Symposium Organizers
Hao Cheng, Drexel University
Wendy Liu, University of California, Irvine
Minglin Ma, Cornell University
Cherie Stabler, University of Florida
Symposium Support
JDRF, International
SM6.1: Fundamental Material Design for Reducing Innate Immune Recognition
Session Chairs
Tuesday PM, April 18, 2017
PCC North, 100 Level, Room 122 C
2:30 PM - *SM6.1.01
Fundamental Material Design to Translational Applications for a Sphere-Templated Porous Polymer that Integrates and Heals in Many Tissues and Sites
Buddy Ratner 1
1 , University of Washington, Seattle, Washington, United States
Show AbstractMillions of medical devices made of synthetic or modified natural materials are implanted in humans each year saving millions of lives and improving the quality of life for millions more. Biomaterial implants all trigger a similar reaction, the foreign body reaction (FBR). For materials that pass routine cytotoxicity testing, the implantation outcome is largely associated with a mild FBR, i.e., a thin, collagenous, avascular, non-adherent foreign body capsule. Another way to describe this is to say that implant is incorporated into a “dead-zone” of acellular scar. The contemporary biomaterials and tissue engineering paradigm would suggest that all synthetic biomaterials and scaffolds (particularly those lacking cellular, biomolecule or biomimetic elements) will give this same fibrotic, avascular healing reaction. In this talk, synthetic, porous biomaterials fabricated by a sphere-templating process will be described that readily integrate into tissue and may stimulate spontaneous reconstruction of tissue. Fundamental materials design for these polymers will be discussed. The desirable in vivo outcomes seem to be driven by macrophages in the M2 polarization. The in vivo results from our group and related results from other groups suggest we are on the cusp of a revolution in healing, biomaterials integration and tissue reconstruction. In fact, this biomaterials-driven healing much resembles outcomes seen in tissue engineering. Because no biological elements (cells, proteins) are used in these materials, translation to specific medical applications is simplified. Progress in translation to clinical practice for these sphere-templated porous biomaterials will be described for ophthalmologic applications, drug delivery and vascular prostheses.
3:00 PM - SM6.1.02
Engineering In Vitro Platforms for Characterization of Immunological Responses to Encapsulated Cells
A. Frei 1 , Y. Li 1 2 , E. Yang 3 , A.B. Bayer 3 4 , Cherie Stabler 1 3
1 J Crayton Pruitt Family Department of Biomedical Engineering, University of Florida, Gainsville, Florida, United States, 2 Department of Pathology, Immunology and Laboratory Medicine, University of Florida, Gainesville, Florida, United States, 3 Diabetes Research Institute, University of Miami, Miami, Florida, United States, 4 Department of Microbiology and Immunology, University of Miami, Miami, Florida, United States
Show AbstractAlginate is extensively used to encapsulate cells with the goal of protecting these foreign transplants from immune recognition and subsequent destruction. Despite their widespread use, the mechanisms by which biomaterials block antigen recognition and how this blocking impacts graft success remains poorly understood. While it is suspected that encapsulation blocks direct antigen recognition, it is unknown whether indirect antigen recognition occurs and whether this leads to robust immune activation. Further, the impact of this indirect immune response on encapsulated graft success has not been characterized. Herein, we generated an in vitro platform capable of delineating these mechanisms using ovalbumin (OVA) as the model antigen. Cells were isolated from membrane-bound ovalbumin (mOVA) mice, encapsulated within alginate, and co-cultured with responders (immune cells from OTI mice), with CD8+ T cell proliferation and activation quantified via FACS. Co-culture of OTI splenocytes with unencapsulated or encapsulated mOVA cells resulted in the robust proliferation and activation of CD8+ T cells, albeit slightly lower for the latter. The response to the encapsulated cells was antigen-specific and alginate independent, with no stimulation observed in control groups with encapsulated or unencapsulated C57BL/6J cells. To parse out the contribution of the responder cell populations on activation, CD8+ T cells were sorted from OTI splenocytes and co-cultured with either unencapsulated or encapsulated mOVA cells. This purified population only responded to unencapsulated mOVA cells. The addition of antigen presenting cells (APCs) to the CD8+ T cell population restored activation to the encapsulated mOVA cells, supporting the hypothesis that indirect antigen processing leads to CD8+ T cell activation by encapsulated grafts. In conclusion, this study finds standard alginate encapsulation blocks direct antigen activation, but not indirect. This work provides an efficient platform to understand basic mechanisms of immune activation for alginate hydrogels, and an efficient screening tool for characterizing new encapsulation approaches.
3:15 PM - SM6.1.00
Immunomodulatory Biomaterials for Controlling the Macrophage Response
Thuy Luu 1 , Jessica Hsieh 1 , Esther Chen 1 , Wendy Liu 1
1 , University of California, Irvine, Irvine, California, United States
Show AbstractThe immune response to implanted materials remains a critical challenge for the development of biomaterials used in medical devices and regenerative medicine. Macrophages play a key role in this response, and can adopt a multitude of functional phenotypes depending on cues in their microenvironment. In addition, these cells are capable of distinguishing foreign from self tissue using specific molecular cues expressed on the surface of cells. Our laboratory combines microscale technologies with biomaterials engineering to control the physical and biochemical properties of the macrophage environment. Our studies show that physical features of the extracellular microenvironment, including adhesion geometry, topology and matrix composition, influence macrophage function and the wound healing response. In addition, we are utilizing biomolecules expressed by host tissue to mask the biomaterial surface, and thus promote immune tolerance. Our ultimate goal is to create biomaterials that control local immune cells and mitigate the host response to implanted devices.
