Amantadine Surface-Modified Silver Nanorods Improves

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Biological and Medical Applications of Materials and Interfaces

Amantadine Surface-Modified Silver Nanorods Improves Immunotherapy of HIV Vaccine against HIV-Infected Cells Weiyu Li, Yekkuni. L. Balachandran, Yanlin Hao, Xie Hao, Runzhi Li, Zhangjie Nan, Hongying Zhang, Yiming Shao, and Ye Liu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b10948 • Publication Date (Web): 07 Aug 2018 Downloaded from http://pubs.acs.org on August 13, 2018

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ACS Applied Materials & Interfaces

Amantadine Surface-Modified Silver Nanorods Improves Immunotherapy of HIV Vaccine against HIV-Infected Cells Weiyu Li1, Yekkuni L. Balachandran2, Yanling Hao3, Xie Hao1, Runzhi Li1, Zhangjie Nan1, Hongying Zhang4 *, Yiming Shao3 *, Ye Liu2, 5 * 1

Beijing Key Laboratory of New Technology in Agricultural Application, National Demonstration Center

for Experimental Plant Production Education, Beijing University of Agriculture, Beijing 102206, China. 2

CAS Key Laboratory for Biological Effects of Nanomaterials and Nanosafety, National Center for

NanoScience and Technology, No. 11 Zhongguancun Beiyitiao, Beijing 100190, P. R. China. 3

State Key Laboratory of Infectious Disease Prevention and Control, National Center for AIDS/STD

Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 100190, China. 4

College of Tobacco Science, Henan Agricultural University, Zhengzhou 450002, China.

5

University of Chinese Academy of Sciences, Beijing 100049, P. R. China.

KEYWORDS: Surface modification, Silver nanorods, HIV vaccine, Immunotherapy, HIV-infected cells

ABSTRACT: Surface modifications can endow nanomaterials with presupposed immunoregulatory functions to optimize vaccine-induced immune responses. In this work, we modified an immunoregulatory molecule, amantadine (Ada), on the outermost layer of PVP-PEG coated silver nanorods (Ada-PVP-PEG silver nanorods). Such amantadine surface-modified silver nanorods promote HIV vaccine-triggered cytotoxic lymphocytes (CTLs) to produce around 8-fold stronger tumor necrosis factor alpha (TNF-α) in vivo. The enhancement of HIV-specific CTLs-derived TNF-α significantly facilitates the death of HIVinfected cells (from 28.86% to 84.19%) and reduces HIV production (around 6-fold). This work supports the critical role of surface modifications of nanomaterials in fundamentally improving the immunotherapy of HIV vaccine against HIV-infected cells.

HIV-infected cells provide almost all resources and environments for HIV survival and generation, such as the storage of viral genes, the expression of viral proteins, as well as the packaging of viral particles.1-3 Killing HIV-infected cells has been a crucial means for decreasing HIV production and inhibiting disease 1 ACS Paragon Plus Environment

