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While first generation SARS-CoV-2 vaccines were effective in slowing the spread and severity of disease during the COVID-19 pandemic, there is a need for vaccines capable of inducing durable and broad immunity against emerging variants of concern. Nanoparticle-based vaccines (i.e., "nanovaccines") composed of polyanhydride nanoparticles and pentablock copolymer micelles have previously been shown to protect against respiratory pathogens, including influenza A virus, respiratory syncytial virus, and Yersinia pestis. In this work, a nanovaccine containing SARS-CoV-2 spike and nucleocapsid antigens was designed and optimized. The optimized nanovaccine induced long-lived systemic IgG antibody responses against wild-type SARS-CoV-2 virus. In addition, the nanovaccine induced antibody responses capable of neutralization and cross-reactivity to multiple SARS-CoV-2 variants (including B.1.1.529) and antigen-specific CD4 + and CD8 + T cell responses. Finally, the nanovaccine protected mice against a lethal SARS-CoV-2 challenge, setting the stage for advancing particle-based SARS-CoV-2 nanovaccines. STATEMENT OF SIGNIFICANCE: First-generation SARS-CoV-2 vaccines were effective in slowing the spread and limiting the severity of COVID-19. However, current vaccines target only one antigen of the virus (i.e., spike protein) and focus on the generation of neutralizing antibodies, which may be less effective against new, circulating strains. In this work, we demonstrated the ability of a novel nanovaccine platform, based on polyanhydride nanoparticles and pentablock copolymer micelles, to generate durable and broad immunity against SARS-CoV-2. These nanovaccines induced long-lasting (> 62 weeks) serum antibody responses which neutralized binding to ACE2 receptors and were cross-reactive to multiple SARS-CoV-2 variants. Additionally, mice immunized with the SARS-CoV-2 nanovaccine showed a significant increase of antigen-specific T cell responses in the draining lymph nodes and spleens. Together, these nanovaccine-induced immune responses contributed to the protection of mice against a lethal challenge of live SARS-CoV-2 virus, indicating that this nanovaccine platform is a promising next-generation SARS-CoV-2 vaccine., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Balaji Narasimhan reports financial support was provided by Coronavirus Aid, Relief, and Economic Security (CARES) Act. Balaji Narasimhan reports a relationship with ImmunoNanoMed Inc. that includes: equity or stocks. Balaji Narasimhan has patent #SARS-CoV-2 nanovaccine pending to Iowa State University Research Foundation. Michael Wannemuehler reports a relationship with ImmunoNanoMed Inc. that includes: equity or stocks. Michael Wannemuehler has patent #SARS-CoV-2 Nanovaccine pending to Iowa State University Research Foundation. Surya K Mallapragada reports a relationship with ImmunoNanoMed Inc. that includes: equity or stocks. Surya K Mallapragada has patent #SARS-CoV-2 nanovaccine pending to Iowa State University Research Foundation. Kathleen A Ross has patent #SARS-CoV-2 nanovaccine pending to Iowa State University Research Foundation. Sean M Kelly has patent #SARS-CoV-2 nanovaccine pending to Iowa State University Research Foundation., (Copyright © 2024. Published by Elsevier Ltd.)

Seasonal influenza A virus infections present substantial costs to both health and economic resources each year. Current seasonal influenza vaccines provide suboptimal protection and require annual reformulation to match circulating strains. In this work, a recombinant equine H3N8 hemagglutinin trimer (rH3 3 ) known to generate cross-protective antibodies and protect animals against sublethal, heterologous virus challenge was used as a candidate vaccine antigen. Nanoadjuvants such as polyanhydride nanoparticles and pentablock copolymer hydrogels have been shown to be effective adjuvants, inducing both rapid and long-lived protective immunity against influenza A virus. In this work, polyanhydride nanoparticles and pentablock copolymer hydrogels were used to provide sustained release of the novel rH3 3 while also facilitating the retention of its structure and antigenicity. These studies lay the groundwork for the development of a novel universal influenza A virus nanovaccine by combining the equine H3N8 rH3 3 and polymeric nanoadjuvant platforms.

Pancreatic cancer (PC) is one of the most lethal malignancies and represents an increasing and challenging threat, especially with an aging population. The identification of immunogenic PC-specific upregulated antigens and an enhanced understanding of the immunosuppressive tumor microenvironment have provided opportunities to enable the immune system to recognize cancer cells. Due to its differential upregulation and functional role in PC, the transmembrane mucin MUC4 is an attractive target for immunotherapy. In the current study we characterized the antigen stability, antigenicity and release kinetics of a MUC4β-nanovaccine to guide further optimization and, in vivo evaluation. Amphiphilic polyanhydride copolymers based on 20 mol % 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane and 80 mol % 1,6-bis(p-carboxyphenoxy)hexane were used to synthesize nanoparticles. Structurally stable MUC4β protein was released from the particles in a sustained manner and characterized by gel electrophoresis and fluorescence spectroscopy. Modest levels of protein degradation were observed upon release. The released protein was also analyzed by MUC4β-specific monoclonal antibodies using ELISA and showed no significant loss of epitope availability. Further, mice immunized with multiple formulations of combination vaccines containing MUC4β-loaded nanoparticles generated MUC4β-specific antibody responses. These results indicate that polyanhydride nanoparticles are viable MUC4β vaccine carriers, laying the foundation for evaluation of this platform for PC immunotherapy., (© 2020 Wiley Periodicals LLC.)

Respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract infection in both young children and in older adults. Despite the morbidity, mortality, and high economic burden caused by RSV worldwide, no licensed vaccine is currently available. We have developed a novel RSV vaccine composed of a prefusion-stabilized variant of the fusion (F) protein (DS-Cav1) and a CpG oligodeoxynucleotide adjuvant encapsulated within polyanhydride nanoparticles, termed RSVNanoVax. A prime-boost intranasal administration of RSVNanoVax in BALB/c mice significantly alleviated weight loss and pulmonary dysfunction in response to an RSV challenge, with protection maintained up to at least 6 mo postvaccination. In addition, vaccinated mice exhibited rapid viral clearance in the lungs as early as 2 d after RSV infection in both inbred and outbred populations. Vaccination induced tissue-resident memory CD4 and CD8 T cells in the lungs, as well as RSV F-directed neutralizing Abs. Based on the robust immune response elicited and the high level of durable protection observed, our prefusion RSV F nanovaccine is a promising new RSV vaccine candidate., (Copyright © 2021 by The American Association of Immunologists, Inc.)

In the course of this study, a series of novel, biodegradable polyanhydrides based on betulin disuccinate and dicarboxylic derivatives of poly(ethylene glycol) were prepared by two-step polycondensation. These copolymers can be used as carriers in drug delivery systems, in the form of microspheres. Betulin and its derivatives exhibit a broad spectrum of biological activity, including cytotoxic activity, which makes them promising substances for use as therapeutic agents. Microspheres that were prepared from betulin based polyanhydrides show promising properties for use in application in drug delivery systems, including inhalation systems. The obtained copolymers release the active substance-betulin disuccinate-as a result of hydrolysis under physiological conditions. The use of a poly(ethylene glycol) derivative as a co-monomer increases the solubility and bioavailability of the obtained compounds. Microspheres with diameters in the range of 0.5-25 µm were prepared by emulsion solvent evaporation method and their physicochemical and aerodynamic properties were analyzed. The morphological characteristics of the microspheres depended on the presence of poly(ethylene glycol) (PEG) segment within the structure of polyanhydrides. The porosity of the particles depended on the amount and molecular weight of the PEG used and also on the speed of homogenization. The most porous particles were obtained from polyanhydrides containing 20% wt. of PEG 600 by using a homogenization speed of 18,000 rpm.