4:00 PM - *SM6.1.03
From Flexible Filomicelles to 'Self' Recognition
Dennis Discher 1
1 , University of Pennsylvania, Philadelphia, Pennsylvania, United States
Show AbstractDrug carriers have traditionally been spherical and, based on early ideas of a cell's glycocalyx, such carriers are designed to be more difficult to clear by using synthetic polymers that are hydrophilic such as PEG. We have explored filamentous shapes and also 'Self' signaling by peptides rather than PEG in various efforts to advance delivery science. In a first example (1), we sought to improve the delivery of aromatic drugs using elongated and flexible filomicelles, and to that end aromatic groups were integrated into the hydrophobic block of a degradable di-block copolymer. These aromatic filomicelles were formed by self-directed assembly of amphiphilic di-block copolymer PEG-PBCL with suitable block ratios. The aromatic core enabled loading with a common chemotherapeutic, Paclitaxel, for tests of release as well as effects on cancer cell lines in vitro and in vivo. For carcinoma lines in vitro and initial tests in vivo, flexible filomicelles with an aromatic core form an efficient drug delivery system that leads to higher cell death than previously reported systems, while inducing aneuploidy in surviving cells. In the second example, we update the idea of the cell surface as including factors that are specifically recognized as 'Self'. ‘Self’ cells are spared due (in part) to a polypeptide found on all cells that marks cells as ‘Self’, limiting their phagocytic clearance in vitro and in vivo – even when displayed on nanoparticles (2). Application to nanoparticle-based tumor imaging will be illustrated, as will application of the pathway to a cell-based therapy for cancer that exploits the ability of phagocytic cells to deeply penetrate into disease sites such as tumors.
(1) P.R. Nair, K. S. Anbazhagan, K.R. Spinler, M.R. Vakili A. Lavasanifar, and D.E. Discher. Filomicelles from aromatic di-block copolymers increase paclitaxel-induced tumor cell death and aneuploidy compared to aliphatic copolymer. Nanomedicine (May/June 2016).
(2) Rodriguez, P.L.; Harada, T.; Christian, D.A.; Pantano, D.A.; Tsai, R.K.; and Discher, D.E. Minimal 'Self' peptides that inhibit phagocytic clearance and enhance delivery of nanoparticles. Science 2013 339: 971-975
4:30 PM - *SM6.1.04
Zwitterionic Polymer Conjugation or Encapsulation of Proteins Enhances Pharmacokinetics and Mitigates Immune Response
Shaoyi Jiang 1
1 , University of Washington, Seattle, Washington, United States
Show AbstractProteins are promising therapeutics with several design challenges, such as their inherent immunogenicity due to their exogenous source or short circulation time. The common solution to such issues is the chemical conjugation of poly(ethylene glycol) (PEG), a process known as PEGylation. However, several studies demonstrated a decrease in protein bioactivity or an increase in the presence of specific antibodies post PEGylation, highlighting the importance of an alternative strategy. In this talk, a number of clinical reports and some critical animal studies regarding pre-existing and treatment-induced anti-PEG antibodies will be summarized. Then, zwitterlation, as an alternative to PEGylation, will be presented. Zwitterionic polymer conjugation and encapsulation of therapeutic enzymes (e.g., uricase for the treatment of gout) and protective enzymes (e.g., enzyme hydrolyzing organophosphates for nerve agent prophylaxis) will be demonstrated for maintained bioactivity, prolonged circulation and minimized immunological response. Finally, various anti-PEG detection methods, including the latest developments, will be provided.
5:00 PM - SM6.1.05
Enhancing Nanocarrier Blood Circulation by Controlling Their Topographical Structures
Hao Zhou 1 , Hao Cheng 1
1 , Drexel University, Philadelphia, Pennsylvania, United States
Show AbstractDrug nanocarriers have been extensively studied for cancer treatment. Despite some development, one major challenges in applying nanomedicines in cancer therapy is the short blood circulation half-life of nanocarriers as they are cleared by the phagocytic cells in the reticuloendothelial system. We have found that a topographical structure of hierarchical polyethylene glycol (PEG) shell is highly efficient in prolonging nanoparticle blood circulation through a kinetic effect. High density PEG shell in the brush regime does not have this effect. In this talk, I will show how the kinetic effect reduces nanoparticle internalization by macrophages under shear flow.
5:15 PM - SM6.1.06
In Vitro Evaluation of Bioactive Implant Materials
Weitian Zhao 1 , Jacques Lemaitre 1 , Paul Bowen 1
1 , École Polytechnique Fédérale de Lausanne, Lausanne Switzerland
Show AbstractArtificial materials which have the ability to spontaneously bond to living bone, when inserted into bone defects, are of great interest as implant materials. It is commonly agreed that the key step in avoiding the typical host response of implants, namely the encapsulation by fibrous tissues, is the spontaneous formation of a hydroxyapatite layer on the material’s surface after implantation. This apatite layer help stimulate the activities of osteoblasts and eventually lead to the direct bone formation on implant surface. This discovery has led to the development of various implants with enhanced fixation and long-term durability.
The process of surface hydroxyapatite formation can also be simulated in an acellular environment using a simulated body fluid (SBF), originally proposed by Kokubo et al. This in vitro test aims to reduce the amount of animal experiments used to evaluate implants and ideally would achieve a similar distinguishing power compared to animal tests. However, the current SBF used lacks critical components of human blood plasma, such as proteins, which play an important role in material-cell interactions. The pH buffering is achieved, instead of using 5% CO2 as in human blood, by artificial buffer TRIS, which might have an effect on calcium phosphate nucleation.
In this project, we investigated the use of carbonate-buffered SBFs and the effect of proteins on the nucleation of hydroxyapatite on chemically treated titanium surfaces. As-polished Ti and clinically-proven bioactive chemically-treated Ti were used as negative and positive control, respectively. It has been found that carbonated SBFs performed as expected for the control groups, but showed enhanced apatite deposition for certain materials. In the presence of 1 g/L protein, represented by bovine serum albumin, a complete inhibition of apatite nucleation was observed using the traditional SBF, while carbonate-buffered SBF exhibited a decreased apatite formation. A kinetic study on the effect of protein concentration was also carried out using carbonate-buffered SBF. It was found that 0.1 g/L of protein does not have a significant effect on apatite formation, 1 g/L of protein only decreased the amount of apatite but did not significantly change the time for nucleation, while 5 g/L of protein fully inhibited the apatite formation, even for a duration of two weeks. These studies could help better design the SBFs and correlate the in vitro apatite forming ability of a tested material to its in vivo bone-bonding behavior. Animal experiments to validate the proposed solution are currently being carried out with evaluations of bone-bonding behavior using torque test as well as the measurement of bone-implant contact area.
