Biodegradable Polyanhydride Research Articles

PSV-1 A Subcutaneous Ear Implant to Deliver an Anaplasmosis Vaccine to Dairy Steers

Abstract Bovine anaplasmosis is the most prevalent tick-transmitted disease of cattle worldwide and a major obstacle to beef production. The objective of the study was to test the ability of a single-dose delivery platform to produce long-lasting immunity against anaplasmosis infections. Twelve Holstein steers were administered a 3-stage, single-dose vaccine against Anaplasma marginale major surface protein 1a. The vaccine consisted of a soluble vaccine administered subcutaneously for immune priming, a biodegradable polyanhydride depot for intermediate slow-release of the vaccine for boosting the immune response, and an immune-isolated vaccine platform for extended antigen release deposited subcutaneously in the ear. Six calves were randomly assigned to two vaccine constructs that featured rods and implants containing a combination of two adjuvants (DEAE-Dextran and Quil-A). The remaining 6 calves were randomly assigned to two vaccine constructs that featured rods and implants containing the same adjuvant (DEAE-Dextran or Quil-A). Twenty months post-implantation, calves were challenged with Anaplasma marginale stabilate and were monitored weekly for signs of fever, decreased packed cell volume (PCV) and bacteremia. Outcome variables (fever, PCV, bacteremia) were analyzed using a linear mixed model (PROC GLMMIX; SAS University Edition v9.04.01, SAS Institute, Cary, NC). Two-tailed chi-squared tests were used to compare need for antibiotic intervention between vaccine constructs. Calves within the combination adjuvant construct group (DEAE-D and Quil-A) exhibited significantly (P = .006) higher PCV than calves within the single adjuvant construct group (DEAE-Dextran or Quil-A). Likewise, calves within the combination adjuvant group required significantly (P = .014) fewer antibiotic interventions compared with calves in the single adjuvant group. Findings of this study suggest that calves exhibited significantly diminished clinical signs of anaplasmosis when vaccine was delivered with a combination of adjuvants as opposed to a single adjuvant in a single-dose delivery platform.

Bovine immune response to leptospira antigen in different novel adjuvants and vaccine delivery platforms

Leptospirosis is a global zoonosis causing significant economic losses for cattle production. Current cattle vaccines against leptospirosis need improvement to provide efficacy against multiple serovars, reduce shedding in urine, and to induce earlier and more robust immune responses. In this study, Leptospira borgpetersenii serovar Hardjo strain 203 antigen was combined with novel adjuvants (a biodegradable polyanhydride compressed rod implant (VPEAR), poly(diaminosulfide) microparticles, a water-oil-water emulsion adjuvant, and aluminum hydroxide) to develop novel vaccines. Cattle were immunized twice, at a 4 week interval, with inoculums containing adjuvants alone or leptospira antigens and immune responses were compared to responses of cattle receiving a commercial monovalent leptospirosis vaccine (Spirovac). All animals were inoculated with a single dose of Spirovac at 20 weeks to assess antigen recall responses. Serum antibody responses were increased (P > 0.05) at 8 and 20 weeks after vaccination in cattle receiving inoculums containing leptospira antigens combined with water-oil-emulsion, poly(diaminosulfide) microparticles (PNSN-MP), or aluminum hydroxide and in cattle vaccinated with Spirovac. Humoral responses were predominantly IgG1 isotypes. Antigen-specific proliferative responses were detected after initial vaccination in cattle vaccinated with Spirovac, PNSN-MP and water-oil-water treatments. Most proliferative responses occurring within CD4+ and gamma delta T cell populations expressing CD45RO and CD25 markers, a response consistent with an effector memory phenotype. Antigen-specific immune responses were not detected in cattle vaccinated with VPEAR after initial inoculation, but were detected in the antigen recall responses. PBMCs from cattle vaccinated with Spirovac, oil-water-oil, or PNSN-MP treatments had increased (P < 0.05) IL-17A release after in vitro stimulation with leptospirosis antigens, whereas all groups produced IFN-γ and IL-17A after in vitro stimulation during the antigen recall response. Our data demonstrates that combining leptospirosis antigens with these adjuvants enhances immunogenicity in cattle.Interpretative Summary: Vaccination of livestock is a key mechanism for minimizing transmission of leptospirosis, a zoonotic disease. Leptospirosis vaccines for cattle need to be improved to provide greater levels of protection from kidney colonization, better immune responses, and protection against multiple serovars. This could be accomplished using new vaccine adjuvants. In this study, several novel adjuvants were evaluated for their ability to induce effective immune responses in cattle to leptospira antigens as compared to currently available vaccines. Data suggested that vaccines containing biodegradable polymer microparticles and oil-emulsion adjuvants induced similar or greater immune responses as compared to a commercial vaccine. Our data suggest these new vaccine formulations warrant further investigation as new vaccine formulations for cattle and other livestock.