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progress.4 Immune attacks from cytotoxic lymphocytes (CTLs), natural killer (NK) cells and complement membrane attack complex (MAC) play important roles in clearing HIV-infected cells in vivo.5-9 Strikingly, HIV-specific CTLs can produce TNF-α to effectively induce the death of HIV-infected cells.10-12 Given such an important role of CTLs-derived TNF-α, we hypothesize that developing a nanomaterial to facilitate CTLs-derived TNF-α will significantly promote the death of HIV-infected cells. Surface modification provides us a powerful tool for artificially endowing engineered nanomaterials with presupposed immunoregulatory functions.13-15 We and others have managed to design multiple surface-modified nanomaterials to regulate HIV vaccine-triggered immune responses in vivo. For instance, Darrell Irvine’s group modified various Toll like receptor agonists on the surface of nanoscale liposomes. This strategy successfully enhanced HIV vaccine-triggered IgG response and T cell response.16 B. Narasimhan reported that polyanhydride-functionalized nanoparticles can activate antigen presenting cells to enhance HIV vaccine-triggered immune responses.17 We also have developed multiple types of surface modifications (such as PVP-PEG dual-modification, polydiallydimethylammonium chloride modification and polyethyleneimine modification) to improve HIV vaccine efficacy.18-20 Strikingly, one of our studies reported that PVP-PEG modified silver nanorods as a safe HIV vaccine adjuvant can effectively induce CTLs to produce multiple cytokines, but not for TNF-α.18 If we can further optimize the surface modification of silver nanorods to enhance CTL-derived TNF-α, we will be able to effectively kill HIVinfected cells. Adamantane derivatives have shown a potent capability for enhancing TNF-α production in vivo and in vitro. For instance, amantadine-treated mice produce a significantly increased TNF-α in serum, in comparison to normal mice.21 Adamantylamide dipeptide derived from bacteria is able to improve T cells to produce multiple types of cytokines (including TNF-α) in mice and monkey.22-25 Vildagliptin, an adamantane-derived drug for treating Type 2 diabetes, can elevate the serum level of TNF-α in male rats.26 Adamantylsulfanyl heterocycles and adamantylaminopyrimidines as potent inducers can also significantly enhance TNF-α production in genetically modified murine melanoma cells.27, 28 In this work, in order to induce HIV vaccine-triggered CTLs to produce stronger TNF-α, we use amantadine/1-adamantylamine (Ada) to further modify PVP-PEG coated silver nanrods.18 Firstly, Ada shows its potential for enhancing TNF-α production in vivo.21 Secondly, Ada has a satisfactory safety and has been approved by Food and Drug Administration (FDA) for their clinical applications.29 Thirdly, the amino group of Ada enables us to conveniently modify this molecule on the surface of sliver nanorods, via a straightforward condensation reaction with carboxyl group. Together, Ada modification on the surface of silver nanorods might promote 2 ACS Paragon Plus Environment

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HIV vaccine-triggered CTLs to produce stronger TNF-α, finally facilitating the death of HIV-infected cells (Figure 1).

Figure 1. The outmost modification of amantadine on silver nanorods facilitates HIV vaccine-triggered CTLs to produce stronger TNF-α to kill HIV-infected cells.

RESULTS AND DISCUSSION Synthesis and characterization of amantadine surface-modified silver nanorods To synthesize amantadine surface-modified silver nanorods, we first prepared PVP-PEG silver nanorods according to our previous protocol.18 We use LA-PEG-COOH to introduce carboxylic groups on the outer layer of PVP-PEG coating.30 Via EDC/NHS coupling, Ada molecules are incorporated on the outmost layer of silver nanorods (Figure 2A). Ada modification on silver nanorods is confirmed by fourier transform infrared spectroscopy (FTIR). FTIR revealed the occurrence of Ada on the surface of sliver nanorods, which showed IR bands centered at 2926 cm-1 (stretching vibrations of C-H) and 1648 and 1570 cm-1 (N-H vibrations) (Figure 2B). X-ray photoelectron spectroscopy (XPS) further demonstrated Ada modification on the surface of PVP-PEG sliver nanorods. By measuring the changes of carbon (C1s), oxygen (O1s) and nitrogen (N1s), we found the characteristic occurrence in C-C bonds (284.7 eV) and C-N bonds (285.5 eV) caused by Ada molecules (Figure 2C). An absence of O1s C=O (532.2 eV) (Figure 2C) and the significant increase in N1s -N- (399.8 eV, Figure S1B) further demonstrated a successful Ada modification on the surface PVP-PEG silver nanorods. We also calculated the number ratio of Ada:PVP:PEG via quantifying 3 ACS Paragon Plus Environment


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the change of nitrogen on the surface of silver nanorods. XPS peak area of nitrogen increased by Ada modification is 1111 (Figure S1), and the percentage of nitrogen in pure Ada is 9.3%. XPS peak area of nitrogen on PVP-PEG silver nanorods is 1296 (Figure S1), and the percentage of nitrogen in pure PVP is 12.6%. Therefore, the number ratio between Ada and PVP molecules is 1.2 : 1 1111 9.3% ⁄1296 12.6% 1.2. Since only PVP, instead of PEG, contributes to the XPS peak area of nitrogen, and the number ratio between PVP and PEG on PVP-PEG silver nanorods is 1 : 2.7 (shown in our previous work18), we thus calculated that the number ratio of Ada : PVP : PEG on the surface of silver nanorods is 1.2 : 1 : 2.7. Transmission electron microscopy (TEM) showed that Ada-PVP-PEG silver nanorods well separated in aqueous solution without aggregation (Figure 2D). Such Ada-PVP-PEG silver nanorods possess negatively charged surface (-14 mV, Figure 2E). The hydrodynamic diameter of Ada-PVP-PEG silver nanorods is around 540 nm (Figure 2F), which is bigger than PVP-PEG silver nanorods (around 320 nm18), suggesting an Ada modified on the outmost layer of silver nanorods.

