Periodic Arrangement of Lipopolysaccharides Nanostructures

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Periodic Arrangement of Lipopolysaccharides Nanostructures Accelerates and Enhances the Maturation Processes of Dendritic Cells Yang Liu, Kang-hsin Wang, Huan-Yuan Chen, Jieren Li, Ted A. Laurence, Sonny Ly, Fu-Tong Liu, and Gang-yu Liu ACS Appl. Nano Mater., Just Accepted Manuscript • DOI: 10.1021/acsanm.7b00254 • Publication Date (Web): 29 Jan 2018 Downloaded from http://pubs.acs.org on January 30, 2018

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ACS Applied Nano Materials

Periodic Arrangement of Lipopolysaccharides Nanostructures Accelerates and Enhances the Maturation Processes of Dendritic Cells

Yang Liu,1 Kang-Hsin Wang,1,2 Huan-Yuan Chen,2,3 Jie-Ren Li,1 Ted A. Laurence,4 Sonny Ly,4 Fu-Tong Liu,2,3 and Gang-Yu Liu1*

1

Department of Chemistry, University of California, Davis, California 95616, United States 2

Department of Dermatology, School of Medicine, University of California, Davis, Sacramento, California 95817, United States 3

4

Institute of Biomedical Sciences, Academia Sinica, Taipei, 8862 Taiwan, ROC

Lawrence Livermore National Laboratory, Livermore, CA 94550, United States

*Author to whom correspondence should be addressed: [email protected]

ACS Paragon Plus Environment

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ABSTRACT: This work reports an important application of nanomaterials consisting of biological ligands, such as lipopolysaccharides (LPS) nanostructures. Upon immobilization onto surfaces as periodic arrays of nanostructures, these surface bound LPS nanostructures significantly impacted the maturation process of bone marrow derived dendritic cells (BMDCs): accelerating the maturation process in comparison to other means of ligand presentations such as LPS solution and monolayers on surfaces. In some cases, LPS nanostructures led to a new maturation phenotype in vitro characterized by hyperdendritized morphology. This level of maturation enhancement was observed for the first time in vitro. Possible mechanisms leading to the acceleration and enhancement of BMDC maturation were also discussed. This work demonstrates an important concept: surface bound nanostructures of ligands provide a new and powerful cue for regulating cellular signaling processes. The fact that primary cells’ signaling processes, such as BMDC maturation, can be altered suggests a new means for programming DCs for immune therapy.

KEYWORDS: nanomaterials, bone marrow derived dendritic cells (BMDC), maturation, lipopolysaccharides (LPS), atomic force microscopy (AFM), scanning electron microscopy (SEM), laser scanning confocal microscopy (LSCM) 1. INTRODUCTION


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Dendritic cells (DCs) are antigen-presenting cells with a unique ability to initiate and modulate primary immune responses.1-5 DCs initially reside and circulate in peripheral tissues in an immature state with high antigen-capturing capability.2, 6 Following tissue damage, immature DCs capture antigens and become activated by pathogens or factors such as inflammatory cytokines. These activated DCs undergo a maturation process and migrate from peripheral tissues to the lymphoid tissues where they present antigens to Tcells which primes the subsequent immune responses.1, 6, 7 Only mature DCs can present antigens to T-cells to regulate the immune behavior of T-cells and ultimately final immune outcomes of the body.1, 2, 8 Therefore, utilizing DCs’ unique antigen-presenting capability, much effort has been invested to pre-expose DCs using designed activation ligands followed by loading with tumor-associated antigens, in vitro or in vivo, in the hope of generating potent and specific antigen presentations to trigger T-cells’ immune responses against the targeted tumor or cancer.9, 10 Prior efforts have identified several important ligands to trigger DC activation, and maturation, wherein the mature DCs would capture and prime downstream T-cell responses.1, 2, 8, 11, 12 The most frequently used stimulant is bacterial lipopolysaccharides (LPS), which can be recognized by Toll-like receptor 4 (TLR4) at DC membrane.1-3 Upon binding to LPS molecules, TLR4 receptors mediate activation cascades into maturation of DCs, characterized by dendritic morphology, enhanced membrane ruffling, and expression of various maturation markers, e.g., clustering of differentiation 86 (CD86).1-3,

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The

dendritic morphology, featured by extending long, delicate dendrites, gives rise to the term “dendritic” cell.2 A net of fine dendrites also contributes to the enhanced immune efficacy, as these dendrites sweep their surroundings to select antigen-specific T cells in lymph


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nodes.13 Higher surface/volume ratio and swept volume increases the probability of capturing T-cells in comparison to that among immature DCs.8, 13-15 Recently, dendrites have also been reported as a requirement for transfer of human immunodeficiency virus type 1 (HIV-1) from DCs to T-cells.16 The ruffling of the DC’s membrane further enhances immune efficacy. Upon initial contact with T-cells by dendrites, changing and roughening of cellular membrane enhances the interactions among DCs and T-cells, and as such facilitates interaction among antigen and T-cells.8 Expression of maturation markers contribute further into T-cell activation. CD86, for example, is a co-stimulatory molecule responsible for constituting “Signal II”, i.e., promoting T cell proliferation in the process of priming T cells.17-21 Therefore, the aforementioned three aspects of DC activation and maturation also serve as important and characteristic readouts for researchers to access the immune efficacy of mature DCs.2, 3, 8, 22 Guided by these important insights, formidable attempts were made to initiate DC maturation followed by specific antigen presentation.3, 7, 23, 24 Typically, DC maturation was generated in vitro by culturing cells in media containing 1 μg/mL of LPS for 24 h.3 LPS originates from bacteria and is widely used for DC maturation.25 This approach is simple, however, it is often used as a binary practice, i.e. lacking tunability, or resulting in either mature or immature DCs. At 1 μg/mL LPS concentration, for example, the CD86 expression remained low during the first 6 h of incubation, progressively increased and peaked at 15 h, then fluctuated slightly but became steady and plateaued at 24 h.26 Although most studies used 1 μg/mL LPS and 24 h as a standard maturation protocol, the CD86 expression actually plateaued as the LPS concentration reached 100 ng/mL.24, 27 In addition to the lack of tunability, the 0.1–1 μg/mL LPS concentrations are too high to be translated


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due to side effects, such as sepsis.28 Besides the most well-known ligand LPS, other ligands include lipopeptides,29 single stranded RNA,30 and methylated oligonucleotides,28 as well as tumor necrosis factor alpha (TNF-α),3, 12 CD40 ligand,31 and interferon (IFN).32 These approaches lead to DC maturation via various pathways and were also used as binary pratice.25 Most of those ligands required 24 – 48 h incubation to mature DCs,25, similar to sLPS, and IFN-α (1000 U/mL) required ~8 h to reach DC maturation.33 In general, using ligand solutions have similar limitations to those of LPS solutions. To enable tunability and translatability, ligand presentation should be simple, enable regulation of DC maturation beyond binary control, and exhibit low toxicity. Toward these application requirements, this work reports an alternative presentation of LPS, e.g., surface bound nanostructures. The rationale behind this approach is the current knowledge of LPS triggered DC maturation mechanism: LPS binding and clustering of TLR4 at DC membrane triggers the downstream maturation cascades.

