Yashdeep Phanse

Polyanhydride microparticles enhance dendritic cell antigen presentation and activation.

The present study was 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 or 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane 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 2h, 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 major histocompatability complex class 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.

Amphiphilic Polyanhydride Nanoparticles Stabilize Bacillus anthracis Protective Antigen

Advancements toward 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.

Rational Design of Pathogen-Mimicking Amphiphilic Materials as Nanoadjuvants

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.

Functionalization of polyanhydride microparticles with di-mannose influences uptake by and intracellular fate within dendritic cells

Innovative vaccine delivery platforms can facilitate the development of effective single-dose treatment regimens to control emerging and re-emerging infectious diseases. Polyanhydride microparticles are promising vaccine delivery vehicles due to their ability to stably maintain antigens, provide tailored release kinetics and function as adjuvants. A major obstacle for the use of microparticle-based vaccines, however, is their limited uptake by dendritic cells (DCs). In this study, we functionalized the microparticle surface with di-mannose in order to target C-type lectin receptors (CLRs) on DCs. Polyanhydride particles based on sebacic acid (SA), 1,6-bis(p-carboxyphenoxy)hexane (CPH) and 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG) were evaluated. Co-incubation of di-mannose-functionalized microparticles up-regulated the expression of CLRs on DCs. More importantly, di-mannose functionalization increased the uptake, as measured by the percentage of cells internalizing particles. The uptake of CPH:SA microparticles increased ∼20-fold, from 0.82% (non-functionalized) to 20.2%, and internalization of CPTEG:CPH microparticles increased ∼7-fold from 1.35% (non-functionalized) to 9.3% upon di-mannose functionalization. Both di-mannose-functionalized and non-functionalized particles trafficked to lysosomes. Together, these studies demonstrate that employing rational vaccine design principles, such as the targeting of CLRs on antigen-presenting cells, can enhance delivery of encapsulated antigens and potentially induce a more robust adaptive immune response.

Analyzing cellular internalization of nanoparticles and bacteria by multi-spectral imaging flow cytometry.

Nanoparticulate systems have emerged as valuable tools in vaccine delivery through their ability to efficiently deliver cargo, including proteins, to antigen presenting cells. 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. 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.

An In Vitro Model of Antibody-Enhanced Killing of the Intracellular Parasite Leishmania amazonensis

Footpad infection of C3HeB/FeJ mice with Leishmania amazonensis leads to chronic lesions accompanied by large parasite loads. Co-infecting these animals with L. major leads to induction of an effective Th1 immune response that can resolve these lesions. This cross-protection can be recapitulated in vitro by using immune cells from L. major-infected animals to effectively activate L. amazonensis-infected macrophages to kill the parasite. We have shown previously that the B cell population and their IgG2a antibodies are required for effective cross-protection. Here we demonstrate that, in contrast to L. major, killing L. amazonensis parasites is dependent upon FcRγ common-chain and NADPH oxidase-generated superoxide from infected macrophages. Superoxide production coincided with killing of L. amazonensis at five days post-activation, suggesting that opsonization of the parasites was not a likely mechanism of the antibody response. Therefore we tested the hypothesis that non-specific immune complexes could provide a mechanism of FcRγ common-chain/NADPH oxidase dependent parasite killing. Macrophage activation in response to soluble IgG2a immune complexes, IFN-γ and parasite antigen was effective in significantly reducing the percentage of macrophages infected with L. amazonensis. These results define a host protection mechanism effective during Leishmania infection and demonstrate for the first time a novel means by which IgG antibodies can enhance killing of an intracellular pathogen.

A systems approach to designing next generation vaccines: combining α-galactose modified antigens with nanoparticle platforms.

Innovative vaccine platforms are needed to develop effective countermeasures against emerging and re-emerging diseases. These platforms should direct antigen internalization by antigen presenting cells and promote immunogenic responses. This work describes an innovative systems approach combining two novel platforms, αGalactose (αGal)-modification of antigens and amphiphilic polyanhydride nanoparticles as vaccine delivery vehicles, to rationally design vaccine formulations. Regimens comprising soluble αGal-modified antigen and nanoparticle-encapsulated unmodified antigen induced a high titer, high avidity antibody response with broader epitope recognition of antigenic peptides than other regimen. Proliferation of antigen-specific CD4+ T cells was also enhanced compared to a traditional adjuvant. Combining the technology platforms and augmenting immune response studies with peptide arrays and informatics analysis provides a new paradigm for rational, systems-based design of next generation vaccine platforms against emerging and re-emerging pathogens.