Development of a subcutaneous ear implant to deliver an anaplasmosis vaccine to dairy steers.

Bovine anaplasmosis is the most prevalent tick-transmitted disease of cattle worldwide and a major obstacle to profitable beef production. Use of chlortetracycline-medicated feed to control active anaplasmosis infections during the vector season has raised concerns about the potential emergence of antimicrobial resistance in bacteria that may pose a risk to human health. Furthermore, the absence of effectiveness data for a commercially available, conditionally licensed anaplasmosis vaccine is a major impediment to implementing anaplasmosis control programs. The primary objective of this study was to develop a single-dose vaccine delivery platform to produce long-lasting protective immunity against anaplasmosis infections. Twelve Holstein steers, aged 11 to 12 wk, were administered a novel 3-stage, single-dose vaccine against Anaplasma marginale, a major surface protein 1a. The vaccine consisted of a soluble vaccine administered subcutaneously (s.c.) for immune priming, a vaccine depot of a biodegradable polyanhydride rod with intermediate slow release of the vaccine for boosting immune response, and an immune-isolated vaccine platform for extended antigen release (VPEAR implant) deposited s.c. in the ear. Six calves were randomly assigned to 2 vaccine constructs (n = 3) that featured rods and implants containing a combination of 2 different adjuvants, diethylaminoethyl (DEAE)-Dextran and Quil-A (Group A). The remaining 6 calves were randomly assigned to 2 vaccine constructs (n = 3) that featured rods and implants containing the same adjuvant (either DEAE-Dextran or Quil A) (Group B). Twenty-one months post-implantation, calves were challenged intravenously with A. marginale stabilate and were monitored weekly for signs of fever, decreased packed cell volume (PCV) and bacteremia. Data were analyzed using a mixed-effects model and chi-squared tests (SAS v9.04.01, SAS Institute, Cary, NC). Calves in Group A had higher PCV than calves in Group B (P = 0.006) at day 35 post-infection. Calves in Group A were less likely to require antibiotic intervention compared with calves in Group B (P = 0.014). Results indicate that calves exhibited diminished clinical signs of anaplasmosis when antigen was delivered with a combination of adjuvants as opposed to a single adjuvant. This demonstrates the feasibility of providing long-lasting protection against clinical bovine anaplasmosis infections using a subcutaneous ear implant vaccine construct.

Biodegradable polyanhydride-based nanomedicines for blood to brain drug delivery.

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.

Improved delivery of the anticancer agent citral using BSA nanoparticles and polymeric wafers.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children, with a 5-year survival rate of between 30 and 65%. Standard treatment involves surgery, radiation treatment, and chemotherapy. However, there is a high recurrence rate, particularly from locoregional spread. We investigated the use of the natural compound citral (3,7-dimethyl-2,6-octadienal), which can be found in a number of plants, but is particularly abundant in lemon grass (Cymbopogon citratus) oil, for activity against immortalized RMS cells. Significant cancer cell death was seen at concentrations above 150 μM citral, and mitochondrial morphological changes were seen after incubation with 10 μM citral. However, since citral is a highly volatile molecule, we prepared albumin particles by a desolvation method to encapsulate citral, as a means of stabilization. We then further incorporated the loaded nanoparticles into a biodegradable polyanhydride wafer to generate a slow release system. The wafers were shown to degrade by 50% over the course of 25 days and to release the active compound. We therefore propose the use of the citral-nanoparticle-polymer wafers for implantation into the tumor bed after surgical removal of a sarcoma as a means to control locoregional spread due to any remaining cancerous cells.

Polyanhydride nanovaccine against swine influenza virus in pigs

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.

