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In JoVE (1)
Other Publications (14)
- Journal of Biomedical Materials Research. Part A
- Biomaterials
- Journal of Pharmaceutical Sciences
- Pharmaceutical Research
- Combinatorial Chemistry & High Throughput Screening
- Biomaterials
- Journal of Biomedical Materials Research. Part B, Applied Biomaterials
- Acta Biomaterialia
- PloS One
- Acta Biomaterialia
- Biomaterials
- Molecular Pharmaceutics
- Scientific Reports
- Biomaterials
Articles by Michael Wannemuehler in JoVE
JoVE Bioengineering
Harvesting Murine Alveolar Macrophages and Evaluating Cellular Activation Induced by Polyanhydride Nanoparticles
1Department of Chemical and Biological Engineering, Iowa State University, 2Department of Veterinary Microbiology and Preventive Medicine, Iowa State University
Herein, we describe protocols for harvesting murine alveolar macrophages, which are resident innate immune cells in the lung, and examining their activation in response to co-culture with polyanhydride nanoparticles.
Other articles by Michael Wannemuehler on PubMed
Single Dose Vaccine Based on Biodegradable Polyanhydride Microspheres Can Modulate Immune Response Mechanism
This study focuses on the development of single dose vaccines based on biodegradable polyanhydride microspheres that have the unique capability to modulate the immune response mechanism. The polymer system employed consists of copolymers of 1,6-bis(p-carboxyphenoxy)hexane and sebacic acid. Two copolymer formulations that have been shown to provide extended release kinetics and protein stability were investigated. Using tetanus toxoid (TT) as a model antigen, in vivo studies in C3H/HeOuJ mice demonstrated that the encapsulation procedure preserves the immunogenicity of the TT. The polymer itself exhibited an adjuvant effect, enhancing the immune response to a small dose of TT. The microspheres provided a prolonged exposure to TT sufficient to induce both a primary and a secondary immune response (i.e., high antibody titers) with high-avidity antibody production, without requiring an additional administration. Antigen-specific proliferation 28 weeks after a single immunization indicated that immunization with the polyanhydride microspheres generated long-lived memory cells and plasma cells (antibody-secreting B cells) that generally do not occur without maturation signals from T helper cells. Furthermore, by altering the vaccine formulation, the overall strength of the T helper type 2 immune response was selectively diminished, resulting in a balanced immune response, without reducing the overall titer. This result is striking, considering free TT induces a T helper type 2 immune response, and has important implications for developing vaccines to intracellular pathogens. The ability to selectively tune the immune response without the administration of additional cytokines or noxious adjuvants is a unique feature of this delivery vehicle that may make it an excellent candidate for vaccine development.
Protein Stability in the Presence of Polymer Degradation Products: Consequences for Controlled Release Formulations
When encapsulating proteins in polymer microspheres for sustained drug delivery there are three stages during which the stability of the protein must be maintained: (1) the fabrication of the microspheres, (2) the storage of the microspheres, and (3) the release of the encapsulated protein. This study focuses on the effects of polymer degradation products on the primary, secondary, and tertiary structure of tetanus toxoid, ovalbumin (Ova), and lysozyme after incubation for 0 or 20 days in the presence of ester (lactic acid and glycolic acid) and anhydride (sebacic acid and 1,6-bis(p-carboxyphenoxy)hexane) monomers. The structure and antigenicity or enzymatic activity of each protein in the presence of each monomer was quantified. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, circular dichroism, and fluorescence spectroscopy were used to assess/evaluate the primary, secondary, and tertiary structures of the proteins, respectively. Enzyme-linked immunosorbent assay was used to measure changes in the antigenicity of tetanus toxoid and Ova and a fluorescence-based assay was used to determine the enzymatic activity of lysozyme. Tetanus toxoid was found to be the most stable in the presence of anhydride monomers, while Ova was most stable in the presence of sebacic acid, and lysozyme was stable when incubated with all of the monomers studied.
Vaccine Adjuvants: Current Challenges and Future Approaches
For humans, companion animals, and food producing animals, vaccination has been touted as the most successful medical intervention for the prevention of disease in the twentieth century. However, vaccination is not without problems. With the development of new and less reactogenic vaccine antigens, which take advantage of molecular recombinant technologies, also comes the need for more effective adjuvants that will facilitate the induction of adaptive immune responses. Furthermore, current vaccine adjuvants are successful at generating humoral or antibody mediated protection but many diseases currently plaguing humans and animals, such as tuberculosis and malaria, require cell mediated immunity for adequate protection. A comprehensive discussion is presented of current vaccine adjuvants, their effects on the induction of immune responses, and vaccine adjuvants that have shown promise in recent literature.
Polymer Chemistry Influences Monocytic Uptake of Polyanhydride Nanospheres
To demonstrate that polyanhydride copolymer chemistry affects the uptake and intracellular compartmentalization of nanospheres by THP-1 human monocytic cells.