Figure 2. Synthesis and characterization of amantadine surface-modified silver nanorods. (A) Schematic representation of the synthesis of Ada-PVP-PEG silver nanorods. (B) FTIR spectrum of PVP-PEG silver nanorods (red), pure Ada (brown), and Ada-PVP-PEG silver nanorods (blue). (C) Comparative XPS

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measurements of C1s, O1s, and N1s region of PVP-PEG silver nanorods (red), pure Ada (brown), and AdaPVP-PEG silver nanorods (blue). The characteristic contribution of Ada molecules on silver nanorods occurs at ~284.7 eV (C-C) and 285.5 eV (C-N) for C1s, and intense N1s at 399.8 eV (-N-) (Figure S1). (D) TEM images of Ada-PVP-PEG silver nanorods under different scale. (E) Zeta potential of Ada-PVP-PEG silver nanorods (-14 mV). (F) Hydrodynamic diameter of Ada-PVP-PEG silver nanorods (540 nm).

Amantadine surface-modified silver nanorods enhance HIV vaccine-triggered CTL-derived TNF-α

Figure 3. HIV-specific CTL-derived TNF-α in mice. (A) Vaccination scheme: each mouse receives three intradermal vaccinations with an interval time of three weeks. (B) We design four mouse groups (six mice per each group). Group 1 (red): each mouse receives 50 µg HIV DNA vaccine and 10 µg Ada-PVP-PEG silver nanorods. Group 2 (blue): each mouse receives 50 µg HIV DNA vaccine and 10 µg PVP-PEG silver nanorods. Group 3 (orange): each mouse receives 50 µg HIV DNA vaccine. Group 4 (blank): mice do not receive any vaccination. We use flow cytometry analysis to gate HIV vaccine-triggered CTLs (CD3+CD8+ T cells) from CD3+



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splenocytes, and quantify the percentage of CTLs that produce HIV-specific TNF-α. The corrected value of CTLs% (TNF-α) = The original value of CTLs% (TNF-α) - The background value of CTLs% (TNF-α) (the value in Group 4). The corrected value of CTLs% (TNF-α) > 0.05 is defined as positive. * means p < 0.05.

We investigated the effect of Ada-PVP-PEG silver nanorods on regulating HIV vaccine-triggered CTLs to produce TNF-α in vivo. Here, we designed four mice groups (six mice per each group). Group 1: 50 µg HIV DNA vaccine and 10 µg Ada-PVP-PEG silver nanorods per one mouse. Group 2: 50 µg HIV DNA vaccine and 10 µg PVP-PEG silver nanorods per one mouse. Group 3: 50 µg HIV DNA vaccine per one mouse. Group 4: Mice do not receive any vaccination (blank group). Mice in Group 1, 2, 3 received three vaccinations via intradermal injection (Figure 3A). We used flow cytometry to quantify the percentage of two major subgroups of T cells which can produce HIV-specific TNF-α (CD3+CD4+ T cells/T helper cells and CD3+CD8+ T cells/CTLs). The corrected value of CTLs% (TNF-α) is obtained using the original value of CTLs% (TNF-α) to subtract the background value of CTLs% (TNF-α) (the value in Group 4). Moreover, the corrected value of CTLs% (TNF-α) > 0.05 is defined as a positive TNF-α response. The corrected value of T helper cells% (TNF-α) in Group 1, 2, 3 is 0.029%, 0.030%, 0.027%, suggesting that T helper cells fail to produce TNF-α in Group 1, 2, 3 (Figure S2). In contrast, 0.581% CTLs produced HIV-specific TNF-α in Group 1 (Figure 3B). It is significantly higher than the percentage of CTLs in Group 2 (0.127%) and Group 3 (0.076%) (Figure 3B). Similarly, increased TNF-α concentration also is observed in serum samples from mouse Group 1 (Figure S3), in comparison with other groups. Together, these results indicated that Ada modification predominantly enhanced HIV vaccine-triggered CTLs, instead of T helper cells, to produce TNF-α. Moreover, we detected the percentage of IL-2 and IFNγ produced by CD3+CD4+ T cells/T helper cells and CD3+CD8+ T cells/CTLs in all four groups (Figure S4). IL-2 derived from T Helper cells significantly increased in Group 1 and Group 2, in comparison to Group 3. CTL-derived IFN-γ in Group 1 and Group 2 showed a higher production than that in Group 3.

