Targeting pathogen recognition receptors on dendritic cells (DCs) offers the advantage of triggering specific signaling pathways to induce a tailored and robust immune response. In this work, we describe a novel approach to targeted antigen delivery by decorating the surface of polyanhydride nanoparticles with specific carbohydrates to provide "pathogen-like" properties that ensure nanoparticles engage C-type lectin receptors on DCs. The surface of polyanhydride nanoparticles was functionalized by covalent linkage of dimannose and lactose residues using an amine-carboxylic acid coupling reaction. Coculture of functionalized nanoparticles with bone marrow-derived DCs significantly increased cell surface expression of MHC II, the T cell costimulatory molecules CD86 and CD40, the C-type lectin receptor CIRE and the mannose receptor CD206 over the nonfunctionalized nanoparticles. Both nonfunctionalized and functionalized nanoparticles were efficiently internalized by DCs, indicating that internalization of functionalized nanoparticles was necessary but not sufficient to activate DCs. Blocking the mannose and CIRE receptors prior to the addition of functionalized nanoparticles to the culture inhibited the increased surface expression of MHC II, CD40 and CD86. Together, these data indicate that engagement of CIRE and the mannose receptor is a key mechanism by which functionalized nanoparticles activate DCs. These studies provide valuable insights into the rational design of targeted nanovaccine platforms to induce robust immune responses and improve vaccine efficacy. The use of vaccine adjuvants to activate the innate immune system is crucial to vaccine effectiveness. 1 Adjuvants can be used to enhance the efficacy of single dose vaccines and reduce the required antigen dose. The use of biodegradable polymer nanoparticles as vaccine delivery vehicles allows for effective delivery of payloads by parental or mucosal administration by protecting the antigen from harsh physiological conditions and enabling transport across biological barriers (e.g., mucus membranes). 2 Polyanhydride nanoparticles have shown excellent potential as vaccine carriers. 3À6 Encapsulation of protein antigens into polyanhydride particles stabilizes them and provides sustained azntigen release; 4,7 these particles also enhance the immune response by acting as an adjuvant. 3 Dendritic cells (DCs) are antigen presenting cells (APCs) that play a major role in connecting the innate and adaptive immune systems, a key step to inducing protective immunity. 8 DCs can sense and internalize antigen by a variety of mechanisms that trigger DC maturation and direct further interactions with other immune cells, including naive T cells. 1,9,10 Pattern recognition receptors (PRRs) on DCs detect the presence of a potential threat by interacting with pathogen-associated molecular patterns (PAMPs). 10,11 In particular, C-type lectin receptors (CLRs) are PRRs with highly conserved carbohydrate-recognition domains that bind sugar moieties (e.g., mannose, fuco...

The present studies were designed to evaluate the adjuvant activity of polyanhydride microparticles prepared in the absence of additional stabilizers, excipients, or immune modulators. Microparticles composed of varying ratios of either 1,6-bis(p-carboxyphenoxy)hexane (CPH) and sebacic acid (SA) or 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG) and CPH were added to in vitro cultures of bone marrow-derived dendritic cells (DCs). Microparticles were efficiently and rapidly phagocytosed by DCs in the absence of opsonization and without centrifugation or agitation. Within 2 h, internalized particles were rapidly localized to an acidic, phagolysosomal compartment. By 48 h, only a minor reduction in microparticle size was observed in the phagolysosomal compartment, indicating minimal particle erosion consistent with being localized within an intracellular microenvironment favoring particle stability. Polyanhydride microparticles increased DC surface expression of MHC II, the co-stimulatory molecules CD86 and CD40, and the C-type lectin CIRE (murine DC-SIGN; CD209). In addition, microparticle stimulation of DCs also enhanced secretion of the cytokines IL-12p40 and IL-6, a phenomenon found to be dependent on polymer chemistry. DCs cultured with polyanhydride microparticles and ovalbumin induced polymer chemistry-dependent antigen-specific proliferation of both CD4+ OT-II and CD8+ OT-I T cells. These data indicate that polyanhydride particles can be tailored to take advantage of the potential plasticity of the immune response, resulting in the ability to induce immune protection against many types of pathogens.

Nanosphere and antigen uptake by monocytes can be directly correlated to the chemistry of the nanosphere. These results demonstrate the importance of choosing polyanhydride chemistries that facilitate enhanced interactions with antigen presenting cells that are necessary in the initiation of efficacious immune responses.

