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...

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In recent years, the incidence of atherosclerotic cardiovascular disease, obesity, and diabetes has increased largely worldwide. In the present work, we evaluated the genoprotective effect of the consumption of flavonoids-rich chocolate on 84 young volunteers. Biochemical indicators related to the prevention and treatment of cardiovascular risk and metabolic syndrome were also determined. A randomized, placebo-controlled, double-blind study was performed in the Autonomous University of Baja California. The treatments comprised the daily consumption of either 2 g of dark chocolate containing 70% cocoa, or 2 g of milk chocolate, for 6 months. The total amount of phenolic compounds and flavonoids was determined in both types of chocolate. Anthropometrical and Biochemical parameters were recorded prior to and after the study. The evaluation of the genotoxicity in buccal epithelial cells was performed throughout the duration of the study. Flavonoids from cocoa in dark chocolate significantly prevented DNA damage, and improved the nucleus integrity of cells. This effect could be related to the antioxidant capacity of the dark chocolate that decreased cellular stress. Biochemical parameters (total cholesterol, triglycerides, and LDL-cholesterol level in blood) and anthropometrical parameters (waist circumference) were improved after six months of daily intake of 2 g of dark chocolate with a 70% of cocoa.

Biodegradable nanoparticles have emerged as a versatile platform for the design and implementation of new intranasal vaccines against respiratory infectious diseases. Specifically, polyanhydride nanoparticles composed of the aliphatic sebacic acid (SA), the aromatic 1,6-bis(p-carboxyphenoxy)hexane (CPH), or the amphiphilic 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG) display unique bulk and surface erosion kinetics and can be exploited to slowly release functional biomolecules (e.g., protein antigens, immunoglobulins, etc.) in vivo. These nanoparticles also possess intrinsic adjuvant activity, making them an excellent choice for a vaccine delivery platform. In order to elucidate the mechanisms governing the activation of innate immunity following intranasal mucosal vaccination, one must evaluate the molecular and cellular responses of the antigen presenting cells (APCs) responsible for initiating immune responses. Dendritic cells are the principal APCs found in conducting airways, while alveolar macrophages (AMɸ) predominate in the lung parenchyma. AMɸ are highly efficient in clearing the lungs of microbial pathogens and cell debris. In addition, this cell type plays a valuable role in the transport of microbial antigens to the draining lymph nodes, which is an important first step in the initiation of an adaptive immune response. AMɸ also express elevated levels of innate pattern recognition and scavenger receptors, secrete pro-inflammatory mediators, and prime naïve T cells. A relatively pure population of AMɸ (e.g., greater than 80%) can easily be obtained via lung lavage for study in the laboratory. Resident AMɸ harvested from immune competent animals provide a representative phenotype of the macrophages that will encounter the particle-based vaccine in vivo. Herein, we describe the protocols used to harvest and culture AMɸ from mice and examine the activation phenotype of the macrophages following treatment with polyanhydride nanoparticles in vitro.

Polyanhydrides are a class of biomaterials with excellent biocompatibility and drug delivery capabilities. While they have been studied extensively with conventional one-sample-at-a-time synthesis techniques, a more recent high-throughput approach has been developed enabling the synthesis and testing of large libraries of polyanhydrides(1). This will facilitate more efficient optimization and design process of these biomaterials for drug and vaccine delivery applications. The method in this work describes the combinatorial synthesis of biodegradable polyanhydride film and nanoparticle libraries and the high-throughput detection of protein release from these libraries. In this robotically operated method (Figure 1), linear actuators and syringe pumps are controlled by LabVIEW, which enables a hands-free automated protocol, eliminating user error. Furthermore, this method enables the rapid fabrication of micro-scale polymer libraries, reducing the batch size while resulting in the creation of multivariant polymer systems. This combinatorial approach to polymer synthesis facilitates the synthesis of up to 15 different polymers in an equivalent amount of time it would take to synthesize one polymer conventionally. In addition, the combinatorial polymer library can be fabricated into blank or protein-loaded geometries including films or nanoparticles upon dissolution of the polymer library in a solvent and precipitation into a non-solvent (for nanoparticles) or by vacuum drying (for films). Upon loading a fluorochrome-conjugated protein into the polymer libraries, protein release kinetics can be assessed at high-throughput using a fluorescence-based detection method (Figures 2 and 3) as described previously(1). This combinatorial platform has been validated with conventional methods(2) and the polyanhydride film and nanoparticle libraries have been characterized with (1)H NMR and FTIR. The libraries have been screened for protein release kinetics, stability and antigenicity; in vitro cellular toxicity, cytokine production, surface marker expression, adhesion, proliferation and differentiation; and in vivo biodistribution and mucoadhesion(1-11). The combinatorial method developed herein enables high-throughput polymer synthesis and fabrication of protein-loaded nanoparticle and film libraries, which can, in turn, be screened in vitro and in vivo for optimization of biomaterial performance.