Symposium Organizers
Hao Cheng, Drexel University
Wendy Liu, University of California, Irvine
Minglin Ma, Cornell University
Cherie Stabler, University of Florida
Symposium Support
JDRF, International
SM6.2: Materials in Controlling Macrophage Polarization
Session Chairs
Wednesday AM, April 19, 2017
PCC North, 100 Level, Room 122 C
9:45 AM - SM6.2.01
Poly (2-methacryloyloxyethyl phosphorylcholine) Encapsulated Oxalate Oxidase—A Potential Anti-Hyperoxaluria Drug with Prolonged Blood Residence and Mitigated Immunogenicity
Ming Zhao 1 , Yunfeng Lu 1
1 , University of California, Los Angeles, Los Angeles, California, United States
Show AbstractAccumulation of oxalate leads to hyperoxaluria and calcium oxalate nephrolithiasis in humans. Classical treatments such as intensive dialysis, extracorporeal shock wave lithotripsy and surgery are less effective owing to their side effects and easy recurrences. As a potential alternative strategy, oxalate oxidase (OXO) catalyzes oxalate oxidation into carbon dioxide and hydrogen peroxide in the presence of oxygen, yet, the use of OXO as a therapeutic drug is impeded by its low purity, short plasma residence and immunogenicity. In this study, we use highly purified OXO and its nonglycosylated variant S49A-OXO overexpressed in Pichia pastoris. We further encapsulate this enzyme within a superhydrophilic poly(phosphorylcholine) network using aqueous in situ polymerization. Through extensive in vitro and in vivo studies, our nanocapsules manifest enhanced catalytic activity and stability, significantly prolonged plasma circulation time, greatly mitigated immunogenicity and overall abated organ accumulation. To our knowledge, this is the first systematic investigation of OXO as an antidote for hyperoxaluria, thereby opening up a new avenue for research and novel clinical strategies against hyperoxaluria related diseases.
10:00 AM - SM6.2.02
High-Water-Content and Resilient PEG-Based Hydrogels and In Vivo Evaluation of Their Biocompatibility
Minglin Ma 1
1 , Cornell University, Ithaca, New York, United States
Show AbstractHydrogels such as polyethylene glycol (PEG)-based ones are broadly used in the biomedical world for wound dressings, tissue scaffolds, cell or drug deliveries and medical implants. However, conventional hydrogels swell under physiological conditions and often become undesirably weak or brittle after swelling, limiting the scope of their applications. We report here a purely synthetic, PEG-based hydrogel that remains resilient (i.e. highly stretchable and recoverable) after maximum swelling. We made the hydrogel by incorporating reversible, swelling-resistant, “dual” hydrogen bonding into a network of highly coiled and water-swellable PEG molecular chains. In its equilibrium swollen state, the hydrogel has a water content of ~ 97.6 wt% and can still be stretched to ~10 times of its original length with a tensile stress of ~ 57 kPa. It also exhibited remarkable recoverability after repeated deformation. To demonstrate the potential applications of this type of hydrogel in biomedicine, we evaluated their biocompatibility in mice and showed their ability to form robust implantable cell encapsulation devices without additional structural support.
10:15 AM - *SM6.2.03
Combinatorial Development of Materials for Islet Transplantation
Daniel Anderson 1
1 , Massachusetts Institute of Technology, Cambridge, Massachusetts, United States
Show AbstractThe fibrotic reaction to implanted biomaterials is a fundamental challenge to the development of immuno-isolation devices. Here we describe our work developing new biomaterials and devices for the purposes of enabling islet transplantation. In particular we describe the development of a large library of synthetic hydrogel materials, and the characterization of their biocompatibility in vivo. Data will be presented on the nature of the immune response to these and conventional biomaterials. Several lead materials have been identified with significantly improved biocompatibility in rodents and primates. When formulated into microcapsules these materials enable functional, long-term islet transplantation in immune competent, diabetic rodents.
11:15 AM - *SM6.2.04
Improving Selective Targeting to Macrophage Subpopulations through Modifying Liposomes with Arginine Based Materials
Kaitlin Bratlie 1
1 , Iowa State University, Ames, Iowa, United States
Show AbstractTwo pathways for activating macrophages (MΦ) exist. One of these routes is termed the classically activated M1 pathway is achieved through exposure to lipopolysaccharide (LPS). M1 MΦs are part of the type 1 T helper (Th1) response and are known as pro-inflammatory cells. The other pathway is reached through interleukin-4 (IL-4) and is known as the alternatively activated M2 pathway. M2 MΦ produce pro-angiogenic factors. Tumor-associated MΦ (TAMs) are of the M2 pathway and promote tumor growth through the release of angiogenic molecules. Our goal is to use liposomal drug delivery systems to selectively deliver drugs to TAMs. These polymers will be eventually used to delivery anti-cancer therapeutics to the tumor.
Liposomes were loaded with doxorubicin and incubated with M(LPS), M(IL-4), and M(0) cells. Dose response curves were fit to a Sigmoidal curve and the half-maximum of inhibitor concentration (IC50) was obtained. The lower IC50 values for M(IL-4) cells compared to M(LPS) cells suggests that modifications H and I could be used to improve targeted delivery to TAMs. PCA was applied to the dataset to determine the relationships between physicochemical properties of the modifiers and the IC50 values. The multidimensional dataset was reduced to a two-dimensional plot to better facilitate analysis of latent relationships. The physicochemical properties were chosen based on previous reports describing attributes of drug molecules such as Lipinski’s rule of five, polar surface area, flexibility, enthalpy, lipophilicity, and charge. Macrophages were observed to have different correlations with these physicochemical properties based on their phenotype. The IC50 values for both M(LPS) and M(0) cells were well aligned on the projection map and were situated between the number of hydrogen bond donors and the number of freely rotating bonds of the modifiers. The M(IL-4) cells were dependent on the zeta potential of the liposomes and the logP (the partition coefficient of the modifier in octanol and water) of the modifiers. Taken together, these insights may elucidate design principles in drug delivery targeted to specific macrophage phenotypes. Further work on larger library is necessary to determine if these relationships between the identified materials properties and IC50 values hold for different macrophage polarizations.