34-36

Therefore, by molecular

level regulation of the spatial arrangement of LPS, one could regulate ligand-TLR4 binding and clustering,37-39 impacting DC maturation. Taking advantage of the high spatial precision offered by today’s nanotechnology,37, 38, 40-43 the arrangement of ligands could be regulated from molecular, to nanometer, and to macroscopic level.40-42, 44-48 The surface bound presentation of ligand nanostructures has shown great promise to regulate cellular responses in other cell types, mostly cell lines.37-39, 49-51 This work tests if/how surface bound nanostructures of LPS ligands impact the activation and maturation of primary cells, e.g., bone marrow derived dendritic cells (BMDCs). Faster and higher efficacy are seen among LPS nanostructures than in soluble ligands. The outcomes were particularly encouraging as the efficacy of nanostructures is demonstrated using primary cells, BMDC,


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instead of cell lines. The underlying mechanism is also discussed to explain the observed efficacy. These findings bring us closer to the “Holy Grail” of programming cellular behavior and function for therapy.

2. MATERIALS AND METHODS 2.1. Reagents. Reagents were used without further purification unless described specifically.

Organosilanes,

including

2-[methoxy(polyethyleneoxy)6-9-

propyl]trichlorosilane (OEG-silane) and octadecyltrichlorosilane (OTS), were purchased from Gelest (Morrisville, PA). Silica microspheres were purchased from Thermo Scientific (Waltham, MA). The spheres were suspended in their original concentration of 2% (w/v, aqueous) until usage. Polished silicon wafers, Si(111) doped with boron, were purchased from Virginia Semiconductor Inc. (Fredericksburg, VA). Lipopolysaccharide (LPS, from Escherichia coli O111:B4, purified by phenol extraction), toluene, sulfuric acid (95.0 – 98.0%), hydrogen peroxide (30% aqueous solution), 37% formaldehyde solution, 25% glutaraldehyde solution, 4% osmium tetroxide (OsO4) solution, hexamethyldisilazane (HMDS), red blood cell lysis buffer, bisbenzimide H 33342 trihydrochloride (Hoechst 33342, ≧ 98%), normal donkey serum, bovine serum albumin (BSA, heat shock fraction, Australia origin, protease free, low fatty acid, low IgG, pH 7, ≧ 98%), and sodium azide (≧ 99.5%) were purchased from Sigma-Aldrich (St. Louis, MO). Ethanol (EtOH, 99.5%) was purchased from KPTEC (King of Prussia, PA). Ammonium hydroxide (NH4OH, 28.0 – 30.0% aqueous solution) and potassium chloride (KCl, 99.7%) were purchased from Fischer Chemicals (Fair Lawn, NJ). Recombinant murine granulocyte macrophage colony – stimulating factor (GM-CSF) was purchased from PeproTech Inc. (Rocky Hill, NJ). Fetal


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bovine serum (FBS, heat-inactivated), Roswell Park Memorial Institute (RPMI) 1640 medium, HEPES buffer solution (1 M), Dulbecco's Phosphate Buffered Saline (DPBS, 1X), and penicillin/streptomycin (10000 U/mL) were purchased from Gibco by Life Technologies (Grand Island, NY). Paraformaldehyde solution (4% in DPBS) was purchased from ChemCruz (Santa Cruz, CA). Nitrogen gas (99.999%) and hydrogen gas (99.95%) were purchased from Praxair, Inc. (Danbury, CT). Two types of eFluor®615 labeled American hamster immunoglobulin G (IgG) were purchased from eBioscience (San Diego, CA): hamster-anti-mouse CD11c eFluor®615 (refer to as anti-CD11c-615, 0.2 mg/mL), and IgG with eFluor®615 label (IgG-615, 0.2 mg/mL) serving as isotype matched control or non-specific antibody. Two types of alexa Fluor®488 labeled rat IgG2aκ were purchased from BioLegend (San Diego, CA): rat-anti-mouse CD86 (referred to as antiCD86-488, 0.5 mg/mL), and rat IgG2aκ (referred to as IgG-488, 0.5 mg/mL) serving as isotype control antibody. C57BL/6 mice were purchased from the Jackson Laboratory (Sacramento, CA). Deionized and ultrapure water was obtained from a MilliQ water system (EMD Millipore, Billerica, MA). Multi-well tissue culture plates (Falcon®, polystyrene, flat bottom with lid, sterilized, non-pyrogenic) were purchased from Corning Incorporated-Life Science (Durham, NC). Carbon conductive adhesives tabs (PELCO TabsTM, 12 mm diameter) and scanning electron microscope (SEM) pin stub mounts (Al, with 12.7 diameter and 8 mm pin height) were purchased from Ted Pella, Inc. (Redding, CA). Petri dishes (50 mm diameter), each with a glass bottom (#1.5 coverslip, 30 mm diameter), were purchased from MatTek Corporation (Ashland, MA). 2.2. Preparation of LPS Nanostructures on Silicon Substrates. Particle lithography followed by self-assembly chemistry was used to first produce periodic structures of


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methyl-terminated nanodomains on silicon surfaces, following previously reported protocols.38, 40, 52 Polished silicon wafers were cleaned by immersion in piranha solution for 1 h and subsequently in basic solution at 70 ºC for 1 h. Piranha solution was prepared by mixing H2SO4 and H2O2 at a volume ratio of 3:1. It is highly corrosive and should be handled with caution. Basic bath was prepared by mixing NH4OH solution, H2O2 solution, and H2O at a volume ratio of 1:1:5. Silicon wafers were then rinsed copiously with water and dried in N2. Silica microspheres were washed, and then separated from solvent by centrifugation to remove additives such as charge stabilizers or surfactants. After cleaning, silica microspheres were re-suspended in aqueous solutions by sonication, and used immediately afterward. A drop (25 μL) of the silica microsphere solution was deposited on a clean 1 cm × 1 cm Si(111) surface, and allowed to spread and dry to produce a mask of closely packed microspheres. Next, the wafer was placed into a sealed Teflon container (100 mL) containing 20 mL toluene solution of OEG-silane and with a concentration of 5 mM for 1 h. During immersion, OEG-silane molecules from solution attached to uncovered interstitial areas. The silica microspheres masks were removed by 2 sonication steps, first in ethanol and then in MilliQ water, for 10 min in each step. These OEG-silane/Si(111) were immersed in 5 mM OTS solution (in toluene) for 5 min, allowing the OTS to attach to the voids on the Si(111) surfaces. Afterward, the samples were cleaned by the aforementioned sonication steps. Finally, the LPS-functionalization was achieved using hydrophobic effect between lipid A of LPS and alkyl chain of the OTS nanopatterns, following a previously reported protocol.53 First, LPS was suspended in a HEPES buffer solution (pH= 7.3, 5 mM HEPES and 100 mM KCl) at a concentration of 2 mg/mL. Then, the OTS nanopatterns prepared