The effect of polyanhydride chemistry in particle-based cancer vaccines on the magnitude of the anti-tumor immune response.

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.

Neuronal protection against oxidative insult by polyanhydride nanoparticle-based mitochondria-targeted antioxidant therapy

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.

Polyanhydride Nanoparticle Delivery Platform Dramatically Enhances Killing of Filarial Worms.

Filarial diseases represent a significant social and economic burden to over 120 million people worldwide and are caused by endoparasites that require the presence of symbiotic bacteria of the genus Wolbachia for fertility and viability of the host parasite. Targeting Wolbachia for elimination is a therapeutic approach that shows promise in the treatment of onchocerciasis and lymphatic filariasis. Here we demonstrate the use of a biodegradable polyanhydride nanoparticle-based platform for the co-delivery of the antibiotic doxycycline with the antiparasitic drug, ivermectin, to reduce microfilarial burden and rapidly kill adult worms. When doxycycline and ivermectin were co-delivered within polyanhydride nanoparticles, effective killing of adult female Brugia malayi filarial worms was achieved with approximately 4,000-fold reduction in the amount of drug used. Additionally the time to death of the macrofilaria was also significantly reduced (five-fold) when the anti-filarial drug cocktail was delivered within polyanhydride nanoparticles. We hypothesize that the mechanism behind this dramatically enhanced killing of the macrofilaria is the ability of the polyanhydride nanoparticles to behave as a Trojan horse and penetrate the cuticle, bypassing excretory pumps of B. malayi, and effectively deliver drug directly to both the worm and Wolbachia at high enough microenvironmental concentrations to cause death. These provocative findings may have significant consequences for the reduction in the amount of drug and the length of treatment required for filarial infections in terms of patient compliance and reduced cost of treatment.

Nanoparticle Chemistry and Functionalization Differentially Regulates Dendritic Cell–Nanoparticle Interactions and Triggers Dendritic Cell Maturation

Biodegradable polyanhydride nanoparticles have been used as vaccine adjuvants and delivery vehicles. Upon parenteral administration, the surface characteristics of these nanoparticles may be modified by serum protein adsorption, likely affecting how the particles interact with antigen presenting cells. These studies investigated the role of the differential adsorption of serum proteins onto carbohydrate‐functionalized and non‐functionalized polyanhydride nanoparticles of different chemistries on interactions with dendritic cells (DCs). Di‐mannose (a carboxymethyl‐modified disaccharide of 1,2‐α‐linked‐d‐mannopyranoside) or glycolic acid was covalently linked to nanoparticles synthesized using copolymers of 1,6‐bis(p‐carboxyphenoxy)hexane (CPH) and sebacic acid (SA) and 1,8‐bis(p‐carboxyphenoxy)‐3,6‐dioxaoctane (CPTEG) and CPH. Serum protein adsorption to CPH:SA nanoparticles enhanced particle internalization, but did not enhance uptake of CPTEG:CPH nanoparticles. When DCs were cultured with nanoparticles, the surface expression of MHC II, CD86, and CD206 was enhanced and mediated by both polymer chemistry and functionalization. Cytokine secretion by DCs was differentially modulated by both polymer chemistry and functionalization. The amount and profile of serum proteins adsorbed indicated that polymer hydrophobicity and functionalization enhanced protein adsorption. Overall polymer chemistry, functionalization, and serum protein adsorption influenced nanoparticle interactions with DCs and elucidating the complex relationships between these parameters and dendritic cell maturation will enable rational design of targeted vaccines and immunotherapies.

Combinatorial Synthesis of and High-throughput Protein Release from Polymer Film and Nanoparticle Libraries

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

Local interstitial delivery of z-butylidenephthalide by polymer wafers against malignant human gliomas