High Throughput Cell-based Screening of Biodegradable Polyanhydride Libraries
A parallel screening method has been developed to rapidly evaluate discrete library substrates of biomaterials using cell-based assays. The biomaterials used in these studies were surface-erodible polyanhydrides based on sebacic acid (SA), 1,6-bis(p-carboxyphenoxy)hexane (CPH), and 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG) that have been previously studied as carriers for drugs, proteins, and vaccines. Linearly varying compositional libraries of 25 different polyanhydride random copolymers (based on CPH:SA and CPTEG:CPH) were designed, fabricated, and synthesized using discrete (organic solvent-resistant) multi-sample substrates created using a novel rapid prototyping method. The combinatorial libraries were characterized at high throughput using infrared microscopy and validated using 1H NMR and size exclusion chromatography. The discrete libraries were rapidly screened for biocompatibility using standard SP2/0 myeloma, CHO and L929 fibroblasts, and J774 macrophage cell lines. At a concentration of 2.8 mg/mL, there was no appreciable cytotoxic effect on any of the four cell lines evaluated by any of the CPH:SA or CPTEG:CPH compositions. Furthermore, the activation of J774 macrophages was evaluated by incubating the cells with the polyanhydride libraries and quantifying the secreted cytokines (IL-6, IL-10, IL-12, and TNFalpha). The results indicated that copolymer compositions containing at least 50% CPH induced elevated amounts of TNFalpha. In summary, the results indicated that the methodologies described herein are amenable to the high throughput analysis of synthesized biomaterials and will facilitate the rapid and rational design of materials for use in biomedical applications.
The Simultaneous Effect of Polymer Chemistry and Device Geometry on the in Vitro Activation of Murine Dendritic Cells
Polyanhydrides are a promising class of biomaterials for use as vaccine adjuvants and as multi-component implants. Their properties can be tailored for such applications as controlled drug release, drug stability, and/or immune regulation (adjuvant effect). Understanding the induction of immunomodulatory mechanisms of this polymer system is important for the design and development of efficacious vaccines and tissue compatible multi-component implantable devices using this polymer system. This study describes the development of a rapid multiplexed method for the investigation of the adjuvanticity of polyanhydride nanospheres and films using murine dendritic cells (DCs). To assess the immune response, cell surface markers including MHC II, CD86, CD40, and CD209 and cytokines including IL-6, IL-12p40, and IL-10 were measured. The DCs incubated with nanospheres displayed enhanced expression of all the surface markers and the production of IL-12p40 compared to DCs incubated with polymer films in a chemistry-dependent manner. This suggests that polyanhydrides of various chemistries and device geometries can be tailored to achieve desired levels of immune cell activation for specific applications. The observed biocompatibility and activation of DCs by polyanhydride devices supports their inclusion in vaccine delivery devices as well as in multi-component medical implants.
Effect of Polymer Chemistry and Fabrication Method on Protein Release and Stability from Polyanhydride Microspheres
The release kinetics and stability of ovalbumin encapsulated into polyanhydride microspheres with varying chemistries were studied. Polymers based on the anhydride monomers sebacic acid (SA), 1,6-bis(p-carboxyphenoxy)hexane (CPH), and 1,8-bis (p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG) were utilized. Microspheres were fabricated using two non-aqueous methods: a solid/oil/oil double emulsion technique and cryogenic atomization. The studies showed that the two fabrication methods did not significantly affect the release kinetics of ovalbumin, even though the burst release of the protein was a function of the fabrication method and the polymer chemistry. Antigenic stability of ovalbumin released from microspheres prepared by cryogenic atomization was studied by western blot analysis. These studies indicate that the amphiphilic CPTEG:CPH polyanhydrides preserved protein structure and enhanced protein stability by preserving the immunological epitopes of released protein.
Encapsulation into Amphiphilic Polyanhydride Microparticles Stabilizes Yersinia Pestis Antigens
The design of biodegradable polymeric delivery systems based on polyanhydrides that would provide for improved structural integrity of Yersinia pestis antigens was the main goal of this study. Accordingly, the full-length Y. pestis fusion protein (F1-V) or a recombinant Y. pestis fusion protein (F1(B2T1)-V10) was encapsulated and released from microparticles based on 1,6-bis(p-carboxyphenoxy)hexane (CPH) and sebacic acid (SA) copolymers and 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG) and CPH copolymers fabricated by cryogenic atomization. An enzyme-linked immunosorbent assay was used to measure changes in the antigenicity of the released proteins. The recombinant F1(B2T1)-V10 was unstable upon release from the hydrophobic CPH:SA microparticles, but maintained its structure and antigenicity in the amphiphilic CPTEG:CPH system. The full-length F1-V was stably released by both CPH:SA and CPTEG:CPH microparticles. In order to determine the effect of the anhydride monomers on the protein structure, changes in the primary, secondary, and tertiary structure, as well as the antigenicity of both Y. pestis antigens, were measured after incubation in the presence of saturated solutions of SA, CPH, and CPTEG anhydride monomers. The results indicated that the amphiphilic environment provided by the CPTEG monomer was important to preserve the structure and antigenicity of both proteins. These studies offer an approach by which a thorough understanding of the mechanisms governing antigenic instability can be elucidated in order to optimize the in vivo performance of biodegradable delivery devices as protein carriers and/or vaccine adjuvants.