Cellular uptake of the liposomes was found to be dependent upon macrophage phenotype and surface modifications. There were also differences in trends between internalization of liposomal FC and the IC50 of liposomal doxorubicin, which were attributed to changes in the ability of doxorubicin to escape the endosome. Two modifications were able to increase the toxicity of encapsulated doxorubicin for M(IL-4) cells over M(LPS) cells, improving targeted deliver to specific macrophage subpopulations.
11:45 AM - *SM6.2.05
Novel Strategies to Modulate the Inflammatory Response to Biomaterials
Kara Spiller 1
1 , Drexel University, Philadelphia, Pennsylvania, United States
Show AbstractBiomaterials implanted into the body immediately elicit an inflammatory response, elicited primarily by macrophages. Macrophages exist on a spectrum of phenotypes, with activities ranging from inflammatory to anti-inflammatory behavior. Biomaterial surface modification strategies designed to modulate macrophage behavior have major implications for strategies to mitigate the foreign body response or to promote tissue regeneration. For example, in a healthy wound healing response to injury, including biomaterial implantation, macrophages must first exhibit pro-inflammatory behavior (also known as M1 activation) and then transition into an anti-inflammatory phenotype (also known as M2) to promote tissue regeneration and homeostasis. In this talk, we will present our work that demonstrates the importance of this sequential activation behavior of macrophages for biomaterial vascularization, integration, and tissue healing, using in vitro models of macrophage-biomaterial interactions, animal models, and clinical samples. We will also present biomaterial surface modification strategies for promoting rapid M1 activation and sustained M2 activation of infiltrating macrophages. Particular emphasis in our group is on surface modification strategies based on controlled affinity interactions between biotinylated proteins and avidin-based proteins. Finally, we will discuss new directions in immunomodulatory biomaterials, including promotion of specific phenotypes of macrophages to promote tissue deposition (M2a) or tissue degradation and remodeling (M2c). Ultimately, understanding and controlling the response of macrophages to implanted biomaterials represents a new and important area of biomaterials design.
12:15 PM - SM6.2.06
Global Gene Expression Analysis of Macrophages in Response to Changing Size and Porosity of Silica Nanoparticles
Mostafa Yazdimamaghani 1 2 , Jiban Saikia 1 , Seyyed Pouya Hadipour Moghaddam 1 2 , Philip J Moos 3 , Hamid Ghandehari 1 2 4
1 Utah Center for Nanomedicine, Nano Institute of Utah, University of Utah, Salt Lake City, Utah, United States, 2 Department of Pharmaceutics and Pharmaceutical Chemistry, University of Utah, Salt Lake City, Utah, United States, 3 Department of Pharmacology and Toxicology, University of Utah, Salt Lake City, Utah, United States, 4 Department of Bioengineering, University of Utah, Salt Lake City, Utah, United States
Show AbstractImmune cell interaction, recognition, and response to nanoparticles (NPs) depend on key physicochemical properties such as surface area, size distribution, charge, surface groups, shape, geometry, and aggregation state, among others. In physiological conditions, non-specific protein adsorption and formation of protein corona change surface properties. The objective of this research is to investigate the influence of size and porosity of silica nanoparticles (SNPs) in mediating protein adsorption, cellular uptake, toxicity, and whole gene expression analysis. Spherical nonporous SNPs of two different diameters, namely 46 ±4.9 nm and 432 ± 18.7 nm were synthesized. To evaluate the effect of porosity, mesoporous spherical SNPs were produced with a diameter of 466 ±86 nm comparable to 432 ± 18.7 nm for nonporous particles. Particles were characterized using transmission electron microscopy (TEM), dynamic light scattering (DLS), scanning electron microscopy (SEM), X-ray diffraction (XRD) techniques, nitrogen adsorption-desorption analysis, and zeta potential measurement. Next, particles were evaluated for toxicity on RAW 264.7 cell lines in the presence and absence of serum protein. We found that the toxicity of SNPs was reduced upon non-specific protein adsorption and formation of protein corona. The smallest size particle adsorbed higher amounts of protein. Detailed analysis of the amount of proteins recovered from each nanoparticle demonstrated similarities in the protein adsorption profile as a function of size and porosity. The mechanism of uptake was highly dependent on size rather than porosity, or the adsorbed proteins. The smallest sized SNPs followed multiple endocytosis pathways whereas for the larger particles the scavenger mediated pathway regulated the uptake along with caveolae-mediated pathway. We utilized RNAseq to generate transcriptional profiles of RAW264.7 macrophages exposed to non-toxic SNP doses. Non-toxic amounts of NPs were dosed to macrophages. We used Qiagen RNeasy minikit protocol RNA to extract RNA, and then Nanodrop spectrophotometer and Agilent Bioanalyzer to ensure high quality RNA, Illumina TruSeq RNA to generate barcoded sample libraries, and DESeq2 to generate the count matrices. DESeq2 and BinReg2 software were used to analyze the data based on both unsupervised and supervised strategies to identify genes with greatest differences among NP treatments. We found that mesoporous silica particles are capable of altering gene expression in macrophages at doses that do not elicit acute cytotoxicity, while gene transcription was not affected by nonporous Stober SNPs. A total of 192 genes’ expressions changed for mesoporous silica particles compared with control. Utilizing GATHER and DAVID software, possible induced pathways for ATP synthesis, membrane and lysosome disruption, and enhanced expression of inflammatory mediators such as tumor necrosis factor superfamily members were observed.
SM6.3: Scaffold and Hydrogel for Immune Modulation
Session Chairs
Wednesday PM, April 19, 2017
PCC North, 100 Level, Room 122 C
2:30 PM - *SM6.3.01
Building a Pro-Regenerative Immune Environment with Biomaterials
Kaitlyn Sadtler 1 , Amy Anderson 1 , Alexis Parillo 1 , Drew Pardoll 1 , Jennifer Elisseeff 1
1 , Johns Hopkins University School of Medicine, Baltimore, Maryland, United States
Show AbstractRegenerative medicine strategies for repairing lost tissues have thus far produced disappointing results with respect to clinical translation and therapeutic efficacy when translated. Historical approaches in the field focused on delivery of stem cells, tissue-specific cells and mobilizing local cells, frequently with the help of a biomaterial scaffold. Tissue-derived scaffolds, or ECM biomaterials, demonstrate excellent regenerative capacity that has been associated with a pro-regenerative macrophage. To further define the immunological mechanism of creating a pro-regenerative environment, we extensively profiled the immunological response to the wound environment and ECM scaffold. We found that Th2 T cells were required for the scaffold stimulation of wound repair through the production of interleukin 4. We are now exploiting this discovery, and the role of the adaptive immune system in biomaterial responses, to stimulate wound healing for a number of tissue trauma applications and further biomaterials design.