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were soaked in the LPS suspension for 1 h at 50 ℃, allowing LPS to immobilize onto the OTS nanostructures. Finally, these LPS nanostructures were rinsed with MilliQ water to remove HEPES and KCl, and loosely bound LPS molecules. The Si pieces containing clean LPS nanostructures were placed individually in multi-well plates, and used immediately for subsequent investigations. 2.3. Preparation of LPS Immobilized on Pure and Binary Mixed Self-Assembled Monolayers. A full-covered LPS monolayer was produced by immobilizing LPS onto selfassembled monolayers (SAMs) of OTS. First, 1 cm × 1 cm pieces, each with a clean Si(111) surface, were immersed into a toluene solution containing 5 mM OTS for 5 min. After being rinsed with ethanol, the OTS SAMs were immersed into the 2 mg/mL LPS suspension for 1 h at 50 ℃. Finally, the LPS/OTS SAMs were rinsed with MilliQ water, then placed individually in multi-well plates for immediate usage. LPS on mixed-SAMs were produced similarly except for the adsorbates, in this case, a mixture of OTS:OEG-silane = 1.5:98.5 with concentrations of 0.075 and 4.925 mM, respectively. The OTS domains randomly distributed in the matrix OEG SAMs, where the LPS molecules attached. After cleaning, LPS/mixed SAMs were placed individually in multi-well plates and used immediately for subsequent investigations. 2.4. Preparation of BMDCs. BMDCs were cultured from C57BL/6 mice by culturing bone marrow cells as described previously.3 In brief, C57BL/6 mice were euthanized by CO2 inhalation following protocols approved by the Institutional Animal Care and Use Committee at University of California, Davis. After removing all muscle tissues from femur and tibia, the bones were washed with DPBS. Then, both ends were cut to expose the lumen and bone marrow was flushed from the bones. Clusters within the marrow


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suspension were disintegrated by vigorous pipetting. After centrifuging at 500 g for 10 min, the cells were collected and suspended in DPBS. Following addition of red blood cell lysate for depletion of erythrocytes, the bone marrow cells were washed with DPBS and resuspended in complete RPMI 1640 medium containing 20 ng/mL recombinant GM-SCF, 10% FBS, and penicillin/streptomycin (100 U/mL). The cells were cultured at 37.0 ℃ in an incubator containing 5% CO2. The medium was replaced once every two days of culture. After 8 days, culture medium was switched to the one containing 10 ng/mL of GM-CSF. After culturing for another 3 days, suspending cells in the medium, i.e., immature BMDCs, were harvested and transferred into multi-well plates for further experiment. The silicon substrates with designed LPS presentations were placed individually at the bottom of multi-well cell culture plates. For each investigation, we also performed control experiments. Two controls were used for this work. For each control, a silicon substrate with clean Si(111) surface was used. First, LPS solutions with designed ligand concentration, referred to as sLPS, were prepared by simply adding 1 μL of 1 mg/mL LPS solution (DPBS based) into the 1 mL culture media to reach a concentration of 1 μg/mL. Upon placing all presentations individually inside multi-well plates, 1 mL of culture media containing the suspending BMDCs (7.5 × 104 mL-1) was added to each well. The plates were first subject to a centrifugation (Marathon 3000R Benchtop Centrifuge, Fisher Scientific, Hampton, NH) at 350 g for 5 min to ensure attachment of BMDCs to surfaces, then placed into incubator at 37 ℃ and 5% CO2 for designed periods before subsequent investigations. 2.5. Immunocytochemistry Staining of BMDCs. Cell samples were labeled using a fluorescent immunocytochemistry (ICC) staining technique, following a modified protocol


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developed by R&D Systems Inc. (Minneapolis, MN). First, a series of reagents were prepared. Donkey serum was diluted with DPBS into a 10% solution and used as a blocking buffer. A DPBS solution containing 1% BSA, 1% donkey serum, and 0.01% sodium azide was used as dilution buffer. A 0.1% BSA solution in DPBS was used as a wash buffer. Hoechst 33342 was dissolved in DPBS into a 0.1 mg/mL solution followed by dilution with dilution buffer into a 1 μg/mL staining solution. The BMDCs on surfaces prepared in section 2.4 were taken out from wells and rinsed with DPBS. After a 30 min immersion in paraformaldehyde solution (4% in DPBS) for fixation, the samples were rinsed with wash buffer 3X, followed by soaking in blocking buffer at room temperature for 45 min. After removing the blocking buffer, the samples were covered with a 160 μL 1:1 mixture of anti-CD11c-615 : anti-CD86-488 with desired concentration. For each staining experiment above, a twin sample was prepared with a 160 μL 1:1 mixture of IgG-615 : IgG-488 with same concentration as isotype matched control. For fresh prepared cell samples, the antibody stock solutions were diluted with dilution buffer into a 1.25 μg/mL solution and used for ICC staining. For post-SEM cell samples, the antibody stock solutions were diluted with dilution buffer into a 10 μg/mL solution and used for ICC staining. After 1 h incubation at 2 – 8 ℃, the cell samples were rinsed two times with wash buffer, followed by covering with 80 μL of 1 μg/mL Hoechst 33342 staining solution to label cell nucleus for 2 min. The cell samples were rinsed with wash buffer and stored in wash buffer for Laser scanning confocal microscope (LSCM) imaging. 2.6. LSCM Imaging of BMDCs. Laser scanning confocal microscope (LSCM, Olympus FluoViewTM FV1000, Olympus America, Central Valley, PA) was used to image the cell samples. For LSCM imaging of samples on silicon substrate, the samples were