Amantadine surface-modified silver nanorods improve immunotherapy of HIV vaccine against HIVinfected cells We evaluated whether such an enhancement of HIV-specific CTLs-derived TNF-α can facilitate the death of HIV-infected cells. Firstly, we constructed a HIV-infected cell model according to the previous study.31 TZM-BL cells (a standard cell line for HIV infection) are cultured in a 96-well plate at a density of 1×104 cells/well. 200 TCID50 HIV (50% Tissue Culture Infective Dose, a unit of virus titer) is added into each well for 2 hours incubation at 37 °C to ensure that TZM-BL cells obtain a sufficient infection by HIV. 6 ACS Paragon Plus Environment

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Secondly, HIV-infected cells (1×104 cells) are co-cultured with CTLs (1×104 cells) for 48 hours with a stimulation of HIV peptide (Figure 4A).32 We use enzyme-linked immuno sorbent assay (ELISA) to detect the supernatant histones to evaluate the death of HIV-infected cells, due to dead cells will leak amounts of histones from cell nucleus into culture medium.33 CTLs from Group 1 induce significantly more histone production, in comparison to CTLs from Group 2 and Group 3 (P < 0.05, Figure 4B). Moreover, along with a gradual increase of CTLs from 1×104 to 1×105 cells, the quantity of histone in cell culture medium also increased (Figure 4C). However, the quantity of histone did not continue to increase when we increase CTLs from 1×105 to 1×106 cells in cellular co-culture system (Figure 4C). These results not only suggested that 1×105 HIV-specific CTLs can sufficiently induce the death of 1×104 HIV-infected cells, but also indicated that leaked histones predominantly derive from dead HIV-infected cells, instead of CTLs, due to no increased histones leak even when the number of CTLs increased 10 folds (from 1×105 to 1×106 cells). To more comprehensively evaluate that CTLs-derived TNF-α predominantly mediate the death of HIV-infected cells, we introduced anti-TNF-α monoclonal antibodies (TNF-α mAbs) to block the function of TNF-α. 5 µg/mL TNF-α mAbs are added into co-culture system of HIV-infected cells (1×104 cells) and CTLs (1×104 cells). The introduction of TNF-α mAbs significantly reduced the death of HIV-infected cells (Figure 4D). Together, these results solidly supported that Ada-PVP-PEG silver nanorods can facilitate HIV vaccine-triggered CTLs to produce TNF-α to promote the death of HIV-infected cells. We used flow cytometry to quantify the death rate of HIV-infected cells. The death rate of HIVinfected cells is calculated by the formula (death rate% = (1 – the percentage of viable HIV-infected cells) × 100%). We define dual negative Annexin V-propidium iodide staining cells as viable cells.34 As shown in Figure 4E, 84.19% (1-15.81%) cells are dead in co-culture system of HIV-infected cells and CTLs from Group 1. In contrast, in the co-culture system of HIV-infected cells and CTLs from Group 2, 3 and 4, the cell death rate is 46.88% (1-53.12%), 28.86% (1-71.14%) and 9.82% (1-90.18%) (Figure 4E). It indicated that Ada-PVP-PEG silver nanorods promoted HIV vaccine-triggered CTLs to cause much more death of HIV-infected cells. This result is consistent with above results based on histone detection (Figure 4B). We further analyzed the death stage of HIV-infected cells (early apoptosis or direct necrosis) induced by amantadine surface-modified silver nanorods. We identify the early apoptotic cells and necrotic cells using Annexin V-alone staining (early apoptosis) and dual Annexin V-propidium iodide staining (necrosis) flow cytometry.34 28.71% and 39.06% cells are at early apoptosis in the co-culture system of HIV-infected cells and CTLs from Group 1 and Group 2. Both are far higher than the percentage of early apoptotic cells in the co-culture system of HIV-infected cells and CTLs from Group 3 (9.08%) and Group 4 (4.4%) (P < 7 ACS Paragon Plus Environment


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0.05, Figure 4E). The percentage of necrotic cells in the co-culture system of HIV-infected cells and CTLs from Group 1 (14.88%) and Group 3 (13.05%) is higher than that in Group 2 (7.65%) and Group 4 (4.69%) (Figure 4E).