3:00 PM - *SM6.3.02
Dendritic Cell-Targeted Immunomodulation for Tolerance
Benjamin Keselowsky 1
1 , University of Florida, Gainesville, Florida, United States
Show Abstract
This presentation will highlight results from our biomaterials and molecular based approaches to direct the immune system toward tolerance. Microparticle-based systems have been designed as injectable vaccines to retrain the immune system to correct aberrant activation toward self-antigens. Hydrogels and microparticles with encapsulated immunomodulatory factors and antigen provide targeted, controlled delivery in vivo to both intracellular and cell surface targets of immune cells, dendritic cells in particular, in order to promote tolerance. Using a microparticle formulation consisting of a combination of suppressive factors we have demonstrated prevention and reversal of type 1 diabetes in non-obese diabetic mice, as well as treatment of multiple sclerosis in an EAE mouse model. Investigations into the immunological mechanisms implicate a role for regulatory T cells and B cells. In addition, we are developing molecular engineering approaches to direct tissue-localized metabolism to guide immune cell function toward tolerogenic phenotypes.
4:30 PM - *SM6.3.03
Mesoporous Silica-Based Cancer Immunotherapy
Jaeyun Kim 1
1 Chemical Engineering, Sungkyunkwan University, Suwon Korea (the Republic of)
Show AbstractFailures of the native immune system to eradicate tumors are mostly due to the immunosuppressive environments created by the tumor. In order to generate robust and durable antitumor adaptive immune responses, it is crucial to use appropriate adjuvants that support the activation of immune responses enough to overcome the immunosuppressive environments. In this presentation I will discuss on the application of mesoporous silica in cancer immunotherapy as an adjuvant and as a building block to form cellular microenvironment for the activation of host dendritic cells. Mesoporous silica microparticles with high aspect ratio were injected to form macroporous structures via spontaneous assembly in the body, which provide a 3D cellular microenvironment where host immune cells could be recruited and activated. The dendritic cell recruitment and subsequent homing to lymph nodes were modulated by sustained release of chemokines and adjuvants, respectively, to invoke antigen-specific cellular and humoral immune responses. The encapsulation of bioactive molecules in mesopores and their sustained release at target site allowed high bioactivity and enhanced half-life of the bioactive molecules compared to the free form in the physiological condition. We also used mesoporous silica nanoparticles loaded with antigen and danger signal molecules to generate mature antigen-presenting dendritic cells for subsequent induction of antigen-specific immunity. These findings suggest that mesoporous silica could be used as an efficient delivery vehicle and an adjuvant to immune cells, and a 3D building block to form cellular microenvironments for cancer immunotherapy.
5:00 PM - *SM6.3.04
Methods of Controlling and Activating Dendritic Cells with Chemical Tools
Aaron Esser-Kahn 1
1 , University of California, Irvine, Irvine, California, United States
Show AbstractRecent research into the immune system has revealed that foreign pathogens are detected through a series of receptors on antigen presenting cells. These receptors are synergistically activated by multiple pathogen associated molecular patterns. Materials scientists have a large role to play in the coordinated design of vaccines and synthetic activators of the immune system. We report on our development of chemical and polymeric tools for interacting with the immune system. Our methods involve the bioconjugation of multiple different PAMPs to polymeric scaffolds. These synergistic PAMP scaffolds have been tested against dendritic cells and we will report on the results. Additionally, we will report on our work coupling these synergistic combinations to cell-surface and other antigen rich environments. We also report on our attempts to control the innate immune system using light to guide responses.
5:30 PM - SM6.3.05
Biomimetic Injectable Macroporous Scaffolds for Immune Cells Modulation
Zhiyuan Fan 1 , Junjie Deng 1 , Peter Y Li 1 , Hao Cheng 1
1 , Drexel University, Philadelphia, Pennsylvania, United States
Show AbstractMacroporous scaffolds have been developed using various biocompatible synthetic and natural materials such aspoly(Lactide-co-Glycolide) (PLGA) and alginate. Different preparation methods including gas foaming, cryogelation, and salt leaching, have been utilized to generate the macroporous structures. By encapsulating different proteins and chemicals within the scaffold matrix, they have demonstrated promising applications in fields such as cancer immunotherapy and tissue regeneration. Recently, we have fabricated a novel injectable, biomimetic macroporous scaffold with the ability to encapsulate and sustainably release proteins. The average pore size is around 50 µm and with the encapsulation ofgranulocyte macrophage colony-stimulating factor (GM-CSF), about 1 million immune cells can be recruited to the scaffolds, including dendritic cells, macrophages, CD4+ T cells and CD8+ T cells. Cytokine expression analysis showed that the recruited cells generate a tolerogenic and anti-inflammatory environment. This novel class of scaffolds may have broad applications in regenerative medicine.
5:45 PM - SM6.3.06
Electroporation of HL-60 White Blood Cell Suspensions Using Nanoporous Membrane Electrodes Platform for Immuno Modulation
Bruce Hinds 1
1 , University of Washington, Seattle, Washington, United States
Show AbstractA flow-through electroporation system, based on a novel nanoporous membrane/electrode design, for the delivery of cell wall-impermeant molecules into model leukocytes, HL-60 promyelocytes, was demonstrated. The ability to apply low voltages to cell populations, with nm-scale concentrated electric field in a periodic array, contributes to high cell viability. With applied biases of 1-4V, delivery of target molecules was achieved with 90% viability and up to 65% transfection efficiency. More importantly, the system allowed electrophoretic pumping of molecules from a micro-scale reservoir across the membrane/electrode system into a micro-fluidic flow channel for transfection of cells, a design that can reduce reagent amount by 8 fold compared to current strategies. The flow-through system, which forces intimate membrane/electrode contact by using a 10µm channel height, can be easily scaled-up by adjusting the micro-fluidic channel geometry and/or the applied voltage pulse frequency to control cell residence times at the cell membrane/electrode interface. The demonstrated system shows promise in clinical applications where low-cost, high cell viability and high volume transfection methods are needed without the risk of viral vectors. In particular genetic modification of freely mobile white blood cells to either target disease cells, express desired protein/enzyme biomolecules or modulate emune response is an important target platform enabled by this device system [ Z. Chen, MA Akenhead, X Sun, H. Sapper, HY Shin, BJ Hinds Adv. Healthcare Materials 2016 5(16) 2105-12].