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placed face-down in a MatTek’s glass-bottom dish and soaked in washing buffer. The LSCM images were collected between 443 and 493 nm under the excitation of 405 nm laser for Hoechst 33342 channel; between 519 and 590 nm under the excitation of 488 nm laser for anti-CD86-488 or IgG-488 channel; between 590 and 690 nm under the excitation of 543 nm laser for anti-CD11c-615 or IgG-615 channel. A series of 2D cross-sectional views were taken from the cell-substrate interface to the top. Intensity projection over Z axis of 2D cross-sectional views was collected to visualize the protein or nucleus distribution. All LSCM images were acquired and analyzed using Olympus FluoView 1.7 software. 2.7. SEM Imaging of BMDCs. Sample preparation for SEM characterization followed known protocols.37, 38 Briefly, a 1:1 mixture of 25% glutaraldehyde and 37% formaldehyde was diluted 10-fold with deionized water and used as a primary fixative. The silicon wafers containing BMDCs were taken out from culture medium and rinsed with DPBS. Then, the wafers were immersed in primary fixative for 30 min. The 4% OsO4 solution was diluted with deionized water into a 1% solution for sample preparation. After 1 h immersion in primary fixative, the samples were rinsed with deionized water 3X followed by soaking in 1% OsO4 solution for 30 min. Cell samples were then subjected to a sequential 10 min immersion-removal of ethanol/water mixtures with an increasing ethanol content of 25, 50, 75, 90, and 100%, respectively. Final dehydration in pure ethanol was repeated three times, followed by addition of HMDS, the drying agent for biological samples as previously reported.54 The surfaces containing cells were then placed onto an SEM pin stub mount via a thin carbon conductive adhesive to improve electrical conductivity. After being transported to the SEM vacuum chamber, the BMDCs cells were


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imaged with a Hitachi S-4100T field emission SEM (FE-SEM) (Hitachi High Technologies America, Inc., Pleasanton, CA), using an accelerating voltage of 2 kV at 10 μA. 2.8 AFM Imaging. SAMs, all surface presentation of LPS, and selected BMDC cells were imaged using atomic force microscopy (AFM). Atomic Force Microscope (MFP-3D, Asylum Research Corp., Santa Barbara, CA) was used for the structural characterization of LPS nanostructures, SAMs, and cells. AFM images of nanostructures were acquired under contact mode with imaging forces of 15 – 25 nN using silicon cantilevers with a spring constant of 0.1 N/m, purchased from Bruker (MSNL cantilever E, Camarillo, CA). AFM images of SAMs were acquired under tapping mode in ambient condition using silicon cantilevers with a spring constant of 0.6 N/m were purchased from Bruker (MSNL cantilever F, Camarillo, CA). The driving frequency was set at the fundamental resonance of the cantilever, 100 to 110 kHz, and the damping was set to 65%. To measure the SAMs’ height or thickness, a 500 nm × 500 nm areas of adsorbates were displaced at 40 – 45 nN under contact mode in ambient condition, followed by imaging at 2000 nm × 2000 nm under tapping mode using the same probe.41 For BMDCs, sample preparation for AFM imaging followed similar protocol to those for SEM characterization, except that OsO4 soaking was skipped. AFM images of BMDCs were acquired under tapping mode in ambient condition with using silicon cantilevers with a spring constant of 0.03 N/m that were purchased from NanoAndMore (CSC38 cantilever B, Watsonville, CA). The driving frequency was set at the fundamental resonance of the cantilever, 10.1 kHz, and the damping was set to 40%.


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For hyper-dendritized BMDCs, the SEM samples were imaged with AFM after being rinsed with ethanol and dried in air. AFM images of hyper-dendritized BMDCs were acquired under contact mode with imaging forces of 3 nN using silicon cantilevers with a spring constant of 0.03 N/m purchased from NanoAndMore (CSC38 cantilever B, Watsonville, CA). The AFM images were acquired using Asylum MFP-3D software developed on the Igor Pro 6.20 platform. 2.9. Quantification of LPS Coverage. The LPS coverage of each LPS nanopattern is defined as area covered by LPS over the total surface area. Using the hexagonal unit mesh of LPS nanofeatures, the LPS coverage = π(D∕2)2/a2 sin120°, where D is the diameter of the LPS nanofeature measured from AFM topographs, and a represents periodicity of the LPS pattern, i.e., the distance between two nearest neighbor nanofeatures, measured from AFM images. Due to the lack of periodicity, the quantification of LPS coverage among LPS/OTS SAMs and LPS/mixed SAMs were carried out by importing the AFM topographs into ImageJ. The LPS features are identified because the LPS region is taller than the surroundings (see section 3.2). The LPS coverage can be quantified from the marked AFM images: areas occupied by LPS area/total image area.

3. RESULTS AND DISCUSSION 3.1 Key Experimental Steps. The key experimental steps are summarized in the schematic diagram in Figure 1. Detailed protocols of each key step are included in Methods. First, periodic nanostructures of LPS were designed and produced on silicon wafer surfaces


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using particle lithography.38, 40, 52 These engineered structures were then placed in culture plates and exposed to BMDCs in RPMI 1640 medium in a culture incubator. A gentle spin of 350 g for 5 min was necessary to speed up the cell adhesion and assure BMDC-LPS contact/interactions. After designated incubation time, BMDCs were fixed to allow characterization of dendrites, membrane ruffling, and CD86 expression, using LSCM, SEM, and AFM, respectively. In comparison, BMDCs activated using conventional approaches, i.e., soluble LPS (sLPS) in designed concentrations, were incubated for the same amount of soaking time, then spun down at 350 g for 5 min onto clean silicon surfaces for LSCM, SEM, and AFM imaging.

Figure 1. Schematic diagram to illustrate the key steps for comparing the activation of BMDCs initiated by surface bound nanostructures of LPS (left) with that by soluble LPS (right). 3.2 Presentations of Lipopolysaccharides. As discussed above, we compared the nanostructure presentation of surface-bound LPS with the conventional means of LPS solutions. First, following the conventional approach reported previously,1-3 LPS solution with concentration of 1 mg/mL in DPBS was directly added into cell culture media reaching a final concentration of 1 μg/mL. Second, a series of surface bound LPS were produced. Hierarchical LPS structures with designed periodicity were produced on silicon surfaces: productions of arrays of OTS inlaid in OEG-silane SAMs via particle