Figure 4. The death of HIV-infected cells caused by HIV vaccine-triggered CTLs. (A) HIV-infected TZM-BL cells are co-cultured with CTLs isolated from mouse splenocytes. 48 hours later, the death of HIV-infected cells is evaluated via quantifying histone (ELISA) or staining Annexin V/Propidium iodide (flow cytometry). (B) 1×104


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HIV-infected cells are co-cultured with 1×104 CTLs from Group 1, 2, 3, or 4 for 48 hours. Histone in cell culture medium is quantified using ELISA. (C) 1×104 HIV-infected cells are co-cultured with different numbers of CTLs (1×104, 5×104, 1×105, 5×105, 1×106 cells) from Group 1, 2, 3, or 4 for 48 hours. Histone in cell culture medium is quantified using ELISA. (D) Anti-TNF-α monoclone antibodys (5 µg/mL) is added into the co-cultured system of HIV-infected cells (1×104 cells) and CTLs (1×104 cells) from Group 1, 2, 3, or 4 for 48 hours. Histone in cell culture medium is quantified using ELISA. (E) Cells in co-cultured system of HIV-infected cells (1×104 cells) and CTLs (1×104 cells) from Group 1, 2, 3 and 4 are stained by Annexin V and Propidium iodide. Flow cytometry analyzes the percentage of cells that are stained by either Annexin V alone or dual Annexin V-Propidium iodide. (F) HIV-infected cells (1×104 cells) are co-cultured with CTLs (1×104 cells) from Group 1, 2, 3, or 4 for 48 hours. HIV p24 antigen, an indicator for HIV production, is quantified using ELISA (dark green). Histone is detected using ELISA (dark brown). (G) The correlation analysis between HIV p24 production and histone leak. * means p < 0.05.

We evaluated the correlation between the death of HIV-infected cells and HIV production. We calculated HIV production via quantifying HIV capside protein (p24) using ELISA. As a necessary component of HIV, the quantity of p24 is positively correlated with HIV production.35 We co-cultured 1×104 HIV-infected cells and 1×104 CTLs for 48 hours. HIV production in co-culture system gradually increased from group 1 to group 4 (group 1 < group 2 < group 3 < group 4) (Figure 4F). In contrast, the percentage of dead HIV-infected cells in co-culture system gradually decreased from group 1 to group 4 (group 1 > group 2 > group 3 > group 4) (Figure 4F). Correlation analysis showed a significantly negative correction (R2 = 0.9763) between the death of HIV-infected cells and HIV production (Figure 4G). These results indicated that the introduction of Ada-PVP-PEG silver nanorods reduced HIV production via promoting the death of HIV-infected cells.

Biosafety of amantadine surface-modified silver nanorods We firstly tested the cytotoxicity of Ada-PVP-PEG silver nanorods against HeLa cells, HUVECs, macrophages and dendritic cells using cell counting kit. Ada-PVP-PEG silver nanorods with different concentrations (10 µg/mL, 50 µg/mL, 100 µg/mL) showed a relatively satisfactory cytotoxicity after 72 hours incubation (Figure 5A). Ada-PVP-PEG silver nanorods also show a tolerable cytolysis using the hemolysis of sheep red blood cells (Figure 5B). In comparison to normal mice, the introduction of AdaPVP-PEG silver nanorods did not significantly affect the growth of mouse weight (Figure 5C) and multiple important physiological indicators (red cells, white cells, platelet, protein total, albumin, hemoglobin, urea, uric acid, alanine aminotransferase, alkaline phosphatase, aspartate transaminase) in mice (Figure 5D). 9 ACS Paragon Plus Environment


Moreover, immunohistochemical analysis of heart, liver, spleen, lung and kidney showed neither infiltration of inflammatory cells nor necrosis of organ tissue in Ada-PVP-PEG silver nanorods treated mice (Figure 5E). These results proved a satisfactory biosafety of Ada-PVP-PEG silver nanorods.