Combinatorial techniques can accelerate the discovery and development of polymeric nanodelivery devices by pairing high-throughput synthesis with rapid materials characterization. Biodegradable polyanhydrides demonstrate tunable release, high cellular internalization, and dose sparing properties when used as nanodelivery devices. This nanoparticle platform shows promising potential for small molecule drug delivery, but the pace of understanding and rational design of these nanomedicines is limited by the low throughput of conventional characterization. This study reports the use of a high-throughput method to synthesize libraries of a newly synthesized, rapidly eroding polyanhydride copolymer based on 1,8-bis( p -carboxyphenoxy)-3,6-dioxaoctane (CPTEG) and sebacic acid (SA) monomers. The high-throughput method enabled efficient screening of copolymer microstructure, revealing weak block-type and alternating architectures. The high-throughput method was adapted to synthesize nanoparticle libraries encapsulating hydrophobic model drugs. Drug release from these nanoparticles was rapid, with a majority of the payload released within 3 days. Drug release was dramatically slowed at acidic pH, which could be useful for oral drug delivery. Rhodamine B (RhoB) release kinetics generally followed patterns of polymer erosion kinetics, while Coomassie brilliant blue (CBB) released the fastest from the slowest degrading polymer chemistry and vice versa. These differences in trends between copolymer chemistry and release kinetics were hypothesized to arise from differences in mixing thermodynamics. A high-throughput method was developed to synthesize polymer-drug film libraries and characterize mixing thermodynamics by melting point depression. Rhodamine B had a negative χ for all copolymers with <30 mol % CPTEG tested, indicating a tendency toward miscibility. By contrast, CBB χ increased, eventually becoming positive near 15:85 CPTEG:SA, with increasing CPTEG content. This indicates an increasing tendency toward phase separation in CPTEG-rich copolymers. These in vitro results screening polymer-drug interactions showed good agreement with in silico predictions from Hansen solubility parameter estimation and were able to explain the observed differences in model drug release trends.

The primary objective of this study was to enhance the antitumor efficacy of a model cancer vaccine through co-delivery of pentaerythritol lipid A (PELA), an immunological adjuvant, and a model tumor antigen, ovalbumin (OVA), separately loaded into polyanhydride particles (PA). In vitro experiments showed that encapsulation of PELA into PA (PA-PELA) significantly enhanced its stimulatory capacity on dendritic cells as evidenced by increased levels of the cell surface costimulatory molecules, CD80/CD86. In vivo experiments showed that PA-PELA, in combination with OVA-loaded PA (PA-OVA), significantly expanded the OVA-specific CD8 + T lymphocyte population compared to PA-OVA alone. Furthermore, OVA-specific serum antibody titers of mice vaccinated with PA-OVA/PA-PELA displayed a significantly stronger shift toward a Th1-biased immune response compared to PA-OVA alone, as evidenced by the substantially higher IgG 2C :IgG 1 ratios achieved by the former. Analysis of E.G7-OVA tumor growth curves showed that mice vaccinated with PA-OVA/PA-PELA had the slowest average tumor growth rate., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Objective: The aim of the study was to formulate, cyclodextrin (CD)-polyanhydride (PA) nanoparticles (CPNs) with rosuvastatin calcium (RCa) in order to enhance the poor oral bioavailability. Methods: CPNs containing RCa/CD complexes were prepared by a modified solvent displacement method and morphological analyses, particle size (PS), polydispersity index (PDI), zeta potential (ZP), encapsulation efficiency (EE), DSC, FT-IR, XRD, 1 H-NMR analyses were performed. In vitro release properties, release kinetics, cytotoxicity, in vitro permeability and pharmacokinetic studies were also studied. The stability of the formulations were evaluated during the storage period of 3 months. Results: The physicochemical studies showed that the RCa/CD complexes were well incorporated into CPNs resulted in nanosized particles (215.22 and 189.13 nm) with homogenous size distribution (PDI: 0.203 and 0.182) with relatively high incorporation capacity (76.11 and 68.18%) for the CPN1 and CPN2 formulations respectively. Sustained release of RCa from CPNs were achieved. The cytotoxicity values showed that the safety of the formulations. According to permeability studies, pure RCa had lowest permeability data (3.08 × 10 -7  cm⋅s -1 P app value) while CPNs gained higher permeability data (1.36 × 10 -5 and 1.12 × 10 -5  cm⋅s -1 P app values) for the CPN1 and CPN2 formulations respectively. CPN2 formulation was selected for pharmacokinetic studies and analyses results demonstrated that approximately 8-fold relative oral bioavailability enhancement compared to the pure RCa was achieved. Conclusion: Considering the analyses results of the study, CPNs can be regarded as suitable, safe, functional oral delivery systems for RCa with enhanced oral bioavailability.

Drug delivery vehicles can improve the functional efficacy of existing antimicrobial therapies by improving biodistribution and targeting. A critical property of such nanomedicine formulations is their ability to control the release kinetics of their payloads. The combination of (and interactions among) polymer, drug, and nanoparticle properties gives rise to nonlinear behavioral relationships and large data space. These factors complicate both first-principles modeling and screening of nanomedicine formulations. Predictive analytics may offer a more efficient approach toward the rational design of nanomedicines by identifying key descriptors and correlating them to nanoparticle release behavior. In this work, antibiotic release kinetics data were generated from polyanhydride nanoparticle formulations with varying copolymer compositions, encapsulated drug type, and drug loading. Four antibiotics, doxycycline, rifampicin, chloramphenicol, and pyrazinamide, were used. Linear manifold learning methods were used to relate drug release properties with polymer, drug, and nanoparticle properties, and key descriptors were identified that are highly correlated with release properties. However, these linear methods could not predict release behavior. Nonlinear multivariate modeling based on graph theory was then used to deconvolute the governing relationships between these properties, and predictive models were generated to rapidly screen lead nanomedicine formulations with desirable release properties with minimal nanoparticle characterization. Release kinetics predictions of two drugs containing atoms not included in the model showed good agreement with experimental results, validating the model and indicating its potential to virtually explore new polymer and drug pairs not included in the training data set. The models were shown to be robust after the inclusion of these new formulations, in that the new inclusions did not significantly change model regression. This approach provides the first step toward the development of a framework that can be used to rationally design nanomedicine formulations by selecting the appropriate carrier for a drug payload to program desirable release kinetics.

Immunosenescence poses a formidable challenge in designing effective influenza vaccines for aging populations. While approved vaccines against influenza viruses exist, their efficacy in older adults is significantly decreased due to the diminished capabilities of innate and adaptive immune responses. In this work, the ability of a combination nanovaccine containing both recombinant hemagglutinin and nucleoprotein to provide protection against seasonal influenza virus infection was examined in young and aged mice. Vaccine formulations combining two nanoadjuvants, polyanhydride nanoparticles and pentablock copolymer micelles, were shown to enhance protection against challenge compared to each adjuvant alone in young mice. Nanoparticles were shown to enhance in vitro activation of dendritic cells isolated from aged mice, while both nanoadjuvants did not induce proinflammatory cytokine secretion which may be detrimental in aged individuals. In addition, the combination nanovaccine platform was shown to induce demonstrable antibody titers in both young and aged mice that correlated with the maintenance of body weight post-challenge. Collectively, these data demonstrate that the combination nanovaccine platform is a promising technology for influenza vaccines for older adults.