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lithography,38, 40, 52, 55 followed by immobilization of LPS onto the OTS nanostructures.53 The immobilization of LPS onto methyl-terminated OTS nanostructures was selective, as the surrounding OEG-silane SAM resisted protein adhesion.56, 57 The hierarchical LPS structures were characterized with nanometer resolution using AFM, as shown in Figure 2. The imaging area shown in Figure 2 is 10 × 10 μm2 to best reveal what individual BMDC “see and interact” upon contacting these engineered surfaces, as the typical diameter of BMDC on surfaces ranges from 8 to 18 μm.3, 58 For each presentation, we measured the total number of LPS nanofeatures, the lateral dimension and apparent height of these LPS features, the surface coverage of LPS, number of features and LPS molecules per BMDC cells. For example, in Figure 2B, there were 196 LPS nanodots (bright features). The LPS nanofeatures measured 92 ± 6 nm wide (full width at half maximum, FWHM), and were 5.9 ± 0.7 nm taller than the surrounding OEG SAMs, consistent with nearly closely packed LPS molecules. The LPS patches were arranged in a hexagonal lattice with the periodicity of 709 ± 5 μm. This structure was referred to as “LPS-Nano2”. The actual LPS coverage, in the case of LPS-Nano2, was relatively low, 1.5%. A typical BMDC covers ~ 100 μm2 area upon attachment to surfaces as indicated by prior studies as well as our observations,2, 3

and each LPS occupies ~2 nm2 area assuming standing up on surfaces.59 Therefore, we

could estimate the number LPS molecules underneath each cell (Table 1). The nanofeature size, periodicity, LPS coverage, and number of LPS per BMDC for all 4 engineered LPS structures were summarized in Table 1. In each experiment, the hierarchical ligand structures covered the entire silicon surface of 1 cm × 1 cm area. For each type of nanostructure, at least 4 sets of lithography were carried out to assure consistency and


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reproducibility. For each set of experiment, the AFM imaging was acquired by randomly selecting 6 locations from the surface.

Figure 2. AFM topographic images of four LPS nanostructures, referred to as (A) LPSNano1, (B) LPS-Nano2, (C) LPS-Nano3, (D) LPS-Nano4, respectively. Inset in (D) shows a zoom-in view of LPS-Nano4. Scale bars were 1 µm, except the inset, which was 200 nm. AFM images of four nanostructures were acquired under contact mode in ambient condition, with imaging force of 25 nN in (A – C), and 15 nN in (D) and inset. TABLE 1. The LPS Presentation, Quantified by Periodicity, Domain Separation, Apparent Height, Feature Size, LPS Coverage, Number of LPS Nanostructures per BMDC, and Number of LPS Molecules per BMDC LPS Presentation

Domain Periodicity Separation (nm) (nm)

Apparent Height (nm)

Feature size (nm)

LPS Covera ge (%)

# of LPS # of LPS NanoMolecules per features per cell cell

LPS-Nano1

1002 ± 5

1002 ± 5

5.6 ± 0.7

102 ± 5

0.9 ± 0.1

101 ± 7

(4.5 ± 0.5) × 105

LPS-Nano2

709 ± 5

709 ± 5

5.9 ± 0.7

92 ± 6

1.5 ± 0.2

201 ± 12

(7.6 ± 0.9) × 105

LPS-Nano3

507 ± 6

507 ± 6

5.7 ± 0.5

82 ± 5

2.3 ± 0.4

504 ± 25

(1.2 ± 0.2) × 106

LPS-Nano4

210 ± 3

210 ± 3

5.2 ± 0.5

72 ± 5

11.0 ± 0.9

1789 ± 45

(5.5 ± 0.5) × 106

LPS/mixed SAM

N/A

24 – 111

1.4 – 3.7

8 – 18

1.0 ± 0.1

N/A

(5.0 ± 0.4) × 105

LPS/OTS SAM

N/A

Continuous

0.4 ± 0.1

N/A

(4.5 ± 0.1) × 107

Continuous

90.3 ± 1.7


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3.3 LPS Nanostructures Led to Faster CD86 Expression than That via Soluble LPS. The time-dependence of CD86 expression using soluble LPS has been thoroughly studied in prior investigations, most of which utilized flow cytometry, also known as fluorescenceactivated cell sorting (FACS).3, 8, 60 Due to the nature of surface bound presentation in our approach, LSCM was utilized to measure CD86 expression. First, we compared the measurements from LSCM and flow cytometry using sLPS activation of BMDCs. In LSCM measurements, three dyes were selected for ICC staining (see Materials and Method for details):3, 61 Hoechst 33342 to highlight nucleus; anti-CD11c-615 to recognize CD11c, a preferential integrin in DC; and anti-CD86-488 to highlight CD86 expression. The first two dyes marked viable BMDCs, while anti-CD86-488 revealed the cells with detectable expression of CD86. Under our sample preparation of BMDCs, > 95% of the BMDCs were CD11c+, which indicated high purity and high viability in our practice. The population of BMDCs with high CD86 expression was measured by counting the anti-CD86-488 cells over those stained by both Hoechst 33342 and anti-CD11c-615. In the case of sLPS, counting over 150 cells, 65.7 ± 6.5% BMDC revealed detectable expression of CD86 after 24 h incubation, which was consistent with that in FACS measurements.3 These measurements and comparisons demonstrate that LSCM provides reliable readout of CD86 expression, which is important for this investigation which requires measurements of CD86 expression at single cell level. The time-dependence of the CD86 expression was shown in Figure 3A, where sLPS (red bar) and LPS-Nano2 (black bar) clearly differed. In the case of sLPS, the CD86 expression increased slowly and non-linearly with incubation time, less than 10% within the first 2 h, reaching 40% after 6 h, and saturated (~70%) after 24 h. The measurements were taken


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based on 5 sets of experiments, counting over 240 cells in each condition. LPS-Nano2, in contrast, led to a much more rapid increase in CD86 expression. Similar CD86 expression was seen during the first couple of hours compared to that in sLPS. The CD86 expression quickly surged to 43% at 4 h, and 76% at 6 h, and then remained roughly plateaued. The measurements were taken based on 2 sets of experiments, counting over 100 cells in each treatment. The corresponding LSCM images were shown in Figure 3B, where the highlevel expression of CD86 was very evident. At 6 h for example, only 4 CD86+ cells out of 16 BMDCs were seen in sLPS, while 16 out of 22 cells were CD86+ in the case of LPSNano2. The fluorescent intensity of the latter was also much higher.

At concentration of

1 μg/mL, which is significantly below the critical micelle formation of 15 μg/mL,62 LPS behave like individual molecules in solution phase. Thus there are ~ 40 LPS molecules in contact with each BMDC at any transient time.

The mobility of LPS molecules in

solution at room temperature could be estimated via ~ 1/8 of root mean square speed of that in ideal gas phase, 1.8 m/s. Considering the soaking time of BMDCs in sLPS , e.g. 24 h, there would be ~4.5 × 1013 LPS molecules in collision with each BMDC in the case of sLPS. In contrast, there were only 7.6 × 105 immobilized LPS molecules exposed to one BMDC in the same period in the case of LPS-Nano2. Therefore, we inferred that it was the presentation in LPS-Nano2, not sheer number of LPS, that led to the high potency in DC maturation than that in sLPS.