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Figure 5. The biocompatibility of amantadine surface-modified silver nanorods in vitro and in vivo. (A) The cytotoxicity of Ada-PVP-PEG silver nanorods in vitro. 1×104 HeLa cells, HUVECs, macrophages or dendritic cells are seeded in a well of 96-well plate. Ada-PVP-PEG silver nanorods with different concentrations (10 µg/mL, 50 µg/mL, 100 µg/mL) are added into these cell culture systems. The viability of HeLa cells, HUVECs, macrophages and dendritic cells is detected at Hour 0, Hour 24, Hour 48, Hour 72 by CCK kit (three measurements). (B) Ada-PVP-PEG silver nanorods with different concentrations (from 0 to 10 µg/mL) are incubated with 1×106 sheep red blood cells for 2 hours at room temperature. Hemolysis % = (sample absorbance - negative control absorbance) / (positive control absorbance - negative control absorbance) ×100 %. Commercial red cell lysis buffer (Invitrogen) is used as a control (black). (C) The increased body weight of mice from three different groups (normal mice, mice injected by PBS, mice injected by Ada-PVP-PEG silver nanorods (10 µg per each mouse)). Six mice per group. (D) The effect of Ada-PVP-PEG silver nanorods on multiple physiological indexes (red cells, white cells, platelet, protein total, albumin, hemoglobin, urea, uric acid, alanine aminotransferase, alkaline phosphatase and aspartate transaminase) in mice. Ada-PVP-PEG silver nanorods (10 µg) are intravenously injected into each mouse (n=6). After 24 hours, the blood and urine samples are collected. The experiments are repeated twice. Normal mice is used as control. (E) 10 µg AdaPVP-PEG silver nanorods are injected into one mouse. 30 days later, pathological sections (H&E staining) from heart, liver, spleen, lung and kidney are observed. Normal mice are used as control. Scale bar: 200 µm.

CONCLUSIONS In conclusion, this work reported that Ada surface modification on sliver nanorods significantly enhanced HIV vaccine-triggered CTLs to produce TNF-α in mice. Such an enhancement of HIV-specific CTLderived TNF-α effectively facilitated the death of HIV-infected cells, thus reducing HIV production. This work highlights the importance of surface modification for determining or shaping immunoregulatory profiles of nanomaterials. This work further inspires us that rationally using surface modification can bring a fundamental improvement for the performance of various prophylactic and therapeutic vaccines.

METHODS Materials Ag NO3, PVP (MW 40000), PEG 600, 11-Mercaptoundecanoic acid, 1-Adamantylamine, EDC, NHS, paraformaldehyde and other chemical reagents are purchased from Sigma-Aldrich. Milli-Q water (resistivity > 18.0 mΩ) is used in all preparations. Monoclonal antibodies (anti-mouse CD3e FITC, anti11 ACS Paragon Plus Environment


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mouse CD8a Alexa Fluor® 700, anti-mouse CD4 APC-eFluor® 780, anti-mouse TNF alpha PE-Cyanine7) for the detection of T cell responses are purchased from eBioscience. Anti-Annexin V monoclonal antibody FITC and Propidium iodide are purchased from Becton Dickinson.

Instruments UV spectroscopy (Shimadzu UV-2450) is used to collect UV-Vis absorbance spectrum of silver nanorods. Transmission electron microscope (FEI Tecnai T20) is used for imaging silver nanorods. Malvern Zetasizer is used to detect Zeta potential and hydrodynamic size distribution of silver nanorods. FTIR (Perkin Elmer Spectrum One) and XPS (Thermo Scientific ESCALAB 250XI) are used to analyze the chemical composition of silver nanorods. Enzyme-linked immunosorbent assay plate reader (Thermo Life Sciences, Hampshire, United Kingdom) is used to detect the optical density (OD) of reaction system. Flow cytometry testing is carried out using Calibur Flow Cytometer (Becton Dickinson).

Synthesis of Ada-PVP-PEG silver nanorods PVP-PEG silver nanorods are prepared as our previous report.18 The mixture of PVP (2.5 mL, 1 M, MW 40000), PEG 600 (25 mL) and AgNO3 (0.5 mL, 1 M) is incubated at 70 °C for 1 hour, then heated up to 100 °C for 20 hours. After cooling back to the room temperature, silver nanorods are precipitated with ethanol. Precipitated silver nanorods are dispersed with bath sonication in 20 °C aqueous solution. Ada modification is finished as follows: PVP-PEG silver nanorods (2 mg, 10 mL aqueous solution) are incubated with 11-mercaptoundecanoic acid (0.1 M, 200 µL) overnight at 4 °C. After a re-dispersion of treated PVP-PEG silver nanorods in 10 mL aqueous solution, adamantylamine (0.2 mg/mL) is linked on PVP-PEG silver nanorods using EDC/NHS coupling (EDC:NHS=1:2). Ada-PVP-PEG silver nanorods are stored at 4 °C.