SM6.4: Poster Session: Materials in Immunology
Session Chairs
Thursday AM, April 20, 2017
Sheraton, Third Level, Phoenix Ballroom
9:00 PM - SM6.4.01
Shapes, Surfaces and Hardness in the Immune Response to MSUM
Alicia Brune 1 , William Petuskey 1
1 , Arizona State University, Tempe, Arizona, United States
Show AbstractWe have been characterizing MSUM (MSU, monosodium urate monohydrate, sodium hydrogen urate monohydrate, sodium urate hydrate) crystals regarding morphologies, surfaces, and mechanical properties. These data bear relevance to immunology in the human body because of the physicochemical properties of particulates affecting responses leading to gout disease. Several observations have been alluded to in the literature that suggest connections between crystal morphology/physical properties and the disease, namely: (a) crystalline, but not amorphous, MSUM triggers inflammation within specific tissues and at specific locations within the body; (b) “pointy” MSUM crystals, obtained via crystallization and investigated as vaccine adjuvants, induce greater immune responses than other morphologies; (c) the nature (e.g., specific orientations) of crystalline MSUM surfaces is linked to how the crystals interact with tissues; (d) MSUM crystals can cause mechanical tissue damage, which in turn, causes enhanced immune response. We examine our data on MSUM mechanical properties, crystal surfaces and morphologies and what might be correlated with possible causal mechanisms of action.
9:00 PM - SM6.4.02
A Sensor for Detecting Protein-Nanoparticle Interaction
Christina Moeslund 1 , Duncan Sutherland 1
1 , iNANO, Aarhus University, Aarhus C Denmark
Show AbstractThe utilization of nanoparticles in commercial products has increased tremendously over the last 20 years. Nanoparticles of metal oxides are found in cosmetics, sunscreen and paint. Furthermore, nanoparticles are gaining momentum as a platform for transporting drugs within the body for personalized medicine. It is known that nanoparticles are instantly covered in biomolecules upon entering the bloodstream, the so-called “protein corona”, which influences the particle’s interaction with cells. However, the lack of options for studying interactions of different types and sizes of nanoparticles with specific proteins has led to a knowledge gap. Many different methods have been used on differing nanoparticles, but the lack of consistency makes it difficult to compare between experiments.
The complement system represents an important collection of proteins that may interact with nanoparticles during exposure to plasma. It is initiated by a number of proteins, including the hexamer protein C1q, which interacts with, amongst others, the Fc-region of antibodies deposited on the foreign surfaces. The curvature of the nanoparticle surface will direct the adsorbing protein layers into a specific 3D geometry which has the potential to alter complement activation.
This poster focuses on the production of a sensor, which can be applied to study protein corona formation at nanoparticles. It is intended to be utilized for different types and sizes of nanoparticles, to verify and quantify their interaction with a specific, but interchangeable, protein, such as complement protein. This can aid in gathering sufficient consistent data about nanoparticle-protein interactions to identify trends in nanoparticle-protein interactions across different size ranges and surface chemistries. The design is based on a protein-repellent surface covered in nanoparticles. This creates a sensor surface where proteins bind almost exclusively to the curved surface of the nanoparticles. The surface can be used for both the unlabelled, quantitative method Surface Plasmon Resonance (SPR) and the labelled and qualitative Total Internal Reflectance Spectroscopy, thereby creating a double-checking environment.
During initial fabrication, it was discovered that Polystyrene and SiO2 nanoparticles will bind directly to a SiO2 surface covered with poly-L-Lysine-g-poly(ethylene glycol) (PLL-PEG). This allows the sensor fabrication to be a very simple and fast 2 step procedure requiring a minimum of equipment. Following fabrication, the protein-nanoparticle interaction was verified with fluorescently labelled Bovine Serum Albumin (BSA) through fluorescence microscopy, and quantified with non-labelled BSA through SPR spectroscopy. This confirms that we have built a simple sensor that can be utilized for deriving complex relationships within protein-nanoparticle interactions. With further development, this sensor will be used to derive the interactions between C1q and a range of nanoparticles.
9:00 PM - SM6.4.03
Au-MnFe2O4 Core-Shell Nanoparticles for Drug Delivery, MR Imaging and Immune Activation
Ravichandran Manisekaran 1 , Goldie Oza 1
1 , CINVESTAV, Mexico City, FDM, Mexico
Show AbstractAn upsurge of expanded interest in the field of Magnetic nanotechnology has led us to allow indepth exploitation of magnetic nanoparticles in nanomedicine. Encapsulating the core made up of magnetic nanoparticles by Gold nanoshell leads to the development of a proficient biocompatible and stabilized drug/delivery system under physiological conditions. This modular design enables Au-MnFe2O4 to perform multiple functions simultaneously, such as in multimodal imaging, drug delivery and real-time monitoring, as well as combined therapeutic approaches. The ability of MNPs to enhance proton relaxation of specific tissues and serve as MR imaging contrast agents is one of the most promising applications of nanomedicine. Moreover, there is a deeper understanding of iron oxide nanoparticles activationg immune system, hence this property is also explored in this report.
In the present work, Au-MnFe2O4 nanoparticles are used as cargo for the docking of anti-cancer drug such as Doxorubicin (DOX) using cysteamine as a linker for the attachment. The attachment could be monitored using UV-visible spectroscopy. Attachments were verified using FTIR, which confirmed the formation of non-covalent interactions. The stability of Au-MnFe2O4 nanoparticles was scrutinized by measuring the flocculation parameter, which was found to be in the range of 0-0.65. Further, zeta potential measurements confirmed the pH of 7.4 at which maximum drug attachment can take place. The amalgamation of the drug along with activated folic acid as a navigational molecule is the critical phase for targeted drug delivery. The drug loading capacity of the Au-MnFe2O4 was found to be 92%. This complex possess T1 contrast MR Imaging. This complex was found to be comparatively non-toxic for normal cells and considerably toxic for Hep-2 cells due to hyperthermal properties of SPIONs and targeted-mechanism of folic acid.