An opportunity exists today for cross-cutting research utilizing advances in materials science, immunology, microbial pathogenesis, and computational analysis to effectively design the next generation of adjuvants and vaccines. This study integrates these advances into a bottom-up approach for the molecular design of nanoadjuvants capable of mimicking the immune response induced by a natural infection but without the toxic side effects. Biodegradable amphiphilic polyanhydrides possess the unique ability to mimic pathogens and pathogen associated molecular patterns with respect to persisting within and activating immune cells, respectively. The molecular properties responsible for the pathogen-mimicking abilities of these materials have been identified. The value of using polyanhydride nanovaccines was demonstrated by the induction of long-lived protection against a lethal challenge of Yersinia pestis following a single administration ten months earlier. This approach has the tantalizing potential to catalyze the development of next generation vaccines against diseases caused by emerging and re-emerging pathogens.

Advancements towards an improved vaccine against Bacillus anthracis, the causative agent of anthrax, have focused on formulations composed of the protective antigen (PA) adsorbed to aluminum hydroxide. However, due to the labile nature of PA, antigen stability is a primary concern for vaccine development. Thus, there is a need for a delivery system capable of preserving the immunogenicity of PA through all the steps of vaccine fabrication, storage, and administration. In this work, we demonstrate that biodegradable amphiphilic polyanhydride nanoparticles, which have previously been shown to provide controlled antigen delivery, antigen stability, immune modulation, and protection in a single dose against a pathogenic challenge, can stabilize and release functional PA. These nanoparticles demonstrated polymer hydrophobicity-dependent preservation of the biological function of PA upon encapsulation, storage (over extended times and elevated temperatures), and release. Specifically, fabrication of amphiphilic polyanhydride nanoparticles composed of 1,6-bis(p-carboxyphenoxy)hexane and 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane best preserved PA functionality. These studies demonstrate the versatility and superiority of amphiphilic nanoparticles as vaccine delivery vehicles suitable for long-term storage.

Complex biological barriers are major obstacles for preventing and treating disease. Nano-carriers are designed to overcome such obstacles by enhancing drug delivery through physiochemical barriers and improving therapeutic indices. This review critically examines both biological barriers and nano-carrier payloads for a variety of drug delivery applications. A spectrum of nano-carriers is discussed that have been successfully developed for improving tissue penetration for preventing or treating a range of infectious, inflammatory, and degenerative diseases.

Nanoparticulate systems have emerged as valuable tools in vaccine delivery through their ability to efficiently deliver cargo, including proteins, to antigen presenting cells [1][2][3][4][5] . Internalization of nanoparticles (NP) by antigen presenting cells is a critical step in generating an effective immune response to the encapsulated antigen. To determine how changes in nanoparticle formulation impact function, we sought to develop a high throughput, quantitative experimental protocol that was compatible with detecting internalized nanoparticles as well as bacteria. To date, two independent techniques, microscopy and flow cytometry, have been the methods used to study the phagocytosis of nanoparticles. The high throughput nature of flow cytometry generates robust statistical data. However, due to low resolution, it fails to accurately quantify internalized versus cell bound nanoparticles. Microscopy generates images with high spatial resolution; however, it is time consuming and involves small sample sizes 6-8 . Multi-spectral imaging flow cytometry (MIFC) is a new technology that incorporates aspects of both microscopy and flow cytometry that performs multi-color spectral fluorescence and bright field imaging simultaneously through a laminar core. This capability provides an accurate analysis of fluorescent signal intensities and spatial relationships between different structures and cellular features at high speed.Herein, we describe a method utilizing MIFC to characterize the cell populations that have internalized polyanhydride nanoparticles or Salmonella enterica serovar Typhimurium. We also describe the preparation of nanoparticle suspensions, cell labeling, acquisition on an ImageStream X system and analysis of the data using the IDEAS application. We also demonstrate the application of a technique that can be used to differentiate the internalization pathways for nanoparticles and bacteria by using cytochalasin-D as an inhibitor of actin-mediated phagocytosis. Video LinkThe video component of this article can be found at http://www.jove.com/video/3884/ Protocol 1. RAW 264.7 Cell Culture 1. Harvest RAW 264.7 cells from their flasks when they reach confluency by scraping them gently with a cell scraper. Count and plate them into a 24-well cell culture dish at a density of 5 x 10 5 cells/well in 0.5 mL complete Dulbecco's Modified Eagle Medium (cDMEM; 10% heat-inactivated fetal bovine serum (FBS), 2 mM Glutamax, and 10 mM HEPES) and incubate overnight at 37°C in a 5% CO2 incubator. Pathogenic Salmonella enterica Serovar Typhimurium 14028 Transformation and Culture1. Introduce recombinant plasmids expressing green fluorescent protein (GFP) to S. Typhimurium by electroporation. First, prepare culture of Salmonella enterica serovar Typhimurium (ATCC 14028) in LB broth and incubate overnight at 37°C with aeration. 2. Next day, pellet 1 mL of bacteria at maximum speed in a microcentrifuge. Wash the cells four times with 1 mL of cold MOPS/glycerol solution (1 mM 3-(N-morpholino)propanesulfonic acid (...

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