Nano-enabled delivery of diverse payloads across complex biological barriers

Complex biological barriers are major obstacles for preventing and treating disease. Nanocarriers 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 nanocarrier payloads for a variety of drug delivery applications. A spectrum of nanocarriers is discussed that have been successfully developed for improving tissue penetration for preventing or treating a range of infectious, inflammatory, and degenerative diseases.

Biodistribution and Toxicity Studies of PRINT Hydrogel Nanoparticles in Mosquito Larvae and Cells

Mosquito-borne diseases continue to remain major threats to human and animal health and impediments to socioeconomic development. Increasing mosquito resistance to chemical insecticides is a great public health concern, and new strategies/technologies are necessary to develop the next-generation of vector control tools. We propose to develop a novel method for mosquito control that employs nanoparticles (NPs) as a platform for delivery of mosquitocidal dsRNA molecules to silence mosquito genes and cause vector lethality. Identifying optimal NP chemistry and morphology is imperative for efficient mosquitocide delivery. Toward this end, fluorescently labeled polyethylene glycol NPs of specific sizes, shapes (80 nm x 320 nm, 80 nm x 5000 nm, 200 nm x 200 nm, and 1000 nm x 1000 nm) and charges (negative and positive) were fabricated by Particle Replication in Non-Wetting Templates (PRINT) technology. Biodistribution, persistence, and toxicity of PRINT NPs were evaluated in vitro in mosquito cell culture and in vivo in Anopheles gambiae larvae following parenteral and oral challenge. Following parenteral challenge, the biodistribution of the positively and negatively charged NPs of each size and shape was similar; intense fluorescence was observed in thoracic and abdominal regions of the larval body. Positively charged NPs were more associated with the gastric caeca in the gastrointestinal tract. Negatively charged NPs persisted through metamorphosis and were observed in head, body and ovaries of adults. Following oral challenge, NPs were detected in the larval mid- and hindgut. Positively charged NPs were more efficiently internalized in vitro than negatively charged NPs. Positively charged NPs trafficked to the cytosol, but negatively charged NPs co-localized with lysosomes. Following in vitro and in vivo challenge, none of the NPs tested induced any cytotoxic effects.

Biodistribution and Trafficking of Hydrogel Nanoparticles in Adult Mosquitoes

Background Nanotechnology offers great potential for molecular genetic investigations and potential control of medically important arthropods. Major advances have been made in mammalian systems to define nanoparticle (NP) characteristics that condition trafficking and biodistribution of NPs in the host. Such information is critical for effective delivery of therapeutics and molecules to cells and organs, but little is known about biodistribution of NPs in mosquitoes. Methodology/Principal Findings PRINT technology was used to construct a library of fluorescently labeled hydrogel NPs of defined size, shape, and surface charge. The biodistribution (organ, tissue, and cell tropisms and trafficking kinetics) of positively and negatively charged 200 nm x 200 nm, 80 nm x 320 nm, and 80 nm x 5000 nm NPs was determined in adult Anopheles gambiae mosquitoes as a function of the route of challenge (ingestion, injection or contact) using whole body imaging and fluorescence microscopy. Mosquitoes readily ingested NPs in sugar solution. Whole body fluorescence imaging revealed substantial NP accumulation (load) in the alimentary tracts of the adult mosquitoes, with the greatest loads in the diverticula, cardia and foregut. Positively and negatively charged NPs differed in their biodistribution and trafficking. Following oral challenge, negatively charged NPs transited the alimentary tract more rapidly than positively charged NPs. Following contact challenge, negatively charged NPs trafficked more efficiently in alimentary tract tissues. Following parenteral challenge, positively and negatively charged NPs differed in tissue tropisms and trafficking in the hemocoel. Injected NPs were also detected in cardia/foregut, suggesting trafficking of NPs from the hemocoel into the alimentary tract. Conclusions/Significance Herein we have developed a tool box of NPs with the biodistribution and tissue tropism characteristics for gene structure/function studies and for delivery of vector lethal cargoes for mosquito control.