Purpose: Salmonellosis is a severe economic threat in poultry and a public health concern. Currently available vaccines are ineffective, and thus, developing effective oral Salmonella vaccine is warranted. Especially, a potent oral vaccine such as the mucoadhesive polyanhydride nanoparticle (PNP) protects the vaccine cargo and delivers to intestinal immune sites to elicit robust mucosal immunity and mitigate Salmonella colonization and shedding., Materials and Methods: We designed a Salmonella subunit vaccine using PNP containing immunogenic Salmonella outer membrane proteins (OMPs) and flagellar (F) protein-entrapped and surface F-protein-coated PNPs (OMPs-F-PNPs) using a solvent displacement method. Using high-throughput techniques, we characterized the OMPs-F-PNPs physicochemical properties and analyzed its efficacy in layer birds vaccinated orally., Results: The candidate vaccine was resistant in acidic microenvironment and had ideal physicochemical properties for oral delivery in terms of particle size, charge, morphology, biocompatibility, and pH stability. In vitro, in vivo, and ex vivo studies showed that F-protein surface-anchored nanoparticles were better targeted to chicken immune cells in peripheral blood and splenocytes and intestinal Peyer's patch sites. In layer chickens inoculated orally with OMPs-F-PNPs, substantially higher OMPs-specific IgG response and secretion of Th1 cytokine IFN-γ in the serum, enhanced CD8 + /CD4 + cell ratio in spleen, and increased OMPs-specific lymphocyte proliferation were observed. OMPs-F-PNPs vaccination also upregulated the expression of toll-like receptor (TLR)-2 and -4, TGF-β, and IL-4 cytokines' genes in chicken cecal tonsils (lymphoid tissues). Importantly, OMPs-F-PNPs vaccine cleared Salmonella cecal colonization in 33% of vaccinated birds., Conclusion: This pilot in vivo study demonstrated the targeted delivery of OMPs-F-PNPs to ileum mucosal immune sites of chickens and induced specific immune response to mitigate Salmonella colonization in intestines., Competing Interests: Disclosure The authors report no conflicts of interest in this work.

An urgent need to deliver therapeutics across the blood-brain barrier (BBB) underlies a paucity of effective therapies currently available for treatment of degenerative, infectious, traumatic, chemical, and metabolic disorders of the nervous system. With an eye toward achieving this goal, an in vitro BBB model was employed to simulate biodegradable polyanhydride nanoparticle-based drug delivery to the brain. Using a combination of confocal microscopy, flow cytometry, and high performance liquid chromatography, we examined the potential of polyanhydride nanoparticles containing the anti-oxidant, mito-apocynin, to be internalized and then transferred from monocytes to human brain microvascular endothelial cells. The efficacy of this nanoparticle-based delivery platform was demonstrated by neuronal protection against oxidative stress. Taken together, this polyanhydride nanoparticle-based delivery system holds promise for enhancing neuroprotection by facilitating drug transport across the BBB. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 2881-2890, 2018., (© 2018 Wiley Periodicals, Inc.)

This review focusses on recent developments of polyanhydrides, a class of degradable synthetic biopolymers. Polyanhydrides have been used as carriers for controlled delivery of drugs. A polyanhydride copolymer of carboxyphenoxy propane and sebacic acid has been used in Gliadel brain tumor implants for the controlled delivery of carmustine or bis-chloroethylnitrosourea. They are easy and inexpensive to synthesize (especially scale up). However, polyanhydrides possess a short shelf-life. Hydrolytic cleavage and anhydride interchanges lower their molecular weights during storage. One of the highlights in recent developments of polyanhydride chemistry is the discovery of alternating copolymers having extended shelf-life. Other highlights include their applications in biomedical electronics, vaccine delivery, and nano/micro particulate delivery systems. This review examines approaches for polyanhydride synthesis followed by their recent developments in biomedical applications., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The aim of this work was to investigate the potential of pegylated poly(anhydride) nanoparticles to enhance the oral bioavailability of docetaxel (DTX). Nanoparticles were prepared after the incubation between the copolymer of methyl vinyl ether and maleic anhydride (Gantrez® AN), poly(ethylene glycol) (PEG2000 or PEG6000) and docetaxel (DTX). The oral administration of a single dose of pegylated nanoparticles to mice provided sustained and prolonged therapeutic plasma levels of docetaxel for up 48-72 h. In addition, the relative oral bioavailability of docetaxel was around 32%. The organ distribution studies revealed that docetaxel underwent a similar distribution when orally administered encapsulated in nanoparticles as when intravenously as Taxotere®. This observation, with the fact that the clearance of docetaxel when loaded into the oral pegylated nanoparticles was found to be similar to that of intravenous formulation, suggests that docetaxel would be released at the epithelium surface and then absorbed to the circulation., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Multicompartmental polymer carriers, referred to as Polyanhydride-Releasing Oral MicroParticle Technology (PROMPT), were formed by a pH-triggered antisolvent precipitation technique. Polyanhydride nanoparticles were encapsulated into anionic pH-responsive microparticle gels, allowing for nanoparticle encapsulation in acidic conditions and subsequent release in neutral pH conditions. The effects of varying the nanoparticle composition and feed ratio on the encapsulation efficiency were evaluated. Nanoparticle encapsulation was confirmed by confocal microscopy and infrared spectroscopy. pH-triggered protein delivery from PROMPT was explored using ovalbumin (ova) as a model drug. PROMPT microgels released ova in a pH-controlled manner. Increasing the feed ratio of nanoparticles into the microgels increased the total amount of ova delivered, as well as decreased the observed burst release. The cytocompatibility of the polymer materials were assessed using cells representative of the GI tract. Overall, these results suggest that pH-dependent microencapsulation is a viable platform to achieve targeted intestinal delivery of polyanhydride nanoparticles and their payload(s).

This work describes the feasibility of poly(anhydride) nanoparticles as carriers for the oral administration of glibenclamide (GB) as well as the in vivo evaluation of their hypolipidemic effect in a C. elegans model. For this purpose, and in order to increase the GB payload, the drug was encapsulated in nanoparticles in presence of cyclodextrins (either βCD or HPβCD). The optimized nanoparticles displayed a size of about 220 nm and a negative zeta potential (-40 mV), with a drug loading up to 52 μg/mg. Small-angle neutron scattering studies suggested an internal fractal-like structure, based on the repetition of spherical blocks of polymeric units (about 5 nm) grouped to form the nanoparticle. X-ray diffraction study confirmed the absence of crystalline GB molecules due to its dispersion into the nanoparticles, either entrapped in the polymer chains and/or included into cyclodextrin cavities. GB-loaded nanoparticles induced a significant reduction in the fat content of C. elegans. This hypolipidemic effect was slightly higher for the nanoparticles prepared with coencapsulated HPβCD (8.2%) than for those prepared with βCD (7.9%) or in the absence of cyclodextrins (7.0%). In summary, the coencapsulation of cyclodextrins into poly(anhydride) nanoparticles could be an interesting strategy to develop new oral formulations of glibenclamide., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Traditionally, vaccination strategies require an initial priming vaccination followed by an antigen boost to generate adequate immunity. Here we describe vaccination against a self-peptide for reproductive sterilization utilizing a three-stage vaccine platform consisting of gonadotropin releasing hormone multiple antigenic peptide (GnRH-MAP) as a soluble injection coupled with subcutaneous administration of polyanhydride-immobilized GnRH-MAP and a cyto-exclusive implant containing GnRH-MAP dendrimer-loaded polyanhydride. This strategy generated and maintained cell-mediated and humoral immunity for up to 41 weeks after a single vaccination in mice with enhanced antibody avidity over time. All intact implants had a grossly visible tissue interface with neovascularization and lymphocytic aggregates. Despite detectable immunity, sterility was not achieved and the immune response did not lead to azoospermia in male mice nor prevent estrus and ovulation in female mice. However, the vaccine delivery device is tunable and the immunogen, adjuvants and release rates can all be modified to enhance immunity. This technology has broad implications for the development of long-term vaccination schemes., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Rational design of adjuvants and delivery systems will promote development of next-generation vaccines to control emerging and re-emerging diseases. To accomplish this, understanding the immune-enhancing properties of new adjuvants relative to those induced by natural infections can help with the development of pathogen-mimicking materials that will effectively initiate innate immune signaling cascades. In this work, the surfaces of polyanhydride nanoparticles composed of sebacic acid (SA) and 1,6-bis(p-carboxyphenoxy) hexane were decorated with an ethylene diamine spacer partially modified with either a glycolic acid linker or an α-1,2-linked di-mannopyranoside (di-mannose) to confer "pathogen-like" properties and enhance adjuvanticity. Co-incubation of linker-modified nanoparticles with dendritic cells (DCs) elicited significant increases in surface expression of MHC I, MHC II, CD86, and CD40, and enhanced secretion of IL-6, IL-12p40, and TNF-α. An 800% increase in uptake of ethylene-diamine-spaced, linker and di-mannose functionalized polyanhydride nanoparticles was also observed. Together, our data showed that linker-functionalized polyanhydride nanoparticles demonstrate similar patterns of uptake, intracellular trafficking, particle persistence, and innate activation as did DCs exposed to Yersinia pestis or Escherichia coli. These results set the stage for rational selection of adjuvant chemistries to induce pathogen-mimicking immune responses. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2762-2771, 2017., (© 2017 Wiley Periodicals, Inc.)