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Figure 3. (A) Population in percentile of CD86+ BMDCs plotted as function of interaction time. The ligand presentations are indicted in the 4 colored bars inside the frame. (B) LSCM images of BMDCs stained with Hoechst 33342 (blue) and anti-CD86-488 (green), respectively. The ligand presentation and interaction time are shown atop and on left of the images, respectively. Scale bars = 50 µm. To investigate if the high efficacy of LPS-Nano2 was due to the surface bound presentation of LPS, we tested and compared this with two other surface bound presentations: near monolayer coverage and similar surface coverage of LPS to that in LPSNano2. The high coverage of surface bound LPS, LPS/OTS SAMs, were prepared using well- known protocols (see Materials and Methods section).53 LPS coverage reached 91% under these preparation conditions from our AFM imaging. The LPS molecules formed nearly closely packed domains separated by boundaries. Accurate height measurement of LPS domains were attained by removal of an area of LPS via nanoshaving at 42 nN shaving force, followed by AFM imaging using tapping mode with the same probe. The thickness of LPS domains measured 1.9 ± 0.2 nm. Under the 91% coverage, which was much higher than that of LPS-Nano2 (1.5%), the population of CD86+ BMDCs increased gradually as shown in Figure 3A (green bar), and reached maximum at 24 h, i.e., following a similar profile to that of sLPS. The second system, LPS/mixed SAM, had 1.0% LPS coverage,


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which was similar to LPS-Nano2 (see also Table 1). Different from the periodic distribution of LPS in Nano2, LPS molecules on the mixed SAMs are randomly distributed following the methyl termini in the SAMs. The LPS patches measured 1.4 – 3.7 nm above surroundings, and from several to tens of nanometers wide. The separation among nearest neighbor LPS domains ranged from 20 – 110 nm. BMDCs on those surfaces resulted in much less CD86 expression than that in LPS-Nano2. As shown in Figure 3A (blue bar), only less than 5% population were CD86+. We underline that all surface presentations exhibit similar mechanical properties as sensed by BMDCs as these samples are produced by immobilizing LPS on SAMs.

The mechanical property for all surface presentations is

similar to that of LPS on SAMs supported by silicon wafers. Therefore, the significant differences are due to the geometry or local distribution of LPS.

These results collectively

demonstrated that the local distribution of LPS in LPS-Nano2 was primarily responsible for the high efficacy and rapid expression of CD86. CD86, acting as co-stimulatory molecule toward T cells’ activation, is critical for mature DCs’ efficacy in priming T cells and its expression is widely used to characterize maturation of DC.17-21 Specifically, the periodic arrangement of LPS nanostructures was critical to the observed faster CD86 expression than conventional means. This observation is significant for future applications where the kinetics matter, such as immunotherapy. The surface bound presentation represents much lower toxicity as the ligands are bound and not transportable, and the total amount of LPS molecules are much smaller than in soluble presentations. 3.4 LPS Nanostructures Led to Faster Morphological Evolution than That via Soluble LPS. As discussed in the introduction, the morphology of BMDC provided a direct


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readout of their maturation status and immunological efficacy in terms of T-cell activation. Pertinent morphological parameters included the number and length of dendrites, and membrane ruffling. Using SEM imaging, we monitored the BMDCs’ morphology as a function of interaction time on various LPS presentations, using a “fix-then-image” approach. First, we monitored the appearance of dendrites as a function of time. SEM images shown in Figure 4 were selected from a representative scenario (> 60% cell population) in each experiment. As shown in Figure 4 (top), LPS-Nano2 led to spreading and dendritization upon contact. After 1 h interaction, the cell shown in Figure 4A already developed at least 7 dendrites. Longer incubation time led to more dendrites at the periphery, as well as longer dendrites (Figure 4B), reaching almost peak values of high density and length at 6 h (Figure 4C): > 45 dendrites ranging from 4 – 13 μm. This highly dendritized morphology remained over our measurement time, as shown after 24 h immersion in Figure 4D, 53 dendrites 2 – 12 μm long. In comparison, sLPS had a much slower morphological evolution, as shown in Figure 4 (second row), where it took 4 times as long as that in LPS-Nano2 to reach the highest number and length. The BMDC shown in Figure 4H exhibits 46 dendrites with lengths of 5 – 15 μm. Similar morphology of mature BMDC obtained using sLPS has been observed in previous studies using SEM22, 58 and optical microscopy imaging2, 8, 31. To check if surface immobilization of ligands (LPS) was responsible for the observations, we tested BMDC on near monolayer of LPS/mixed SAMs, which had similar LPS coverage as LPS-Nano2 (1.0% and 1.5%, respectively). The BMDCs on these LPS surfaces failed to initiate or develop dendrites, as shown in Figure 4 (fourth row).

Cellular viability during the 24 hr tests period was tested via trypan blue

assays and > 95% BMDCs remained viability for all presentations tested.


22

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Figure 4. SEM images of BMDCs upon interaction with LPS-Nano2 (4A-4D), sLPS (4E4M), LPS/OTS SAM (4I-4L), and LPS/mixed SAM (4M-4P).

The 4 characteristic time

points, 1, 4, 6 and 24 hrs, at which SEM images were acquired are indicated atop each column.

A red frame in each row marks the highest degree of dendritization. Scale bars

= 12 μm.

To obtain three-dimensional (3D) information on the dendrites, the morphology of the BMDCs were also characterized using AFM, which was label-free and enabled 3D measurement in individual dendrites. A representative AFM image of a BMDC upon 6 h interaction with LPS-Nano2 is shown in Figure 5A, where 65 dendrites surrounding the periphery of the cell are clearly visible. A typical dendrite, as shown in Figure 5B, originated from the cell body and extended outwards with gradually reduced diameter. This dendrite was 9.4 μm long. At the base and in fixed form, the dendrite measured 28 nm in height and 255 nm in width. As it stretched out, the height and width gradually decreased to 17 nm. The height and width of individual dendrites were consistent with previous AFM


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study of mature BMDCs using sLPS presentation,61 while more and longer dendrites were present for BMDCs activated by nanostructures of LPS.