Characterization of Ada-PVP-PEG silver nanorods The morphology of Ada-PVP-PEG silver nanorods are observed using a transmission electron microscope (FEI Tecnai T20). Hydrodynamic size distribution and Zeta potential of Ada-PVP-PEG silver nanorods are tested by Malvern Zetasizer at room temperature. Surface modification of Ada-PVP-PEG silver nanorods are determined using both FTIR (Perkin Elmer Spectrum One) and XPS (Thermo Scientific ESCALAB 250XI). Lyophilized Ada-PVP-PEG silver nanorods are pressed with potassium bromide for FITR detection. A drop of Ada-PVP-PEG silver nanorods solution (on a clean aluminum foil) is dried under a 12 ACS Paragon Plus Environment

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vacuum condition for XPS detection. The distribution of nitrogen, oxygen and carbon is quantified via Xray photoelectron spectrometer.

The construction of HIV DNA vaccine The antigen of vaccine is HIV Gag that is from a predominant epidemic strain in China named CN54 (derived from Chinese isolate 97CN001, B/C recombinant strain). HIV Gag gene is cloned into commercial pcDNA 3.1 eukaryotic expression vector (invitrogen). The construction of HIV DNA vaccine are verified by sequencing (invitrogen), and purified using Qiagen endotoxin-free DNA extraction kit (Va-lencia, CA).

Vaccination Animal experiments are approved by the Animal Ethics Committee of National Center for Nanoscience and Technology and carried out according to guidelines from the Committee of Welfare and Ethics of Laboratory Animals in Beijing. Bal B/C mice (6-8 weeks old) receive three vaccinations via intradermal injection at an interval of three weeks. Two weeks later after the final injection, mouse spleens and blood are harvested for the next flow cytometric assay (TNF-α production of T cells) and ELISA assay (the concentration of TNF-α in serum). We designed four mice groups (6 mice per each group). Group 1: Mice received three vaccinations with 50 µg HIV DNA vaccine and 10 µg Ada-PVP-PEG silver nanorods. Group 2: Mice received three vaccinations with 50 µg HIV DNA vaccine and 10 µg PVP-PEG silver nanorods. Group 3: Mice received three vaccinations with 50 µg HIV DNA vaccine alone. Group 4: Mice without any vaccination (blank group).

Flow cytometry analysis CTL-derived TNF-α production: Spleens are isolated from mice two weeks later after finishing all injections. Fresh splenocytes are stimulated with HIV peptides (2 µg/mL) for 4-6 hours at 37 °C and 5% CO2. Splenocytes are stained with two anti-mouse surface marker antibodies (anti-mouse CD3e, CD4, CD8) for 30 minutes at 4 °C. After fixing with 2% paraformaldehyde for 15 minutes at 4 °C, splenocytes are stained with monoclonal antibodies against intracellular TNF-α for 30 minutes at 4 °C. Samples are analysized by Calibur flow cytometer (Becton Dickinson). The analysis of HIV-infected cells: 5 µL anti-Annexin V monoclonal antibody and 5 µL propidium iodide are added into cell co-cultured system (1-5×105 cells) and keep at room temperature for 15 minutes.



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Cells are washed three times using PBS, and are re-suspended using PBS at a concentration of 1×105 cells/mL. Cells are analyzed by Calibur flow cytometer (Becton Dickinson).

Cell isolation Mouse spleens are harvested and grinded in sterile cell culture plates. 2 mL red cell lysis buffer is added into 1×107 splenocytes and keep 5 minutes to sufficiently remove red cells. Cells are centrifuged at 1000 rpm for 5 minutes and re-suspended in DMEM cell culture medium (10% FBS). Lymphocytes resuspended in DMEM culture medium are isolated using commercial separation kit (mouse plasmacytoid CD8+ T cell isolation kit, mouse plasmacytoid DC isolation kit, mouse APC/macrophage positive selection kit; STEMCELL Technologies) according to the protocol.