Enhanced bioactive anti-oxidant formulations are critical for treatment of inflammatory diseases, such as atherosclerosis. A hallmark of early atherosclerosis is the uptake of oxidized low density lipoprotein (oxLDL) by macrophages, which results in foam cell and plaque formation in the arterial wall. The hypolipidemic, anti-inflammatory, and antioxidative properties of polyphenol compounds make them attractive targets for treatment of atherosclerosis. However, high concentrations of antioxidants can reverse their anti-atheroprotective properties and cause oxidative stress within the artery. Here, we designed a new class of nanoparticles with anti-oxidant polymer cores and shells comprised of scavenger receptor targeting amphiphilic macromolecules (AMs). Specifically, we designed ferulic acid-based poly(anhydride-ester) nanoparticles to counteract the uptake of high levels of oxLDL and regulate reactive oxygen species generation (ROS) in human monocyte derived macrophages (HMDMs). Compared to all compositions examined, nanoparticles with core ferulic acid-based polymers linked by diglycolic acid (PFAG) showed the greatest inhibition of oxLDL uptake. At high oxLDL concentrations, the ferulic acid diacids and polymer nanoparticles displayed similar oxLDL uptake. Treatment with the PFAG nanoparticles downregulated the expression of macrophage scavenger receptors, CD-36, MSR-1, and LOX-1 by about 20-50%, one of the causal factors for the decrease in oxLDL uptake. The PFAG nanoparticle lowered ROS production by HMDMs, which is important for maintaining macrophage growth and prevention of apoptosis. Based on these results, we propose that ferulic acid-based poly(anhydride ester) nanoparticles may offer an integrative strategy for the localized passivation of the early stages of the atheroinflammatory cascade in cardiovascular disease., Statement of Significance: Future development of anti-oxidant formulations for atherosclerosis applications is essential to deliver an efficacious dose while limiting localized concentrations of pro-oxidants. In this study, we illustrate the potential of degradable ferulic acid-based polymer nanoparticles to control macrophage foam cell formation by significantly reducing oxLDL uptake through downregulation of scavenger receptors, CD-36, MSR-1, and LOX-1. Another critical finding is the ability of the degradable ferulate-based polymer nanoparticles to lower macrophage reactive oxygen species (ROS) levels, a precursor to apoptosis and plaque escalation. The degradable ferulic acid-based polymer nanoparticles hold significant promise as a means to alter the treatment and progression of atherosclerosis., (Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Poly(anhydride) are unstable and prone to hydrolytic degradation and depolymerisation via anhydride interchange. They are stored at -20°C, packed under inert atmosphere until use. We synthesized a new poly(anhydride) from ricinoleic (RA) and sebacic (SA) acid with alternating ester-anhydride structure that is stable at 25°C for over 18months. The copolymer is also stable in chloroform solution and under γ-irradiation. The polymer hydrolyses through anhydride cleavage lasting ~7days to form oligoesters, which are stable for >30days. The release of gentamycin from the synthesized alternate polymer matrix is sustained compared to the random copolymer., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Molecularly imprinted polymers (MIPs) with selective affinity for protein biomarkers could find extensive utility as environmentally robust, cost-efficient biomaterials for diagnostic and therapeutic applications. In order to develop recognitive, synthetic biomaterials for prohibitively expensive protein biomarkers, we have developed a molecular imprinting technique that utilizes structurally similar, analogue proteins. Hydrogel microparticles synthesized by molecular imprinting with trypsin, lysozyme, and cytochrome c possessed an increased affinity for alternate high isoelectric point biomarkers both in isolation and plasma-mimicking adsorption conditions. Imprinted and non-imprinted P(MAA-co-AAm-co-DEAEMA) microgels containing PMAO-PEGMA functionalized polycaprolactone nanoparticles were net-anionic, polydisperse, and irregularly shaped. MIPs and control non-imprinted polymers (NIPs) exhibited regions of Freundlich and BET isotherm adsorption behavior in a range of non-competitive protein solutions, where MIPs exhibited enhanced adsorption capacity in the Freundlich isotherm regions. In a competitive condition, imprinting with analogue templates (trypsin, lysozyme) increased the adsorption capacity of microgels for cytochrome c by 162% and 219%, respectively, as compared to a 122% increase provided by traditional bulk imprinting with cytochrome c. Our results suggest that molecular imprinting with analogue protein templates is a viable synthetic strategy for enhancing hydrogel-biomarker affinity and promoting specific protein adsorption behavior in biological fluids. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1565-1574, 2017., (© 2017 Wiley Periodicals, Inc.)

A progressive loss of neuronal structure and function is a signature of many neurodegenerative conditions including chronic traumatic encephalopathy, Parkinson's, Huntington's and Alzheimer's diseases. Mitochondrial dysfunction and oxidative and nitrative stress have been implicated as key pathological mechanisms underlying the neurodegenerative processes. However, current therapeutic approaches targeting oxidative damage are ineffective in preventing the progression of neurodegeneration. Mitochondria-targeted antioxidants were recently shown to alleviate oxidative damage. In this work, we investigated the delivery of biodegradable polyanhydride nanoparticles containing the mitochondria-targeted antioxidant apocynin to neuronal cells and the ability of the nano-formulation to protect cells against oxidative stress. The nano-formulated mitochondria-targeted apocynin provided excellent protection against oxidative stress-induced mitochondrial dysfunction and neuronal damage in a dopaminergic neuronal cell line, mouse primary cortical neurons, and a human mesencephalic cell line. Collectively, our results demonstrate that nano-formulated mitochondria-targeted apocynin may offer improved efficacy of mitochondria-targeted antioxidants to treat neurodegenerative disease., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The goal of this research was to study the effect of polyanhydride chemistry on the immune response induced by a prophylactic cancer vaccine based on biodegradable polyanhydride particles. To achieve this goal, different compositions of polyanhydride copolymers based on 1,8-bis-(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG), 1,6-bis-(p-carboxyphenoxy)-hexane (CPH), and sebacic anhydride (SA) were synthesized by melt polycondensation, and polyanhydride copolymer particles encapsulating a model antigen, ovalbumin (OVA), were then synthesized using a double emulsion solvent evaporation technique. The ability of three different compositions of polyanhydride copolymers (50:50 CPTEG:CPH, 20:80 CPTEG:CPH, and 20:80 CPH:SA) encapsulating OVA to elicit immune responses was investigated. In addition, the impact of unmethylated oligodeoxynucleotides containing deoxycytidyl-deoxyguanosine dinucleotides (CpG ODN), an immunological adjuvant, on the immune response was also studied. The immune response to cancer vaccines was measured after treatment of C57BL/6J mice with two subcutaneous injections, seven days apart, of 50μg OVA encapsulated in particles composed of different polyanhydride copolymers with or without 25μg CpG ODN. In vivo studies showed that 20:80 CPTEG:CPH particles encapsulating OVA significantly stimulated the highest level of CD8 + T lymphocytes, generated the highest serum titers of OVA-specific IgG antibodies, and provided longer protection against tumor challenge with an OVA-expressing thymoma cell line in comparison to formulations made from other polyanhydride copolymers. The results also revealed that vaccination with CpG ODN along with polyanhydride particles encapsulating OVA did not enhance the immunogenicity of OVA. These results accentuate the crucial role of the copolymer composition of polyanhydrides in stimulating the immune response and provide important insights on rationally designing efficacious cancer vaccines., Statement of Significance: Compared to soluble cancer vaccine formulations, tumor antigens encapsulated in biodegradable polymeric particles have been shown to sustain antigen release and provide long-term protection against tumor challenge by improving the immune response towards the antigen. Treatment of mice with cancer vaccines based on different polyanhydride copolymers encapsulating OVA resulted in stimulation of tumor-specific immune responses with different magnitudes. This clearly indicates that polyanhydride chemistry plays a substantial role in stimulating the immune response. Vaccination with 20:80 CPTEG:CPH/OVA, the most hydrophobic formulation, stimulated the strongest cellular and humoral immune responses and provided the longest survival outcome without adding any other adjuvant. The most important finding in this study is that the copolymer composition of polyanhydride particle-based vaccines can have a direct effect on the magnitude of the antitumor immune response and should be selected carefully in order to achieve optimal cancer vaccine efficacy., (Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

