Brenda R. Carrillo-Conde

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.

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.

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.

Single immunization with a suboptimal antigen dose encapsulated into polyanhydride microparticles promotes high titer and avid antibody responses

Microparticle adjuvants based on biodegradable polyanhydrides were used to provide controlled delivery of a model antigen, ovalbumin (Ova), to mice. Ova was encapsulated into two different polyanhydride microparticle formulations to evaluate the influence of polymer chemistry on the nature and magnitude of the humoral immune response after administration of a suboptimal dose. Subcutaneous administration of a single dose of polyanhydride microparticles containing 25 μg of Ova elicited humoral immune responses that were comparable in magnitude to that induced by soluble doses of 400-1600 μg Ova. In contrast, the avidity of the Ova-specific antibodies was greater in mice administered the microparticle formulations in comparison to the higher soluble doses. Finally, the microparticle delivery system primed an anamnestic immune response as evidenced by the significant increases in Ova-specific antibody when mice were administered an antigenic challenge of 25 μg of Ova at 12 weeks post-vaccination. Together, these results indicate that encapsulation of antigens into polyanhydride microparticles facilitates isotype switching, establishes immunologic memory, and the humoral response was characterized by a higher quality antibody 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.

Characterizing the antitumor response in mice treated with antigen-loaded polyanhydride microparticles.

Delivery of vaccine antigens with an appropriate adjuvant can trigger potential immune responses against cancer leading to reduced tumor growth and improved survival. In this study, various formulations of a bioerodible amphiphilic polyanhydride copolymer based on 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG) and 1,6-bis(p-carboxyphenoxy) hexane (CPH) with inherent adjuvant properties were evaluated for antigen-loading properties, immunogenicity and antitumor activity. Mice were vaccinated with 50:50 CPTEG:CPH microparticles encapsulating a model tumor antigen, ovalbumin (OVA), in combination with the Toll-like receptor-9 agonist, CpG oligonucleotide 1826 (CpG ODN). Mice treated with OVA-encapsulated CPTEG:CPH particles elicited the highest CD8(+) T cell responses on days 14 and 20 when compared to other treatment groups. This treatment group also displayed the most delayed tumor progression and the most extended survival times. Particles encapsulating OVA and CpG ODN generated the highest anti-OVA IgG(1) antibody responses in mice but these mice did not show significant tumor protection. These results suggest that antigen-loaded CPTEG:CPH microparticles can stimulate antigen-specific cellular responses and could therefore potentially be used to promote antitumor responses in cancer patients.

Protein adsorption on biodegradable polyanhydride microparticles.

The in vitro adsorption of plasma proteins on polyanhydride microparticles based on sebacic acid (SA), 1,6-bis(p-carboxyphenoxy)hexane (CPH), and 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG) was studied. Three model proteins from bovine serum (albumin (BSA), immunoglobulin G (IgG), and fibrinogen (Fg)) were used. The adsorption was studied using X-Ray Photoelectron Spectroscopy and gel electrophoresis. 2D electrophoresis was used to study the adsorption of plasma proteins from bovine serum. Differences in the amount of protein adsorbed were detected as a function of the following: (i) copolymer composition and (ii) specific protein studied. A direct correlation between polymer hydrophobicity and protein adsorbed was observed and higher quantities of Fg and IgG were absorbed. In vitro release studies were performed with ovalbumin-encapsulated microparticles that were incubated with Fg; these studies showed a reduction in the amount of ovalbumin released from the microparticles when Fg is adsorbed on the surface. An understanding of protein adsorption patterns on parenteral delivery devices is valuable in optimizing their in vivo performance.

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.

Chemistry-dependent adsorption of serum proteins onto polyanhydride microparticles differentially influences dendritic cell uptake and activation

The delivery of antigen-loaded microparticles to dendritic cells (DCs) may benefit from surface optimization of the microparticles themselves, thereby exploiting the material properties and introducing signals that mimic pathogens. Following in vivo administration microparticle surface characteristics are likely to be significantly modified as proteins are quickly adsorbed onto their surface. In this work we describe the chemistry-dependent serum protein adsorption patterns on polyanhydride particles and the implications for their molecular interactions with DCs. The enhanced expression of MHC II and CD40 on DCs after incubation with amphiphilic polyanhydride particles, and the increased secretion of IL-6, TNF-α, and IL-12p40 by hydrophobic polyanhydride particles exemplified the chemistry-dependent activation of DCs by sham-coated particles. The presence of proteins such as complement component 3 and IgG further enhanced the adjuvant properties of these vaccine carriers by inducing DC maturation (i.e. increased cell surface molecule expression and cytokine secretion) in a chemistry-dependent manner. Utilizing DCs derived from complement receptor 3-deficient mice (CR3(-/-) mice) identified a requirement for CR3 in the internalization of both sham- and serum-coated particles. These studies provide valuable insights into the rational design of targeted vaccine platforms aimed at inducing robust immune responses and improving vaccine efficacy.