghi, O. M.; Stoddart, J. F. Rigid-Strut-Containing Crown Ethers and [2]Catenanes for Incorporation into Metal–Organic Frameworks. Chemistry. 2009, 15, (48), 13356-13380. (42) Izunobi, J. U.; Higginbotham, C. L. Microstructure Characterization and Thermal Analysis of

Hybrid

Block

Copolymer

α-methoxy-poly(ethylene

glycol)-block-poly[ε-

(benzyloxycarbonyl)-l-lysine] for Biomedical Applications. J. Mol. Struct. 2010, 977, 153-164. (43) Synatschke, C. V.; Nomoto, T.; Cabral, H.; Förtsch, M.; Toh, K.; Matsumoto, Y.; Miyazaki, K.; Hanisch, A.; Schacher, F. H.; Kishimura, A.; Nishiyama, N.; Müller, A. H. E.; Kataoka, K. Multicompartment Micelles with Adjustable Poly(ethylene glycol) Shell for Efficient in Vivo Photodynamic Therapy. ACS Nano. 2014, 8, (2), 1161-1172. (44) Fong, B.; Turksen, S.; Russo, P. S.; Stryjewski, W. Colloidal Crystals of SilicaHomopolypeptide Composite Particles. Langmuir. 2004, 20, 266-269. (45) Zhang, L. F.; Eisenberg, A. Morphogenic Effect of Added Ions on Crew-Cut Aggregates of Polystyrene-b-poly(acrylic acid) Block Copolymers in Solutions. Macromolecules. 1996, 29, (27), 8805-8815. (46) Zheng, C.; Zhang, X. G.; Sun, L.; Zhang, Z. P.; Li, C. X. Biodegradable and redoxresponsive chitosan/poly(L-aspartic acid) submicron capsules for transmucosal delivery of proteins and peptides. J. Mater. Sci.: Mater. Med. 2013, 24, (4), 931-939. (47) Lu, Y.; Sun, W.; Gu, Z. Stimuli-responsive Nanomaterials for Therapeutic Protein Delivery. J. Controlled Release. 2014, 194, 1-19. (48) Wu, F.; Wuensch, S. A.; Azadniv, M.; Ebrahimkhani, M. R.; Crispe, I. N. Galactosylated LDL Nanoparticles: A Novel Targeting Delivery System to Deliver Antigen to Macrophages and Enhance Antigen Specific T Cell Responses. Mol. Pharmaceutics. 2009, 6, (5), 1506-1517.


35


Page 36 of 37

(49) Lanzavecchia, A.; Sallusto, F. Regulation of T Cell Immunity by Dendritic Cells. Cell. 2001, 106, 263-266. (50) Gao, J. X.; Madrenas, J.; Zeng, W.; Cameron, M. J.; Zhang, Z.; Wang, J. J.; Zhong, R.; Grant, D. CD40-deficient Dendritic Cells Producing Interleukin-10, but not Interleukin-12, Induce T-cell Hyporesponsiveness in Vitro and Prevent Acute Allograft Rejection. Immunology. 1999, 98, (2), 159-170. (51) Vries, I. J. M. D.; Lesterhuis, W. J.; Scharenborg, N. M.; Engelen, L. P.; Ruiter, D. J.; Gerritsen, M. J. P.; Croockewit, S.; Britten, C. M.; Torensma, R.; Adema, G. J. Maturation of Dendritic Cells Is a Prerequisite for Inducing Immune Responses in Advanced Melanoma Patients. Clin. Cancer Res. 2003, 9, 5091-5100. (52) Zhou, Q. Q.; Zhang, Y. L.; Du, J.; Li, Y.; Zhou, Y.; Fu, Q. X.; Zhang, J. G.; Wang, X. H.; Zhan, L. S. Different-Sized Gold Nanoparticle Activator/Antigen Increases Dendritic Cells Accumulation in Liver-Draining Lymph Nodes and CD8+ T Cell Responses. ACS Nano. 2016, 10, (2), 2678-2692. (53) Dranoff, G. Cytokines in Cancer Pathogenesis and Cancer Therapy. Nat. Rev. Cancer. 2004, 4, 11-22. (54) Celluzzi, C. M.; Mayordomo, J. I.; Storkus, W. J.; Lotze, M. T.; Falo, L. Peptide-Pulsed Dendritic Cells Induce Antigen-Specific CTL-Mediated Protective Tumor Immunity. J. Exp. Med. 1996, 183, 283-287.


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Graphical Abstract:


37

Conjugated Polymer Brush Based on Poly(L-lysine) with Efficient

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