Moreover, we have also studied the effect of core-shell nanoparticles on monocytes and macrophages. This preliminary work is done on whole blood to comprehend whether this complex possess the capability of stimulation of immune system. Later this work will be explored further to understand effective macrophage polarization towards the tumour microenvironment. This will potentiate the effective way of cáncer immunotherapy.
9:00 PM - SM6.4.04
Development of a Universal and Recyclable Virus Deactivation System for Protection against Airborne Transmissible Pathogenic Aerosols
Ilaria Rubino 1 , Hyo-Jick Choi 1
1 Chemical and Materials Engineering, University of Alberta, Edmonton, Alberta, Canada
Show AbstractAirborne transmission has a critical role in the spread of respiratory diseases. Currently used respiratory protection devices in health care systems, such as N95 respirators, rely on filtration of the pathogenic particles. Although not designed to protect the wearer against aerosols, surgical masks have been increasingly adopted for this purpose, as observed during past experiences of severe acute respiratory syndrome (SARS), H1N1 swine flu in 2009, and Middle East respiratory syndrome (MERS). In the absence of a system to deactivate the pathogens collected on the filters, safety concerns arise on the risk of secondary infection and transmission from the contaminated masks. To this end, we propose a universal and reusable virus negation system, which efficiently deactivates viral aerosols on a filter and provides complete protection against diverse substrains.
In this work, we report the development of a safe and highly effective influenza virus deactivation system based on salt recrystallization process. To demonstrate the concept, we functionalized the surface of polypropylene fibers of mask filters with sodium chloride salt. Formation of the salt coating was confirmed with scanning electron microscopy (SEM)/energy dispersive x-ray (EDX) mapping and x-ray diffraction analysis (XRD). Filtration efficiency and virus deactivation on salt-coated filters were determined in vitro following exposure to 2.5–4 μm aerosols of A/California/04/2009 influenza virus, and protective efficacy was studied in vivo. Virus destabilization on salt-coated fibers was examined by measuring hemagglutinin activity and viral infectivity change. In parallel, physical destruction of virus was investigated by transmission electron microscopy. Furthermore, broad-spectrum protection of salt-coated filters against multiple subtypes of viral aerosols was evaluated by investigating both lethal infectivity by penetrated virus in vivo and infectivity by virus collected on filters during filtration in vitro using A/Puerto Rico/08/1934 and A/Vietnam/1203/2004.
Results showed that salt-functionalized filters have significantly superior filtration efficiency compared to commercial surgical mask filters and guaranteed complete protection against influenza virus lethal infection. Virus collected on the surface of salt-coated filters showed rapid infectivity loss, demonstrating disruption of virus. Additionally, performance of salt-coated filters was not compromised by prolonged exposure to harsh environmental conditions (37°C and 70% relative humidity). Therefore, our proof-of-concept virus deactivation system can guarantee development of recyclable and universal respiratory protection devices for infection control during pandemics and epidemics.
Symposium Organizers
Hao Cheng, Drexel University
Wendy Liu, University of California, Irvine
Minglin Ma, Cornell University
Cherie Stabler, University of Florida
Symposium Support
JDRF, International
SM6.5: Nanomaterials for Immune Modulation
Session Chairs
Thursday AM, April 20, 2017
PCC North, 100 Level, Room 122 C
9:45 AM - *SM6.5.01
Self-Assembled Nanogels as Vaccine Delivery Systems
Kazunari Akiyoshi 1 2
1 , Kyoto University, Kyoto Japan, 2 , JST ERATO, Kyoto Japan
Show AbstractNanogels can be used as new biologics DDS by efficiently trapping biomacromolecules such as DNA, siRNA, peptides and proteins. We first reported physically cross-linked nanogels by self-assembly of hydrophobized polysaccharides (self-Nanogel). The attractive characteristics of the amphiphilic self-Nanogels (for example, cholesteryl pullulan (CHP)) is molecular chaperone like function, which enables them to capture various proteins and to release in the native form.1 Self-Nanogel acts as efficient protein delivery system.2 We report here self-Nanogel applications as immunological drug delivery systems for mainly cancer vaccines and nasal vaccines.
Cancer vaccines with complex of protein antigens and CHP self-Nanogel were the first practical application of nanogels as vehicles for antigen delivery system. 2 It was found that vaccines consisting of nanogels could be administered repeatedly to humans without serious adverse effects, and nanogel vaccines induced antigen-specific cellular and humoral immunity. Recently, a novel cancer vaccine using CHP self-Nanogels as carriers for long peptide antigens (LPAs) including multiple peptide epitopes recognized by CD4+ and CD8+ T cells was reported. 3
Nasal vaccination is one of the most attractive immunological dosage for delivering vaccine antigens directly onto the mucosa to induce a protective immune response. Cationic CHP self-Nanogels were effective in penetrating the nasal mucosa and resulted in successful nasal vaccines in mice. 4 Recently, nasal vaccines using pneumococcal surface protein A (PspA) as an antigen were carried out in mice and nonhuman primates. 5 Both experiments using mice and macaques suggested that PspA-specific antibodies, including IgG and IgA in serum, nasal washes, were effectively induced via the cCHP nanogel.
Reference
1) Y. Sasaki, K. Akiyoshi, The Chemical Record, 10 (2010) 366-376.
2) Y. Tahara, K. Akiyoshi, Adv. Drug Deliv. Rev. 95 (2015) 65-76.
3) D. Muraoka, et al., ACS nano, 8 (2014) 9209-9218.
4) T. Nochi, et al., Nature Materials, 9 (2010) 572-578.
5) Y. Fukuyama, et.al., Mucosal Immunology, 8, (2015) 1144-1153.