We have shown that the natural compound z-butylidenephthalide (Bdph), isolated from the chloroform extract of Angelica sinensis, has antitumor effects. Because of the limitation of the blood-brain barrier, the Bdph dosage required for treatment of glioma is relatively high. To solve this problem, we developed a local-release system with Bdph incorporated into a biodegradable polyanhydride material, p(CPP-SA; Bdph-Wafer), and investigated its antitumor effects. On the basis of in vitro release kinetics, we demonstrated that the Bdph-Wafer released 50% of the available Bdph by the sixth day, and the release reached a plateau phase (90% of Bdph) by the 30th day. To investigate the in situ antitumor effects of the Bdph-Wafer on glioblastoma multiforme (GBM), we used 2 xenograft animal models-F344 rats (for rat GBM) and nude mice (for human GBM)-which were injected with RG2 and DBTRG-05MG cells, respectively, for tumor formation and subsequently treated subcutaneously with Bdph-Wafers. We observed a significant inhibitory effect on tumor growth, with no significant adverse effects on the rodents. Moreover, we demonstrated that the antitumor effect of Bdph on RG2 cells was via the PKC pathway, which upregulated Nurr77 and promoted its translocation from the nucleus to the cytoplasm. Finally, to study the effect of the interstitial administration of Bdph in cranial brain tumor, Bdph-Wafers were surgically placed in FGF-SV40 transgenic mice. Our Bdph-Wafer significantly reduced tumor size in a dose-dependent manner. In summary, our study showed that p(CPP-SA) containing Bdph delivered a sufficient concentration of Bdph to the tumor site and effectively inhibited the tumor growth in the glioma.

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.

Intracranial microcapsule drug delivery device for the treatment of an experimental gliosarcoma model

Controlled-release drug delivery systems are capable of treating debilitating diseases, including cancer. Brain cancer, in particular glioblastoma multiforme (GBM), is an extremely invasive cancer with a dismal prognosis. The use of drugs capable of crossing the blood–brain barrier has shown modest prolongation in patient survival, but not without unsatisfactory systemic, dose-limiting toxicity. Among the reasons for this improvement include a better understanding of the challenges of delivery of effective agents directly to the brain tumor site. The combination of carmustine delivered by biodegradable polyanhydride wafers (Gliadel ®), with the systemic alkylating agent, temozolomide, allows much higher effective doses of the drug while minimizing the systemic toxicity. We have previously shown that locally delivering these two drugs leads to further improvement in survival in experimental models. We postulated that microcapsule devices capable of releasing temozolomide would increase the therapeutic capability of this approach. A biocompatible drug delivery microcapsule device for the intracranial delivery of temozolomide is described. Drug release profiles from these microcapsules can be modulated based on the physical chemistry of the drug and the dimensions of the release orifices in these devices. The drug released from the microcapsules in these experiments was the clinically utilized chemotherapeutic agent, temozolomide. In vitro studies were performed in order to test the function, reliability, and drug release kinetics of the devices. The efficacy of the temozolomide-filled microcapsules was tested in an intracranial experimental rodent gliosarcoma model. Immunohistochemical analysis of tissue for evidence of DNA strand breaks via terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was performed. The experimental release curves showed mass flow rates of 36 μg/h for single-orifice devices and an 88 μg/h mass flow rate for multiple-orifice devices loaded with temozolomide. In vivo efficacy results showed that localized intracranial delivery of temozolomide from microcapsule devices was capable of prolonging animal survival and may offer a novel form of treatment for brain tumors.

Preparation and Characterization of Biodegradable Electrospun Polyanhydride Nano/Microfibers

The biodegradable polyanhydride copolymers P(CPP-SA) composed of p-carboxyhenoxy propane (CPP) and sebacic acid (SA) at weight ratios of 20:80, 35:65 and 50:50 were polymerized by a melt polycondensation process without catalyst. The copolymers were characterized by fourier transform infrared spectroscopy (FT-IR), 1H-nuclear magnetic resonance (1H-NMR), Ubbelohde viscometer, differential scanning calorimetry (DSC) and wide angle X-ray powder-diffraction (XRD). P(CPP-SA) nano/microfibers were the first time to be fabricated by electrospinning. The copolymers hold an excellent fibre-forming performance and the diameter range of 80-3,200 nm can be obtained. The in vitro degradation of the polyanhydride copolymers was evaluated in form of the nano/microfibers by investigating the change of fibrous morphology, weight loss and pH change of degradation medium. The experimental results showed that degradation rate was fast in the fist day and slow in the following period, furthermore the degradation rate decreased with the increase of the content of CPP in copolymers. Therefore, the electrospun polyanhydride nano/microfibers exhibited strong potential as drug delivery vehicle and tissue engineering scaffold.

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.