Figure 5. (A) AFM topographic image of a representative BMDCs after 6 h interaction with LPS-Nano2. (B) A zoom-in view of the region marked by orange rectangle in (A). Scale bars are 10 μm and 2 μm in (A) and (B), respectively. All AFM images were acquired under tapping mode in ambient condition: driving frequency = 10.1 kHz at 40% damping. Using the total length of dendrites to quantify the degree of maturation of BMDCs, the results for all 4 nanostructures of LPS were summarized in Figure 6. In each presentation, more than 300 BMDCs were imaged and measured. Comparing LPS-Nano1 with LPSNano2, the latter had higher efficacy in forming dendrites. This trend seemed very reasonable, as the higher LPS coverage, and larger number of LPS nanopatches in LPSNano2 were likely responsible. Comparing LPS-Nano2 with LPS-Nano3 and LPS-Nano4, the efficacy of forming dendrites decreased, even though the LPS coverage and number of LPS nanopatches increased. This trend seemed counter-intuitive at first glance. In fact, this observation suggested that other factors, in addition to the LPS coverage and number of nanofeatures underneath each BMDC, also impacted the activation of BMDCs. A plausible explanation for the observed trends will be discussed under mechanism in a later section.


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Figure 6. Total length of dendrites per BMDC plotted as a function of time for the four LPS-Nano presentations. For each presentation, more than 300 BMDCs were imaged and measured. Average of these measurements was represented as column value while the standard error of the mean was indicated in the error bar. To visualize membrane ruffling due to DC activation or maturation, higher resolution images were acquired by zooming into the central membrane region. Representative highresolution SEM images were shown in Figure 7, from which membrane features, e.g., ridges and ruffles, were clearly visualized. After a long contact period (24 h) in sLPS, as shown in Figure 7H, the surface was dominated by bright lines, resembling “cooked spaghetti on a plate”. These features were consistent with ridges among mature DCs observed in previous study.22, 58 At 1 h, for example, the BMDC on LPS-Nano2 already exhibited high coverage of ridges. In contrast, much shorter and curlier ridges appeared for BMDCs stimulated using sLPS. While the ridges of the BMDCs on LPS/mixed SAM appeared longer than that in sLPS, the density was much lower than that on LPS-Nano2. In the case of LPS-Nano2, the length, density and roughness continued to increase with time, reaching highest level at 6 h (Figure 7, top, red frame). The sLPS showed a similar but slower trend, reaching similar morphology at 24 h. The membrane features for BMDCs on LPS/mixed SAM surfaces significantly differed: much reduced ridge density and


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number. In fact, these observations indicated that the BMDCs might not be activated at all. As also addressed in the introduction, the ruffling of mature DCs’ membrane plays an important role in facilitating interactions antigens and T-cells.8 The dendritic morphology and ruffles were regarded as characteristic features of DCs’ maturation.2, 3, 8, 22 These comparisons indicate that the periodic presentation of surface bound LPS nanostructures was the key for faster maturations, and the resulting morphologies shall favor T cell capturing and high priming efficacy.

Figure 7. SEM images of representative BMDCs upon interaction with LPS-Nano2 (first row), sLPS (second row), LPS/OTS SAM (third row), and LPS/mixed SAM (fourth row) for (A, E, I, M) 1 h, (B, F, J, M) 4 h, (C, G, K, O) 6 h, and (D, H, L, P) 24 h. A red frame indicates the highest degree of ruffling in each presentation. Scale bars = 1 μm. 3.5 Hyper-Dendritized BMDCs were Observed in vitro upon Activation by LPS Nanostructures. To verify the robustness of the high efficacy of LPS nanostructures in BMDC activation, we performed large scale SEM imaging for two samples: BMDCs on LPS-Nano2 and LPS-Nano3. In both cases, more than 230 SEM images were acquired and


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stitched together to cover a 1 cm × 0.2 cm area, where ~500 BMDCs resided. In both cases, we verified that the morphology and trends shown in Figures 4 and 7 above were valid. AFM imaging was also carried out by selecting 6 representative regions to further verify the roughness of BMDC membrane. In the exercise of large area SEM imaging above, a minority population caught our attention, in the case of 0.5 h interactions with LPS-Nano3. A hyper-dendritized morphology of BMDCs is shown in Figure 8A. The BMDC spread over 50 × 70 μm2. The central region of the cell measured 8 µm wide and 1.5 µm in height, surrounded by dendrites. These dendrites were highly branched, as shown in Figure 8B. One could describe them as up to six branchlets counting from the origin outward, as illustrated in Figure 8C. The primary branchlet (red) originated from the cell body branched into 15 secondary branchlets (brown). Following each secondary branchlet, new tertiary branches (green) were seen. Continuing this exercise revealed 4th (blue), 5th (purple), and 6th (black) generations of branchlets. The morphology is responsible for the name of dendritic cells, however, such a hyper-branched morphology represents the first of its kind in vitro, i.e., a new phenotype. On a 1 × 1 cm2 silicon substrate, counting over 1000 BMDCs, 6 cells exhibit hyper-dendritized morphology. In another experiment, and among 200 of cells counted, one highly branched BMDC was seen. The hyper-dendritized BMDCs were also observed upon 1 h interaction with LPS-Nano2 in two sets of experiments, from among hundreds of cells examined.


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Figure 8. (A) AFM topographic image of a BMDC with hyper-dendritized morphology. (B) Zoom-in view of two branches as indicated by the red frame in (A). (C) Following the longest dendrite branch in (B) to count branching, up to 5th generation of branching was seen, as illustrated in the schematic diagram. Scale bars=10 μm. Both AFM images were acquired under contact mode in ambient condition, with imaging force of 3 nN. To avoid potential contamination by other cell types or artifacts, these hyperdendritized cells were subjected to multiple tests of ICC staining (see Method for details). As shown in Figure 9A and 9B, the new phenotype was both Hoechst 33342+ and CD11c+, in other words, was BMDC, not contaminants. The high level of CD86 expression, shown in Figure 9C, further indicated that the new phenotype is mature BMDC. To the best of our knowledge, this was the first time mature BMDCs with highly dendritic morphology were generated in vitro. The degree of branching in this case exceeded that observed in vivo.13 Dendrite formation, length and degree of branching directly related to the efficacy of DC in priming T cells.13, 14 Our success in generating such hyper-dendritized mature BMDCs in vitro and in 30 min, was a significant step for realizing DC based immunotherapy.9, 10 Work is in progress to understand the key factors leading to this phenotype, then to increase the population of hyper-dendritized BMDCs.


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Figure 9. LSCM images of the hyper-dendritized BMDC shown in Figure 8 after being stained with nuclear, DC, and maturation dyes, respectively, as indicated under each image. Fluorescence emission was shown individually: (A) Hoechst 33342, (B) anti-CD11c-615, and (C) anti-CD86-488. Scale bars = 10 μm.