10:15 AM - *SM6.5.02
Multi-Stage Drug Delivery System for Enhanced Payload Delivery to Lymph Node Cells
Alex Schudel 1 , Cody Higginson 1 , M.G. Finn 1 , Susan Thomas 1
1 , Georgia Institute of Technology, Atlanta, Georgia, United States
Show AbstractTargeted drug delivery to lymph nodes (LN), tissues where organized lymphocyte accumulation in the body occurs, has the potential to improve the treatment of a variety of pathologies, such as B and T cell malignancies and immunodeficiency viral infections, as well as enhance the efficacy of vaccines and cancer immunotherapy. To date, size-based principles of lymphatic-mediated transport have emerged as an effective means to achieve facile LN delivery of formulated agents after injection in the periphery. However, within the LN, solutes and particulates are filtered based on size, with smaller molecules penetrating more deeply into LN follicles and the paracortex than large particulates by virtue of their increased diffusivity and uptake within LN conduits. Herein, we report the design and implementation of a multistage lymphatic delivery and payload release system combining 30 nm thiolated poly(propylene sulfide) nanoparticles that exhibit prodigious uptake into lymphatics after administration with the thiol-reactive oxanorbornadiene (OND) linkage chemistry that degrades in a pH- and polarity-insensitive manner at programmable half-lives ranging from hours to days to release small-molecule cargo. Post administration in the skin, nanoparticle tethering dramatically enhances model cargo accumulation within LN draining the site of injection that when released via the OND penetrates deeply throughout the collagenous LN interstitium due to its increased diffusivity relative to polymer nanoparticles. As a result, small molecule cargo is delivered to cell subtypes localized within the LN cortex and paracortex at high levels, a 10- to 1000-fold relative to free cargo alone or cargo tethered to the nanoparticles via an irreversible linkage. These data suggest that by utilizing an engineered multi-stage system to overcome barriers to both lymphatic and lymph node uptake, payload can be delivered to previously undruggable cell subpopulations resident within lymph nodes and has the potential to dramatically enhance drug bioactivity and resulting therapeutic effect.
11:15 AM - *SM6.5.03
Cell Membrane-Coated Nanoparticles as a Vaccine Delivery Platform
Liangfang Zhang 1
1 , University of California, San Diego, La Jolla, California, United States
Show AbstractCell-derived nanoparticles have been garnering increased attention due to their ability to mimic many of the natural properties displayed by their source cells. This top-down engineering approach can be applied towards the development of novel therapeutic strategies owing to the unique interactions enabled through the retention of complex antigenic information. Herein, I report on the biological functionalization of polymeric nanoparticles with a layer of membrane coating derived from cancer cells. The resulting core-shell nanostructures, which carry the full array of cancer cell membrane antigens, offer a robust platform with applicability towards multiple modes of anticancer therapy. Specifically, by coupling the particles with an immunological adjuvant, the resulting formulation can be used to promote a tumor-specific immune response for use in vaccine applications. Moreover, taking advantage of the cargo carrying capacity of nanoparticles, it is possible to load a large amount of adjuvant, a necessity for generating detectable antitumor responses. Effective nanoparticle engineering enables association of both antigen and adjuvant on the same particle, further boosting the potency of the formulation.
11:45 AM - SM6.5.04
Engineering an Effective Immune Adjuvant by Designed Control of Shape and Crystallinity of Aluminum Oxyhydroxide Nanoparticles
Tian Xia 1
1 , University of California, Los Angeles, Los Angeles, California, United States
Show AbstractAdjuvants based on aluminum salts (Alum) are commonly used in vaccines to boost the immune response against infectious agents. However, the detailed mechanism of how Alum enhances adaptive immunity and exerts its adjuvant immune effect remains unclear. Other than being comprised of micrometer-sized aggregates that include nanoscale particulates, Alum lacks specific physicochemical properties to explain activation of the innate immune system, including the mechanism by which aluminum-based adjuvants engage the NLRP3 inflammasome and IL-1β production. This is putatively one of the major mechanisms required for an adjuvant effect. Because we know that long aspect ratio nanomaterials trigger the NLRP3 inflammasome, we synthesized a library of aluminum oxyhydroxide (AlOOH) nanorods to determine whether control of the material shape and crystalline properties could be used to quantitatively assess NLRP3 inflammasome activation and linkage of the cellular response to the material’s adjuvant activitiesin vivo. Using comparison to commercial Alum, we demonstrate that the crystallinity and surface hydroxyl group display of AlOOH nanoparticles quantitatively impact the activation of the NLRP3 inflammasome in human THP-1 myeloid cells or murine bone marrow-derived dendritic cells (BMDCs). Moreover, these in vitro effects were correlated with the immunopotentiation capabilities of the AlOOH nanorods in a murine OVA immunization model. These results demonstrate that shape, crystallinity, and hydroxyl content play an important role in NLRP3 inflammasome activation and are therefore useful for quantitative boosting of antigen-specific immune responses. These results show that the engineered design of aluminum-based adjuvants in combination with dendritic cell property–activity analysis can be used to design more potent aluminum-based adjuvants.
12:00 PM - SM6.5.05
A Novel Method for High-Throughput Discovery of Neo-Antigens and Corresponding T-Cell Receptors
Songming Peng 1 , Jesse Zaretsky 2 , Michael Bethune 1 , James Heath 1
1 , California Institute of Technology, Pasadena, California, United States, 2 , University of California, Los Angeles, Westwood, California, United States
Show AbstractImmunotherapies that boost the ability of endogenous T cells to destroy cancer cells have demonstrated therapeutic efficacy in several human malignancies. Cytotoxic T cells actively recognize peptide antigens, which are displayed on major histocompatibility complexes (MHCs) on the surface of the malignant cells, to play a central role in cancer immunotherapy. However, efficient methods for identifying the tumor antigens and the corresponding T cell receptors are still limited. We report here a DNA-barcode nanoparticle and microfluidics-based method to sort and enumerate neoantigen-specific T cells, at an unprecedented level of sensitivity. The method also permits the matching of a single neoantigen to a specific CD8+ T cell with the TCR a/b gene sequence. The multiplexing capability of DNA allows us to simultaneously analyze up to 64 neo-antigens. The resultant information informs an immunotherapy regimen that is specific for that patient, which can be used to custom-design effective ACT therapies.