We have recently demonstrated the effectiveness of an influenza A virus (IAV) subunit vaccine based on biodegradable polyanhydride nanoparticles delivery in mice. In the present study, we evaluated the efficacy of ∼200nm polyanhydride nanoparticles encapsulating inactivated swine influenza A virus (SwIAV) as a vaccine to induce protective immunity against a heterologous IAV challenge in pigs. Nursery pigs were vaccinated intranasally twice with inactivated SwIAV H1N2 (KAg) or polyanhydride nanoparticle-encapsulated KAg (KAg nanovaccine), and efficacy was evaluated against a heterologous zoonotic virulent SwIAV H1N1 challenge. Pigs were monitored for fever daily. Local and systemic antibody responses, antigen-specific proliferation of peripheral blood mononuclear cells, gross and microscopic lung lesions, and virus load in the respiratory tract were compared among the groups of animals. Our pre-challenge results indicated that KAg nanovaccine induced virus-specific lymphocyte proliferation and increased the frequency of CD4 + CD8αα + T helper and CD8 + cytotoxic T cells in peripheral blood mononuclear cells. KAg nanovaccine-immunized pigs were protected from fever following SwIAV challenge. In addition, pigs immunized with the KAg nanovaccine presented with lower viral antigens in lung sections and had 6 to 8-fold reduction in nasal shedding of SwIAV four days post-challenge compared to control animals. Immunologically, increased IFN-γ secreting T lymphocyte populations against both the vaccine and challenge viruses were detected in KAg nanovaccine-immunized pigs compared to the animals immunized with KAg alone. However, in the KAg nanovaccine-immunized pigs, hemagglutination inhibition, IgG and IgA antibody responses, and virus neutralization titers were comparable to that in the animals immunized with KAg alone. Overall, our data indicated that intranasal delivery of polyanhydride-based SwIAV nanovaccine augmented antigen-specific cellular immune response in pigs, with promise to induce cross-protective immunity., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Quantitative and qualitative toxicological analyses of crosslinked, surface-eroding polyanhydrides (PAHs) made from thiol-ene "click" polymerizations are reported. The cytotoxicity of these PAHs was investigated against three skin-based cell types; melanoma (A-375), human dermal fibroblast adult (HDFa), and 3T3-J2 (mouse fibroblast) cells, thus providing insight into the potential for these PAHs to be used in dermal drug delivery applications. Apoptosis was evaluated quantitatively and qualitatively using MTT assay and fluorescence microscopic imaging as indication of cytotoxicity. Upon exposure of A-375 and HDFa cells to high concentrations (4000 mg/L) of crosslinked PAH, the respective morphologies remained relatively unchanged compared with nonexposed cells. The 3T3-J2 cell type was more sensitive towards the PAH, exhibiting minimal deformation of cell morphology at 4000 mg/L. The MTT assay and fluorescence imaging revealed that this PAH and its degradation products are highly cytocompatible at high concentrations and cytotoxicity observed is dosage/time dependent. Further, the PAH did not induce inhibition of tested cells' proliferation at high polymer concentration up to 2000 mg/L. The IC50 (concentration of the crosslinked PAH required to inhibit 50% cell viability) for HDFa and A-375 cells was determined to be 4300 ± 70 and 8500 ± 50 mg/L, respectively. The high cytocompatibility of this type of crosslinked PAH, in addition to their degradation products, towards these skin cells (standard and cancer cell types) suggests that the polymer may be viable for dermal-based drug delivery to normal and cancerous diseased tissues. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1936-1945, 2016., (© 2016 Wiley Periodicals, Inc.)

Pinosylvin is a natural stilbenoid known to exhibit antibacterial bioactivity against foodborne bacteria. In this work, pinosylvin is chemically incorporated into a poly(anhydride-ester) (PAE) backbone via melt-condensation polymerization, and characterized with respect to its physicochemical and thermal properties. In vitro release studies demonstrate that pinosylvin-based PAEs hydrolytically degrade over 40 d to release pinosylvin. Pseudo-first order kinetic experiments on model compounds, butyric anhydride and 3-butylstilbene ester, indicate that the anhydride linkages hydrolyze first, followed by the ester bonds to ultimately release pinosylvin. An antibacterial assay shows that the released pinosylvin exhibit bioactivity, while in vitro cytocompatibility studies demonstrate that the polymer is noncytotoxic toward fibroblasts. These preliminary findings suggest that the pinosylvin-based PAEs can serve as food preservatives in food packaging materials by safely providing antibacterial bioactivity over extended time periods., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Polyanhydride nanoparticles have emerged as a versatile delivery platform, due to their ability to encapsulate diverse drugs, immunogens, antibodies, and proteins. However, mechanistic studies on the effects of particle chemistry interactions with immune cells have yet to be described. Understanding the mechanism by which these particles are internalized by immune cells will enable rational selection of delivery vehicles for specific applications. In the present study, the internalization, mechanism(s) of uptake by monocytes, and intracellular fate of polyanhydride nanoparticles were evaluated using copolymers based on 1,6-bis(p-carboxyphenoxy)hexane (CPH), sebacic acid (SA), and 1,8-bis(p-carboxyphenoxy)3,6-dioxaoctane (CPTEG). The results showed that 20:80 CPH:SA and 20:80 CPTEG:CPH nanoparticles were internalized to a greater extent by monocytes as compared to the 50:50 CPH:SA and 50:50 CPTEH:CPH nanoparticles. Further, cytochalasin-D treatment of cells inhibited uptake of all the particles, regardless of chemistry, indicating that actinmediated uptake is the primary mechanism of cellular entry for these particles. The insights gained from these studies were used to identify lead nanoparticle formulations to enhance treatment of intracellular bacterial infections. The use of doxycycline-loaded nanoparticles exhibited enhanced therapeutic efficacy compared to soluble drug in treating monocyte monolayers infected with the virulent intracellular pathogen Brucella abortus. Altogether, these studies demonstrate how rational design and selection of nanoscale delivery platforms can be used for a wide spectrum of biomedical applications.