Design of a Protective Single-dose Intranasal Nanoparticle-based Vaccine Platform for Respiratory Infectious Diseases
Despite the successes provided by vaccination, many challenges still exist with respect to controlling new and re-emerging infectious diseases. Innovative vaccine platforms composed of adaptable adjuvants able to appropriately modulate immune responses, induce long-lived immunity in a single dose, and deliver immunogens in a safe and stable manner via multiple routes of administration are needed. This work describes the development of a novel biodegradable polyanhydride nanoparticle-based vaccine platform administered as a single intranasal dose that induced long-lived protective immunity against respiratory disease caused by Yesinia pestis, the causative agent of pneumonic plague. Relative to the responses induced by the recombinant protein F1-V alone and MPLA-adjuvanted F1-V, the nanoparticle-based vaccination regimen induced an immune response that was characterized by high titer and high avidity IgG1 anti-F1-V antibody that persisted for at least 23 weeks post-vaccination. After challenge, no Y. pestis were recovered from the lungs, livers, or spleens of mice vaccinated with the nanoparticle-based formulation and histopathological appearance of lung, liver, and splenic tissues from these mice post-vaccination was remarkably similar to uninfected control mice.
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.
Activation of Innate Immune Responses in a Pathogen-mimicking Manner by Amphiphilic Polyanhydride Nanoparticle Adjuvants
Techniques in materials design, immunophenotyping, and informatics can be valuable tools for using a molecular based approach to design vaccine adjuvants capable of inducing protective immunity that mimics a natural infection but without the toxic side effects. This work describes the molecular design of amphiphilic polyanhydride nanoparticles that activate antigen presenting cells in a pathogen-mimicking manner. Biodegradable polyanhydrides are well suited as vaccine delivery vehicles due to their adjuvant-like ability to: 1) enhance the immune response, 2) preserve protein structure, and 3) control protein release. The results of these studies indicate that amphiphilic nanoparticles possess pathogen-mimicking properties as evidenced by their ability to activate dendritic cells similarly to LPS. Specific molecular descriptors responsible for this behavior were identified using informatics analyses, including the number of backbone oxygen moieties, percent of hydroxyl end groups, polymer hydrophobicity, and number of alkyl ethers. Additional findings from this work suggest that the molecular characteristics mediating APC activation are not limited to hydrophobicity but vary in complexity (e.g., presentation of oxygen-rich molecular patterns to cells) and elicit unique patterns of cellular activation. The approach outlined herein demonstrates the ability to rationally design pathogen-mimicking nanoparticle adjuvants for use in next-generation vaccines against emerging and re-emerging diseases.
Mannose-functionalized "pathogen-like" Polyanhydride Nanoparticles Target C-type Lectin Receptors on Dendritic Cells
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.
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.
Tailoring the Immune Response by Targeting C-type Lectin Receptors on Alveolar Macrophages Using "pathogen-like" Amphiphilic Polyanhydride Nanoparticles
C-type lectin receptors (CLRs) offer unique advantages for tailoring immune responses. Engagement of CLRs regulates antigen presenting cell (APC) activation and promotes delivery of antigens to specific intracellular compartments inside APCs for efficient processing and presentation. In these studies, we have designed an approach for targeted antigen delivery by decorating the surface of polyanhydride nanoparticles with specific carbohydrates to provide pathogen-like properties. Two conserved carbohydrate structures often found on the surface of respiratory pathogens, galactose and di-mannose, were used to functionalize the surface of polyanhydride nanoparticles and target CLRs on alveolar macrophages (AMϕ), a principle respiratory tract APC. Co-culture of functionalized nanoparticles with AMϕ significantly increased cell surface expression of MHC I and II, CD86, CD40 and the CLR CIRE over non-functionalized nanoparticles. Di-mannose and galactose functionalization also enhanced the expression of the macrophage mannose receptor (MMR) and the macrophage galactose lectin, respectively. This enhanced AMϕ activation phenotype was found to be dependent upon nanoparticle internalization. Functionalization also promoted increased AMϕ production of the pro-inflammatory cytokines IL-1β, IL-6 and TNF-α. Additional studies demonstrated the requirement of the MMR for the enhanced cellular uptake and activation provided by the di-mannose functionalized nanoparticles. Together, these data indicate that targeted engagement of MMR and other CLRs is a viable strategy for enhancing the intrinsic adjuvant properties of nanovaccine adjuvants and promoting robust pulmonary immunity.