Cell death and HIV production detection TZM-BL cells (1×104 cells per each well) are prepared in a 96-wells plate overnight. Live HIV (200 TCID50 / 50% Tissue Culture Infective Dose) is added into each well for 2 hours incubation at 37 °C. CTLs (1×104 cells, 5×104 cells, 1×105 cells, 5×105 cells, 1×106 cells) are co-cultured with HIV-infected TZM-BL cells (1×104 cells). 96-wells plates (Costar, Corning, NY) are coated with 0.1 µg/ml anti-histone H2B monoclonal antibody (or anti-HIV p24 monoclonal antibody) in phosphate buffer solution (PBS) at 4 °C overnight. ELISA plates are washed five times with naked PBS, followed by PBS with 3% bull serum albumin (BSA) for 2 hours at 37 °C. 100 µl supernatant from HIV-infected TZM-BL cells culture system is added into each well. After incubating for 1 hour at room temperature, plates are washed with naked PBS five times, and are incubated with ALP-labeled anti-histone H2A-H2B dimer monoclonal antibody (or ALP-labeled antiHIV p24 monoclonal antibody) for 1 hour at 37 °C. Plates are washed with naked PBS five times. Tetramethylbenzidine (TMB) substrate (100 µl each well) is added and incubated for 10 minutes. The reaction is stopped by adding 25 µl 2M H2SO4. The optical density (OD) of reaction system is measured at 450 nm by enzyme-linked immunosorbent assay plate reader.

Cell viability HeLa cell, HUVECs, macrophages and dendritic cells (1×104 cells per each well) are seeded in DMEM cell culture medium (10% fetal bovine serum/FBS, 1% penicillin/streptomycin) overnight at 37 °C and 5 % CO2. Ada-PVP-PEG silver nanorods (10 µg/mL, 50 µg/mL, 100 µg/mL) are added into cell culture 14 ACS Paragon Plus Environment

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medium, and are incubated for 72 hours at 37 °C and 5 % CO2. The viability of cells is detected using commercial CCK-8 kit.

Hemolysis 1×106 sheep red blood cells are co-cultured with Ada-PVP-PEG silver nanorods with different concentrations (from 0 to 10 µg/ml) for 2 hours at room temperature. We centrifuge the cell co-culture system at 1000 rpm/min for 5 minutes and transfer supernatant into a 96-well plate. The OD value of each well is read by a microplate reader (570 nm). The percentage of hemolytic sheep red blood cells is calculated: hemolysis % = [the absorbance value of sample − the absorbance value of negative control] / [the absorbance value of positive control − the absorbance value of negative control] × 100%.

The detection of physiological indicators Each mouse receives 10 µg Ada-PVP-PEG silver nanorods via intravenous injection. After 24 hours, mouse blood samples are harvested. Multiple important physiological indicators in mouse blood (red cells, white cells, platelet, protein total, albumin, hemoglobin, urea, uric acid, alanine aminotransferase, alkaline phosphatase and aspartate transaminase) are analyzed by routine blood examination instrument (CoulterJT) and biochemical detector (Roche cobas 6000). Blood samples from normal mice are used as blank control.

Immunohistochemical analysis Mice are intravenously injected with 10 µg PVP-PEG silver nanorods. 30 days later, liver, kidney, spleen, heart and lungs are isolated. Pathological sections derived from these mouse organs are prepared using H&E staining. Pathological sections are diagnosed and imaged using an optical microscopy with a phototaking function (Leica).

Statistics Values are expressed as means ± standard deviations (SD). Analysis of differences in means between groups is conducted by one-way analysis of variance (ANOVA). P < 0.05 is considered significant.



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ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Details of experimental procedures. The authors declare no competing financial interests.

AUTHOR INFORMATION Corresponding Author [email protected], [email protected], [email protected]

ACKNOWLEDGEMENTS We thank Scientific Research Improvement Project of Beijing University of Agriculture (Project GZL2018001), Beijing Outstanding Talent Training for Young Backbone Individual Projects (Project 2016000020124G049), Research Fund for Young Scientists of BUA (Project SXQN201805), National Science Foundation of China (31500816) and the Ministry of Science and Technology of China (2018ZX10731101-001-014) for financial support.

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Amantadine Surface-Modified Silver Nanorods Improves

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