Camptothecin (CPT), a molecule that shows powerful anticancer activity, is still not used in clinic due to its high hydrophobicity and poor active form's stability. In order to solve these drawbacks, the combination between poly(anhydride) nanoparticles and cyclodextrins was evaluated. CPT-loaded nanoparticles, prepared in the presence of 2-hydroxypropyl-β-cyclodextrin, (HPCD-NP) displayed a mean size close to 170nm and a payload of 50μg per mg (25 times higher than the one of the control nanoparticles). CPT was not released from nanoparticles under gastric conditions. However, under intestinal conditions, about 50% of the drug content was released as a burst, whereas the remained drug was released following a zero-order kinetic. Pharmacokinetic studies revealed that the CPT plasma levels, from orally administered nanoparticles, were high and sustained up to 48h. The CPT oral bioavailability was 7-fold higher than the value obtained with the control, whereas its clearance was significantly lower than for the aqueous suspension. These observations may be directly related to a prolonged residence time of nanoparticles in close contact with the intestinal epithelium, the presence of the cyclodextrin that decreases the CPT transformation into its inactive form and the generation of an acidic microenvironment during the degradation of the poly(anhydride) that would prevent the transformation of the active lactone into the inactive carboxylate conformation., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Biodegradable polymeric nanoparticle-based subunit vaccines have shown promising characteristics by enhancing antigen presentation and inducing protective immune responses when compared with soluble protein. Specifically, polyanhydride nanoparticle-based vaccines (i.e., nanovaccines) have been shown to successfully encapsulate and release antigens, activate B and T cells, and induce both antibody- and cell-mediated immunity towards a variety of immunogens. One of the characteristics of strong thymus-dependent antibody responses is the formation of germinal centers (GC) and the generation of GC B cells, which is part of the T helper cell driven cellular response. In order to further understand the role of nanovaccines in the induction of antigen-specific immune responses, their ability to induce germinal center B cell formation and isotype switching and the effects thereof on serum antibody responses were investigated in these studies. Polyanhydride nanovaccines based on 1,6-bis(p-carboxyphenoxy)hexane and 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane were used to subcutaneously administer a viral antigen. GC B cell formation and serum antibody responses induced by the nanovaccines were compared to that induced by alum-based vaccine formulations. It was demonstrated that a single dose of polyanhydride nanovaccines resulted in the formation of robust GCs and serum antibody in comparison to that induced by the alum-based formulation. This was attributed to the sustained release of antigen provided by the nanovaccines. When administered in a multiple dose regimen, the highest post-immunization titer and GC B cell number was enhanced, and the immune response induced by the nanovaccines was further sustained. These studies provide foundational information on the mechanism of action of polyanhydride nanovaccines.

Salicylic acid (SA) based biodegradable polyanhydrides (PAHs) are of great interest for drug delivery in a variety of diseases and disorders owing to the multi-utility of SA. There is a need for the design of SA-based PAHs for tunable drug release, optimized for the treatment of different diseases. In this study, we devised a simple strategy for tuning the release properties and erosion kinetics of a family of PAHs. PAHs incorporating SA were derived from related aliphatic diacids, varying only in the chain length, and prepared by simple melt condensation polymerization. Upon hydrolysis induced erosion, the polymer degrades into cytocompatible products, including the incorporated bioactive SA and diacid. The degradation follows first order kinetics with the rate constant varying by nearly 25 times between the PAH obtained with adipic acid and that with dodecanedioic acid. The release profiles have been tailored from 100% to 50% SA release in 7 days across the different PAHs. The release rate constants of these semi-crystalline, surface eroding PAHs decreased almost linearly with an increase in the diacid chain length, and varied by nearly 40 times between adipic acid and dodecanedioic acid PAH. The degradation products with SA concentration in the range of 30-350 ppm were used to assess cytocompatibility and showed no cytotoxicity to HeLa cells. This particular strategy is expected to (a) enable synthesis of application specific PAHs with tunable erosion and release profiles; (b) encompass a large number of drugs that may be incorporated into the PAH matrix. Such a strategy can potentially be extended to the controlled release of other drugs that may be incorporated into the PAH backbone and has important implications for the rational design of drug eluting bioactive polymers.

We develop a theoretical model to explain the long induction interval of water intake that precedes the onset of erosion due to degradation caused by hydrolysis in the recently synthesized and studied cross-linked polyanhydrides. Various kinetic mechanisms are incorporated in the model in an attempt to explain the experimental data for the mass loss profile. Our key finding is that the observed long induction interval is attributable to the nonlinear dependence of the degradation rate constants on the local water concentration, which essentially amounts to the breakdown of the standard rate-equation approach, potential causes for which are then discussed. Our theoretical results offer physical insights into which microscopic studies will be required to supplement the presently available macroscopic mass-loss data in order to fully understand the origin of the observed behavior.

Surface eroding and semicrystalline polyanhydrides, with tunable erosion times and drug delivery pharmacokinetics largely dictated by erosion, are produced easily with thiol-ene "click" polymerization. This strategy yields both linear and cross-linked network polyanhydrides that are readily and fully cured within minutes using photoinitiation, can contain up to 60% crystallinity, and have tensile moduli up to 25 MPa for the compositions studied. Since they readily undergo hydrolysis and exhibit the oft-preferred surface erosion mechanism, they may be particularly useful in drug delivery applications. The polyanhydrides were degraded under pseudophysiological conditions and cylindrical samples (10 mm diameter × 5 mm height) were completely degraded within ∼10 days, with the mass-time profile being linear for much of this time after a ∼24 h induction period. Drug release studies, using lidocaine as a model, showed pharmacokinetics that displayed a muted burst release in the early stages of erosion, but then a delayed release profile that is closely correlated to the erosion kinetics. Furthermore, cytotoxicity studies of the linear and cross-linked semicrystalline polyanhydrides, and degradation products, against fibroblast cells indicate that the materials have good cytocompatibility. Overall, cells treated with up to 2500 mg/L of the semicrystalline polyanhydrides and degradation products show >90% human dermal fibroblast adult (HDFa) cell viability indicative of good cytocompatibility.

Carbohydrate functionalization of nanoparticles allows for targeting of C-type lectin receptors. This family of pattern recognition receptors expressed on innate immune cells, such as macrophages and dendritic cells, can be used to modulate immune responses. In this work, the in vivo safety profile of carbohydrate-functionalized polyanhydride nanoparticles was analyzed following parenteral and intranasal administration in mice. Polyanhydride nanoparticles based on 1,6-bis-(p-carboxyphenoxy)hexane and 1,8-bis-(p-carboxyphenoxy)-3,6-dioxaoctane were used. Nanoparticle functionalization with di-mannose (specifically carboxymethyl-α-D-mannopyranosyl-(1,2)-D-mannopyranoside), galactose (specifically carboxymethyl-β-galactoside), or glycolic acid induced no adverse effects after administration based on histopathological evaluation of liver, kidneys, and lungs. Regardless of the polymer formulation, there was no evidence of hepatic or renal damage or dysfunction observed in serum or urine samples. The histological profile of cellular infiltration and the cellular distribution and kinetics in the lungs of mice administered with nanoparticle treatments followed similar behavior as that observed in the lungs of animals administered with saline. Cytokine and chemokine profiles in bronchoalveolar lavage fluid indicated surface chemistry dependence on modest secretion of IL-6, IP-10, and MCP-1; however, there was no evidence of any deleterious histopathological changes. Based on these analyses, carbohydrate-functionalized nanoparticles are safe for in vivo applications. These results provide foundational information towards the evaluation of the capabilities of these surface-modified nanoparticles as vaccine delivery formulations.

Salicylic acid (SA)-based physically crosslinked pH-sensitive hydrogels were developed for oral insulin delivery using various ratios of salicylate-based poly(anhydride-ester) and poly(acrylic acid) (PAA). Pore size, swelling behavior, insulin loading, insulin and SA release rates of the gels were varied by changing PAA concentration. About 50% of insulin was incorporated within all of the hydrogels, with about 4-8% of insulin released in acidic conditions (pH 1.2) over 2 h. In pH simulating the intestine (pH 6.8), 90% of the insulin and 70% of SA were released within 24 h from the hydrogel system. These results suggest that hydrogels enable pH-dependent protein delivery and can be used for oral insulin and SA delivery to benefit diabetes patients.

H5N1 avian influenza is a significant global concern with the potential to become the next pandemic threat. Recombinant subunit vaccines are an attractive alternative for pandemic vaccines compared to traditional vaccine technologies. In particular, polyanhydride nanoparticles encapsulating subunit proteins have been shown to enhance humoral and cell-mediated immunity and provide protection upon lethal challenge. In this work, a recombinant H5 hemagglutinin trimer (H5₃) was produced and encapsulated into polyanhydride nanoparticles. The studies performed indicated that the recombinant H5₃ antigen was a robust immunogen. Immunizing mice with H5₃ encapsulated into polyanhydride nanoparticles induced high neutralizing antibody titers and enhanced CD4(+) T cell recall responses in mice. Finally, the H5₃-based polyanhydride nanovaccine induced protective immunity against a low-pathogenic H5N1 viral challenge. Informatics analyses indicated that mice receiving the nanovaccine formulations and subsequently challenged with virus were similar to naïve mice that were not challenged. The current studies provide a basis to further exploit the advantages of polyanhydride nanovaccines in pandemic scenarios.