Amphiphilic polyanhydrides for protein stabilization and release

The overall goal of this research is to design novel amphiphilic biodegradable systems based on polyanhydrides for the stabilization and sustained release of peptides and proteins. Accordingly, copolymers of the anhydrides, 1,6-bis(p-carboxyphenoxy)hexane (CPH) and 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG), which are monomer-containing oligomeric ethylene glycol moieties, have been synthesized. Microspheres of different CPTEG:CPH compositions have been fabricated by two non-aqueous methods: solid/oil/oil double emulsion and cryogenic atomization. The ability of this amphiphilic polymeric system to stabilize model proteins (i.e., lysozyme and ovalbumin) was investigated. The structure of both the encapsulated as well as the released protein was monitored using gel electrophoresis, circular dichroism, and fluorescence spectroscopy. It was found that the CPTEG:CPH system preserves the structural hierarchy of the encapsulated proteins. Activity studies of the released protein indicate the CPTEG:CPH system retains the biological activity of the released protein. These results are promising for future in vivo studies, which involve the design of novel biodegradable polyanhydride carriers for the stabilization and sustained release of therapeutic peptides and proteins.

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.

Parallel Synthesis and High Throughput Dissolution Testing of Biodegradable Polyanhydride Copolymers

We have demonstrated that polycondensation reactions can be carried out in a combinatorial fashion and that the polymer library can be screened at high throughput using a rapid prototyping technique to fabricate multiwell substrates. A linearly varying compositional library of 100 different biodegradable polyanhydride random copolymers that are promising carriers for controlled drug delivery was designed, fabricated, and characterized by IR microscopy within a few hours. The polyanhydride copolymer library was based on 1,6-bis(p-carboxyphenoxy)hexane (CPH) and sebacic anhydride (SA) and was characterized with infrared microspectroscopy to determine the composition within each well. Since degradation and release rates depend on copolymer composition, we also developed new high-throughput methods to investigate drug release from this library of copolymers by designing specific wells for each task. A subset of this library was chosen, and a substrate was designed and fabricated to enable the synthesis and monitoring of dye dissolution from a range of polyanhydride copolymers in a parallel fashion using a CCD camera. Multisample substrates were fabricated with a novel rapid prototyping method that consists of an organic solvent-resistant array of 10 x 10 microwells of 2-muL volume each. The libraries were deposited with a custom-built liquid dispensing system consisting of a series of computer-controlled volume-dispensing pumps and XYZ motion stages. The parallel dye dissolution study displayed a decreasing rate of release with increasing CPH content. This result agrees with previously published data for dye release from poly(CPH-co-SA) copolymers. The methodology described in this work is amenable to numerous applications in the arenas of high-throughput polymer synthesis and characterization.

Microsphere size, precipitation kinetics and drug distribution control drug release from biodegradable polyanhydride microspheres

A thorough understanding of the factors affecting drug release mechanisms from surface-erodible polymer devices is critical to the design of optimal delivery systems. Poly(sebacic anhydride) (PSA) microspheres were loaded with three model drug compounds (rhodamine B, p-nitroaniline and piroxicam) with a range of polarities (water solubilities). The drug release profiles from monodisperse particles of three different sizes were compared to release from polydisperse microspheres. Each of the model drugs exhibited different release mechanisms. Drug distribution within the polymer was investigated by laser scanning confocal microscopy and scanning electron microscopy. Rhodamine, the most hydrophilic compound investigated, was localized strongly toward the microsphere surface, while the much more hydrophobic compound, piroxicam, distributed more evenly. Furthermore, all three compounds were most uniformly distributed in the smallest microspheres, most likely due to the competing effects of drug diffusion out of the nascent polymer droplets and the precipitation of polymer upon solvent extraction, which effectively “traps” the drug in the polymer matrix. The differing drug distributions were manifested in the drug release profiles. Rhodamine was released very quickly independent of microsphere size. Thus, extended release profiles may not be obtainable if the drug strongly redistributes in the microspheres. The release of p-nitroaniline was more prolonged, but still showed little dependence on microsphere size. Hence, when water-soluble drugs are encapsulated with hydrophobic polymers, it may be difficult to tailor release profiles by controlling microsphere size. The piroxicam-loaded microspheres exhibit the most interesting release profiles, showing that release duration can be increased by decreasing microsphere size, resulting in a more uniform drug distribution.