4. DISCUSSION 4.1. Possible Mechanism for the Observed Rapid Maturation and HyperDendritized Phenotype of BMDCs.

Based on the BMDC responses to LPS

nanostructures and knowledge of cellular signaling processes leading to DC maturation, possible mechanisms were deduced to account for the rapid maturation and hyper-branched phenotype. It is known that 2 LPS molecules bind to 2 TLR4 receptors to form dimers, and multiple dimers cluster at cellular membrane, initiating downstream signaling cascades leading to DC maturation.11,

34-36, 63

In the nanostructure presentations of LPS, each

nanofeature consists over 2000 LPS molecules or could enable more than 1000 dimer complexes.

At least 3 cascades were established: (a) cytoskeletal rearrangement via small

guanosine triphosphate hydrolase (GTPases) of Rho family, resulting in dendrite and ruffle formation,15,

23, 64

acute inflammatory cytokines production, such as TNF-α, myeloid

differentiation primary-response protein 88 (MyD88)-dependent pathways through earlyphase activation of nuclear factor-κB (NF-κB);11, 65-67 (b) upregulation of CD86 expression,


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activated due to late-phase activation of NF-κB via MyD88-independent pathways;11 and (c) acute inflammatory cytokines production, such as TNF-α, myeloid differentiation primary-response protein 88 (MyD88)-dependent pathways through early-phase activation of nuclear factor-κB (NF-κB).11, 65, 67 As discussed in previous sections, BMDC maturation initiated by LPS nanostructures were consistent with the two well-known cascades, (a) and (b) above, both of which are initiated by clustering of LPS-TLR4 dimers. In addition, maturation initiated by LPS nanostructures are faster than that in sLPS and even monolayer of LPS on surfaces. The rapid BMDC maturation by nanopatterned LPS were relatively straightforward to rationalize. As shown in Figure 2, and schematically represented in Figure 10A, LPS nanostructures presented a large number of ligands to each BMDC at the same time, in contrast to sLPS where ligand molecules had to collide and bind TLR4 individually. As such, the LPS nanostructures exhibited higher efficacy to initiate LPS-TLR4 clusters that that occurring in sLPS. In addition, individual LPS nanofeatures were taller than the surrounding at nanometer level (see also Table 1), and as such exhibited higher efficacy than monolayer LPS to bind TLR4 inlaid in BMDC membrane. Similar observations were seen for antigen binding to mast cells.37, 38 Therefore, LPS nanostructures exhibited higher efficacy in forming LPS-TLR4 clusters than monolayer LPS, where the receptor binding units were imbedded in the monolayer. This enhancement was analogous to a higher concentration of reactant(s) leading to faster reaction kinetics. Results shown in Figure 6 revealed new insights in this TLR4 initiated DC maturation, i.e., detailed spatial arrangement of these LPS-TLR4 at micro- and nanometer scales also impacted the


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maturation process. Since nanotechnology enables active arrangement of ligands, regulation of the signaling process is now feasible. The morphology shown in Figures 8 and 9 was less than straightforward to rationalize because this hyper-dendritized phenotype in vitro has not been previously reported. A plausible explanation involves the TNF-α molecules, which were released due to BMDC activation.1, 11, 65 In the case of sLPS induced DC maturation, these TNF-α molecules diffused into culture media, and had little impact on BMDCs subsequently due to insignificant concentration. In the case of LPS-Nano2, however, some of the secreted TNFα could be trapped at the interface of BMDC membrane and the LPS nanostructures, as shown in Figure 10B. These TNF-α molecules could not diffuse afar from the BMDC due to the entrapment. If allowed to interact with BMDC, these TNF-α were known to form homo-trimmer and bind to TNF receptor (TNFR)68 and could also trigger DC maturation,2, 3, 64

via autocrine effect. The probability of trapping these TNF-α molecules near BMDCs

was highly dependent on the local environment, e.g., probability of releasing factors to the interfacial pockets, entrapment, and autocrine. It was, therefore, foreseeable that certain geometries, e.g., LPS-Nano2, might prove more potent in trapping TNF-α. It was also reasonable to observe a small population of the hyper-dendritized mature BMDCs, as the same ligands arrangement did not necessarily translate into the same autocrine effect as more local factors come into play.


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Figure 10. Schematic diagram illustrating possible mechanism leading to (A) rapid maturation; and (B) hyper-dendrized phenotype, among BMDCs in contact with LPS nanostructures. 4.2.

Potential for Future Applications. DC activation and maturation represents an

important initial step for DC based therapy.69 With a high degree of activity and maturity, the subsequent steps of therapy development shall be on solid ground: i.e., T cell priming efficacy tests; and anti-tumor immunity test in vivo by injection of activated DC and designed antigen into living bodies of animal or patients.69, 70 In comparison to the approach of using soluble ligands, surface bound nanostructures have the advantages of avoiding overdose, enabling programming DC activation, for example, by varying of the geometry of the nanostructures (Figure 6), and high efficacy of DC activation (e.g. Figure 8).

4.

CONCLUSIONS

This work demonstrated that surface bound nanostructures of ligands provide a new and high efficacy means to regulate primary dendritic cells’ activation and maturation. By periodical presentation of LPS nanostructures, the BMDCs maturation process is


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significantly accelerated in comparison to the conventional presentation of soluble LPS and monolayers of LPS. Similar to known morphology of DCs, the morphology of these activated BMDCs exhibit high density and long dendrites at the cellular periphery, and a high degree of membrane roughness. Both characteristics suggest high efficacy in capturing and priming T-cells. The degree of acceleration and roughening reveals dependence on the geometry of the nanostructures, which suggest feasibility of regulating DC maturation by design. A new BMDC phenotype, characterized by hyper-dendritized morphology, was also captured in vitro for the first time. The facts that primary cells’ signaling processes such as BMDC maturation can be accelerated and even altered in the case of a new phenotype, suggest that nanomaterials of ligands provide a new and powerful means for regulating cellular signaling processes and functions. Work is in progress to investigate the impact of local presentations in a 3D environment.

The concepts proven

in this work shall pave the ways for applications in nanomaterial and DC based immunotherapy.

Conflict of Interest: The authors declare no competing financial interest. Acknowledgement. We would like to thank Dr. Ming Zhang for his assistance in the initial SEM imaging; Drs. Arpad Karsai, Qingbao Yang, and Ms. Jiali Zhang for discussions; and Ms. Susan Stagner for her assistance in the preparation of the manuscript. This work was supported by National Institutes of Health (1R21CA176850-01), and the Gordon and Betty Moore Foundation. YL is supported in part by UCD-LLNL Graduate Student Mentorship Award.


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TOC FIGURE


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Periodic Arrangement of Lipopolysaccharides Nanostructures

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