Functionalized polyanhydride nanoparticles for improved treatment of mitochondrial dysfunction.

Parkinson's disease (PD) is a devastating neurodegenerative disease affecting a large proportion of older adults. Exposure to pesticides like rotenone is a leading cause for PD. To reduce disease progression and prolong life expectancy, it is important to target disease mechanisms that contribute to dopaminergic neuronal atrophy, including mitochondrial dysfunction. Achieving targeted mitochondrial delivery is difficult for many therapeutics by themselves, necessitating higher therapeutic doses that could lead to toxicity. To minimize this adverse effect, targeted nano-carriers such as polyanhydride nanoparticles (NPs) can protect therapeutics from degradation and provide sustained release, enabling fewer administrations and lower therapeutic dose. This work expands upon the use of the polyanhydride NP platform for targeted drug delivery by functionalizing the polymer with a derivative of triphenylphosphonium called (3-carboxypropyl) triphenylphosphonium (CPTP) using a novel method that enables longer CPTP persistence on the NPs. The extent to which neurons internalized both nonfunctionalized and functionalized NPs was tested. Next, the efficacy of these nanoformulations in treating rotenone-induced mitochondrial dysfunction in the same cell line was evaluated using a novel neuroprotective drug, mito-metformin. CPTP functionalization significantly improved NP internalization by neuronal cells. This was correlated with significant protection by CPTP-functionalized, mito-metformin encapsulated NPs against rotenone-induced mitochondrial dysfunction. However, nonfunctionalized, mito-metformin encapsulated NPs and soluble mito-metformin administered at the same dose did not significantly protect cells from rotenone-induced toxicity. These results indicate that the targeted NP platform can provide enhanced dose-sparing and potentially reduce the occurrence of systemic side-effects for PD therapeutics.

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.

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.

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.

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

Nanoparticulate systems have emerged as valuable tools in vaccine delivery through their ability to efficiently deliver cargo, including proteins, to antigen presenting cells. Internalization of nanoparticles (NP) by antigen presenting cells is a critical step in generating an effective immune response to the encapsulated antigen. To determine how changes in nanoparticle formulation impact function, we sought to develop a high throughput, quantitative experimental protocol that was compatible with detecting internalized nanoparticles as well as bacteria. To date, two independent techniques, microscopy and flow cytometry, have been the methods used to study the phagocytosis of nanoparticles. The high throughput nature of flow cytometry generates robust statistical data. However, due to low resolution, it fails to accurately quantify internalized versus cell bound nanoparticles. Microscopy generates images with high spatial resolution; however, it is time consuming and involves small sample sizes. Multi-spectral imaging flow cytometry (MIFC) is a new technology that incorporates aspects of both microscopy and flow cytometry that performs multi-color spectral fluorescence and bright field imaging simultaneously through a laminar core. This capability provides an accurate analysis of fluorescent signal intensities and spatial relationships between different structures and cellular features at high speed. Herein, we describe a method utilizing MIFC to characterize the cell populations that have internalized polyanhydride nanoparticles or Salmonella enterica serovar Typhimurium. We also describe the preparation of nanoparticle suspensions, cell labeling, acquisition on an ImageStream(X) system and analysis of the data using the IDEAS application. We also demonstrate the application of a technique that can be used to differentiate the internalization pathways for nanoparticles and bacteria by using cytochalasin-D as an inhibitor of actin-mediated phagocytosis.

Protein adsorption on biodegradable polyanhydride microparticles.

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

Different approaches for determination of the attachment degree of polyethylene glycols to poly(anhydride) nanoparticles.

PURPOSE: The aim of the study was to find an appropriate method for determination of the attachment degree of polyethylene glycol (PEG) to poly(anhydride) nanoparticles. METHODS: The nanoparticles were modified with hydroxy-functionalized or with amino-functionalized PEGs. Three methods for their determination were applied and examined: in particular colorimetry, nuclear magnetic resonance ( (1) H-NMR), and elemental analysis (EA). RESULTS: The attachment degrees determined by (1) H-NMR and colorimetry were similar. The associated amounts of PEGs estimated on the basis of EA differed significantly than those determined by colorimetry and (1) H-NMR. CONCLUSION: The colorimetric determination was considered fast and simple technique, but (1) H-NMR contributed to more precise results.

Preparation and characterization of protein-loaded polyanhydride microspheres.