Acute respiratory infections represent a significant portion of global morbidity and mortality annually. There is a critical need for efficacious vaccines against respiratory pathogens. To vaccinate against respiratory disease, pulmonary delivery is an attractive route because it mimics the route of natural infection and can confer both mucosal and systemic immunity. We have previously demonstrated that a single dose, intranasal vaccine based on polyanhydride nanoparticles elicited a protective immune response against Yersinia pestis for at least 40 weeks after immunization with F1-V. Herein, we investigate the effect of nanoparticle chemistry and its attributes on the kinetics and maturation of the antigen-specific serum antibody response. We demonstrate that manipulation of polyanhydride nanoparticle chemistry facilitated differential kinetics of development of antibody titers, avidity, and epitope specificity. The results provide new insights into the underlying role(s) of nanoparticle chemistry in providing long-lived humoral immunity and aid in the rational design of nanovaccine formulations to induce long-lasting and mature antibody responses.

First-line cancer chemotherapy necessitates high parenteral dosage and repeated dosing of a combination of drugs over a prolonged period. Current commercially available chemotherapeutic agents, such as Doxil and Taxol, are only capable of delivering single drug in a bolus dose. The aim of this study is to develop dual-drug-loaded, multilayered microparticles and to investigate their antitumor efficacy compared with single-drug-loaded particles. Results show hydrophilic doxorubicin HCl (DOX) and hydrophobic paclitaxel (PTX) localized in the poly(dl-lactic-co-glycolic acid, 50:50) (PLGA) shell and in the poly(l-lactic acid) (PLLA) core, respectively. The introduction of poly[(1,6-bis-carboxyphenoxy) hexane] (PCPH) into PLGA/PLLA microparticles causes PTX to be localized in the PLLA and PCPH mid-layers, whereas DOX is found in both the PLGA shell and core. PLGA/PLLA/PCPH microparticles with denser shells allow better control of DOX release. A delayed release of PTX is observed with the addition of PCPH. Three-dimensional MCF-7 spheroid studies demonstrate that controlled co-delivery of DOX and PTX from multilayered microparticles produces a greater reduction in spheroid growth rate compared with single-drug-loaded particles. This study provides mechanistic insights into how distinctive structure of multilayered microparticles can be designed to modulate the release profiles of anticancer drugs, and how co-delivery can potentially provide better antitumor response., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: This report is the second part of the previously published study on the impact of light/chemical hardening technology and a newly formulated composite graft material for crestal augmentation during immediate implant placement., Methods: A total of 48 implants were placed into the sockets of the mesial roots of freshly extracted mandibular premolar teeth in three minipigs. Crestal areas and intrabony spaces were randomly augmented with light-hardened graft materials including a composite graft consisting of polymethylmethacrylate, polyhydroxylethylmethacrylate, and calcium hydroxide (PPCH) plus polyanhydride (PA); PPCH graft; and PA graft, or left untreated. Distal sockets not receiving implants and the sockets of first molars (n = 60) were randomly treated with one of the graft materials or left empty. In addition, two molar sockets were treated with the original PPCH graft material. Quantitative microcomputed tomography (micro-CT) was used to assess alveolar bone structure and tissue compositions. Histologic evaluations included descriptive histology to assess the peri-implant wound healing, as well as histomorphometric measurements to determine bone-to-implant contact (BIC)., Results: Both trabecular and cortical bone measurements by micro-CT did not reveal any significant differences among the groups. Sites augmented with PPCH+PA resulted in significantly greater BIC surface than PPCH alone and no-graft-treated implants (P <0.05) histologically. Stained ground sections showed complete bone formation between bone and implant surface in the PPCH+PA group, whereas sites without augmentation showed large gaps between bone and implant surfaces, indicating a slower bone apposition and less BIC surface compared to all other groups. Similar to implant sections, all materials showed positive outcome on trabecular and cortical bone formation in extraction sockets with an intact crestal cortical bone., Conclusion: Histologic evaluations supported the previous findings on implant stability and function and confirmed that PPCH+PA provides a greater BIC with a well-organized implant-bone interface and is useful in crestal augmentation during immediate implant placement.

Salicylate-based poly(anhydride-esters) (PAEs) chemically incorporate salicylic acid (SA) into the polymer backbone, which is then delivered in a controlled manner upon polymer hydrolysis. In this work, a salicylate-based PAE is a carrier to encapsulate and deliver insulin. Polymer microspheres were formulated using a water/oil/water double-emulsion solvent evaporation technique. The microspheres obtained had a smooth surface, high protein encapsulation efficiency, and relatively low emulsifier content. Insulin was released in vitro for 15 days, with no signs of aggregation or unfolding of the secondary structure. The released insulin also retained bioactivity in vitro. Concurrently, SA was released from the microspheres with polymer degradation and anti-inflammatory activity was observed. Based upon these results, the formulated microspheres enable simultaneous delivery of insulin and SA, both retaining bioactivity following processing., (© 2013 Wiley Periodicals, Inc.)

In the last decade, peanut allergy has increased substantially. Significant differences in the prevalence among different countries are attributed to the type of thermal processing. In spite of the high prevalence and the severe reaction induced by peanuts, there is no immunotherapy available. The aim of this work was to evaluate the potential application of poly(anhydride) nanoparticles (NPs) as immunoadjuvants for peanut oral immunotherapy. NPs loaded with raw or roasted peanut proteins were prepared by a solvent displacement method and dried by either lyophilization or spray-drying. After physicochemical characterization, their adjuvant capacity was evaluated after oral immunization of C57BL/6 mice. All nanoparticle formulations induced a balanced T(H)1 and T(H)2 antibody response, accompanied by low specific IgE induction. In addition, oral immunization with spray-dried NPs loaded with peanut proteins was associated with a significant decrease in splenic T(H)2 cytokines (interleukin 4 [IL-4], IL-5, and IL-6) and enhancement of both T(H)1 (gamma interferon [IFN-γ]) and regulatory (IL-10) cytokines. In conclusion, oral immunization with poly(anhydride) NPs, particularly spray-dried formulations, led to a pro-T(H)1 immune response., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

This work illustrates a two-step strategy for the fabrication of polymer/drug nanoparticles. Utilizing solvent/non-solvent precipitation and gaseous basification, composite nanoparticles with 0-100% drug loadings are fabricated. Drug release kinetics are dictated by nanoparticle composition allowing future tuning for therapeutic applications., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Carvacrol, thymol, and eugenol are naturally occurring phenolic compounds known to possess antimicrobial activity against a range of bacteria, as well as antioxidant activity. Biodegradable poly(anhydride-esters) composed of an ethylenediaminetetraacetic acid (EDTA) backbone and antimicrobial pendant groups (i.e., carvacrol, thymol, or eugenol) were synthesized via solution polymerization. The resulting polymers were characterized to confirm their chemical composition and understand their thermal properties and molecular weight. In vitro release studies demonstrated that polymer hydrolytic degradation was complete after 16 days, resulting in the release of free antimicrobials and EDTA. Antioxidant and antibacterial assays determined that polymer release media exhibited bioactivity similar to that of free compound, demonstrating that polymer incorporation and subsequent release had no effect on activity. These polymers completely degrade into components that are biologically relevant and have the capability to promote preservation of consumer products in the food and personal care industries via antimicrobial and antioxidant pathways.

Biodegradable polyanhydrides possess unique features like those that they can predominantly undergo surface erosion, and the payloads can be released by a steady speed. However, there is little work that has been published to describe the polyanhydride micelles with redox-responsiveness as a nanocarrier for drug delivery. In this study, we develop one type of new amphiphilic polyanhydride copolymer containing disulfide bonds between the hydrophilic and hydrophobic segments. The copolymer can self-assemble into stable micelles with well-defined core-shell structure and a uniform size distribution with an average diameter of 69 nm. The disassembly behaviors of the micelles triggered by glutathione are evaluated from the changes of the micellar size, morphology and molecular weight. An approximate zero-order in vitro drug release mode with a fast speed can be achieved in a reducing and acid environment similar with that of tumor cells. In vitro cytotoxicity analysis demonstrate that the Cur-loaded micelles are of great efficiency in inhibiting the growth of cancer cells due to the rapidly intracellular delivery of therapeutic agent. Both the qualitative and quantitative results of the antitumor activity in 4T1 tumor-bearing BALB/c mice reveal that the redox-responsive micelles have a more significant therapeutic effect to artificial solid tumor compared to the redox-insensitive micelles. This study provides a new insight into the biomedical application of polyanhydrides in drug delivery., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Novel poly(anhydride-ester)-b-poly(ethylene glycol) copolymers (PAE-b-PEGs) were synthesized by esterization of methyl poly(ethylene glycol) and poly(anhydride-ester), which were obtained by the melt polycondensation of alpha,omega-acetic anhydride-terminated poly(L-lactic acid), and characterized by 1H-NMR and gel permeation chromatography. The two poly(anhydride-ester)-b-poly(ethylene glycols) (denoted as PAE-b-PEG2k and PAE-b-PEG5k) thus obtained can self-assemble in water to form micelles with hydrodynamic diameters of 92.5 and 97.5 nm above their critical micelle concentrations of 3.78 and 2.36 microg/mL, respectively. The curcumin-loaded PAE-b-PEG2k and PAE-b-PEG5k micelles were prepared by the solid dispersion method, and they could encapsulate approximately 7% (w/w) curcumin. The diameters of the micelles were stable for 5 days. Curcumin is released faster from the micelles at pH 5.0 than at pH 7.4. Curcumin is released from the micelles at a fast rate during the initial 12 h, followed by a zero-order release during the subsequent 200 h, both at pH 5.0 and 7.4. The IC50 values of the curcumin-loaded PAE-b-PEG2k and PAE-b-PEG5k micelles against HeLa cells are 12.41 and 15.31 microg/mL, respectively, which is lower than that of free curcumin (25.90 microg/mL). The PAE-b-PEG2k micelles are taken up faster than the PAE-b-PEG5k micelles by HeLa cells. Curcumin-loaded micelles can induce G2/M phase cell cycle arrest and apoptosis of HeLa cells.

The functionalization of polymeric nanoparticles with ligands that target specific receptors on immune cells offers the opportunity to tailor adjuvant properties by conferring pathogen mimicking attributes to the particles. Polyanhydride nanoparticles are promising vaccine adjuvants with desirable characteristics such as immunomodulation, sustained antigen release, activation of antigen presenting cells (APCs), and stabilization of protein antigens. These capabilities can be exploited to design nanovaccines against viral pathogens, such as HIV-1, due to the important role of dendritic cells (DCs) and macrophages in viral spread. In this work, an optimized process was developed for carbohydrate functionalization of HIV-1 antigen-loaded polyanhydride nanoparticles. The carbohydrate-functionalized nanoparticles preserved antigenic properties upon release and also enabled sustained antigen release kinetics. Particle internalization was observed to be chemistry-dependent with positively charged nanoparticles being taken up more efficiently by DCs. Up-regulation of the activation makers CD40 and CD206 was demonstrated with carboxymethyl-α-d-mannopyranosyl-(1,2)-d-mannopyranoside functionalized nanoparticles. The secretion of the cytokines IL-6 and TNF-α was shown to be chemistry-dependent upon stimulation with carbohydrate-functionalized nanoparticles. These results offer important new insights upon the interactions between carbohydrate-functionalized nanoparticles and APCs and provide foundational information for the rational design of targeted nanovaccines against HIV-1.

The use of bioadhesive polymers as nanodevices has emerged as a promising strategy for oral delivery of therapeutics. In this regard, poly(anhydride) nanoparticles have shown great potential for oral drug delivery and vaccine purposes. However, despite extensive research into the biomedical and pharmaceutical applications of poly(anhydride) nanoparticles, there are no studies to evaluate the interaction of these nanoparticles at a cellular level. Therefore, the main objectives of this study were to evaluate the cytotoxicity as well as the cell interaction of different poly(anhydride) nanoparticles: conventional (NP), nanoparticles containing 2-hydroxypropyl-beta-cyclodextrin (NP-HPCD) and nanoparticles coated with poly(ethylene glycol) 6000 (PEG-NP). For this purpose, nanoparticles were prepared by solvent displacement method and labelled with BSA-FITC. Nanoparticles displayed a size about 175 nm with negative surface charge. Cytotoxicity studies were developed by MTS and LDH assays in HepG2 and Caco-2 cells. Results showed that only in HepG2 cells, NP and NP-HPCD induced significant cytotoxicity at the highest concentrations (1 and 2 mg/mL) and incubation times (48 and 72 h) tested. Studies to discriminate between cytoadhesion and cytoinvasion were performed at 4 degrees C and 37 degrees C in Caco-2 cell line as intestinal cell model. Nanoparticles showed cytoadhesion to the cell surface but not internalization; PEG-NP was the most bioadhesive followed by NP-HPCD and NP as demonstrated by flow cytometry. Finally, cellular localization of particles by fluorescence confocal microscopy confirmed the association of these nanoparticles with cells. Thus, this study demonstrated the safety of NP, NP-HPCD and PEG-NP at cellular level and its bioadhesive properties within 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., (Copyright © 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Diabetes mellitus (DM) involves metabolic changes that can impair bone repair, including a prolonged inflammatory response. A salicylic acid-based poly(anhydride-ester) (SA-PAE) provides controlled and sustained release of salicylic acid (SA) that locally resolves inflammation. This study investigates the effect of polymer-controlled SA release on bone regeneration in diabetic rats where enhanced inflammation is expected. Fifty-six Sprague-Dawley rats were randomly assigned to two groups: diabetic group induced by streptozotocin (STZ) injection or normoglycemic controls injected with citrate buffer alone. Three weeks after hyperglycemia development or vehicle injection, 5mm critical sized defects were created at the rat mandibular angle and treated with SA-PAE/bone graft mixture or bone graft alone. Rats were euthanized 4 and 12weeks after surgery, then bone fill percentage in the defect region was assessed by micro-computed tomography (CT) and histomorphometry. It was observed that bone fill increased significantly at 4 and 12weeks in SA-PAE/bone graft-treated diabetic rats compared to diabetic rats receiving bone graft alone. Accelerated bone formation in normoglycemic rats caused by SA-PAE/bone graft treatment was observed at 4weeks but not at 12weeks. This study shows that treatment with SA-PAE enhances bone regeneration in diabetic rats and accelerates bone regeneration in normoglycemic animals., (© 2013.)

Pneumococcal surface protein A (PspA) is a choline-binding protein which is a virulence factor found on the surface of all Streptococcus pneumoniae strains. Vaccination with PspA has been shown to be protective against a lethal challenge with S. pneumoniae, making it a promising immunogen for use in vaccines. Herein the design of a PspA-based subunit vaccine using polyanhydride nanoparticles as a delivery platform is described. Nanoparticles based on sebacic acid (SA), 1,6-bis-(p-carboxyphenoxy)hexane (CPH) and 1,8-bis-(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG), specifically 50:50 CPTEG:CPH and 20:80 CPH:SA, were used to encapsulate and release PspA. The protein released from the nanoparticle formulations retained its primary and secondary structure as well as its antigenicity. The released PspA was also biologically functional based on its ability to bind to apolactoferrin and prevent its bactericidal activity against Escherichia coli. When the PspA nanoparticle formulations were administered subcutaneously to mice they elicited a high titer and high avidity anti-PspA antibody response. Together these studies provide a framework for the rational design of a vaccine against S. pneumoniae based on polyanhydride nanoparticles., (Copyright © 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)