Poly(1,3-bis-(p-carboxyphenoxy propane)-co-(sebacic anhydride) (P(CPP-SA)) have the anhydride bonds in copolymer backbone, which are available for degradation on the base of passive hydrolysis. This chemical structure made it degraded within a short time in linear degradation rate. For this property, polyanhydrides are one of the most suitable biodegradable polymers employed as drug carriers. This paper aimed at researching the erosion and degradation of P(CPP-SA) microspheres with CPP/SA monomer ratios of 20:80, 35:65 and 50:50. In vitro protein release from the microspheres was also investigated in this paper. Human serum albumin (HSA) was used as the model protein. In this research, the microspheres degradation and drug release rate from microspheres can be adjusted by altering the CPP/SA ratios of P(CPP-SA). The features of surface erosion were observed in SEM. The structural integrity of HSA extracted from microspheres was detected by gel permeation chromatography, compared with native HSA. The results showed HSA remained its molecule weight after encapsulated.

Hydroxy fatty acid based polyanhydride as drug delivery system: synthesis, characterization, in vitro degradation, drug release, and biocompatibility.

Low molecular weight hydroxy fatty acid based polyanhydrides were synthesized by one pot method, a variable of typical melt-condensation and characterized by FTIR, NMR, DSC, and GPC. Polymer degrades by both surface and bulk erosion as trailed by weight loss, anhydride loss and surface morphology. Control over drug release was accessed with drugs featuring different aqueous solubility, that is, methotrexate (hydrophobic) and 5-fluorouracil (hydrophilic). Effect of loading, at 5, 10, and 20% w/w of methotrexate on release profiles was also studied and negligible effect was discovered. Biocompatibility of polymers was evaluated in SD rats after SC injection of the polymer. Histopathology revealed initial inflammation of the tissues near the injection site however healed with time. Overall, these polymers were found good to control the release of the entrapped drug and were found biocompatible in preliminary in vivo study. Due to their low melting temperatures they can be injected locally (SC or intratumorally) to from regional in situ depot and have a great potential as a drug carrier for localized delivery of anticancer drugs.

Recent developments in biodegradable synthetic polymers.

This chapter reviews recent developments in biodegradable synthetic polymers focusing on tailoring polymer structures to meet material specification for emerging applications such as tissue engineered products and therapies. Major classes and new families of synthetic polymers are discussed with regard to synthesis, properties and biodegradability, and known degradation modes and products are summarized based on studies reported during the past 10-15 years. Polyesters and their copolymers, polyurethanes, polyphosphazenes, polyanhydrides, polycarbonates, polyesteramides and recently developed injectable polymer systems based on polypropylenefumarates, polyurethanes and acrylate/urethane systems are reviewed. Polyesters such as polyglycolides, polylactides and their copolymers still remain as the major class of synthetic biodegradable polymers with products in clinical use. Although various copolymerization methods have addressed needs of different applications, release of acidic degradation products, processing difficulties and limited range of mechanical properties remains as major disadvantages of this family of polymers. Injectable polymers based on urethane and urethane/acrylate have shown great promise in developing delivery systems for tissue engineered products and therapies.

Biodegradation of poly(anhydride-esters) into non-steroidal anti-inflammatory drugs and their effect on Pseudomonas aeruginosa biofilms in vitro and on the foreign-body response in vivo.

The ability of poly(anhydride-esters) composed of non-steroidal anti-inflammatory drugs that biodegrade to salicylic acid (SA) and adipic acid to prevent colonization by Pseudomonas aeruginosa and their effects on the foreign-body response were studied in vitro and in vivo, respectively. Soluble SA in bacterial medium at concentrations up to 300 mg/L did not affect the growth rate or viability of P. aeruginosa, indicating that SA does not exhibit a direct toxicity effect on the bacterium. Batch degradation rates of the salicylate-based polymer in the presence of an actively growing bacterial culture only marginally (14%) increased relative to polymer degradation rates in sterile medium. Short-term (3h) bacterial adhesion studies in agitated batch systems indicated a 47% reduction in the rate of P. aeruginosa adhesion relative to a control polymer that does not release SA upon biodegradation. Long-term (3-day) biofilm accumulation studies indicated a dramatic reduction in biofilm formation on salicylate-based polymer versus controls. A recombinant P. aeruginosa pMHLAS, containing a fluorescent reporter gene prior to the las regulon, was employed to determine whether salicylate-based polymer prevents biofilm formation by the released SA inhibiting quorum sensing pathways. Long-term biofilm accumulation studies with P. aeruginosa pMHLAS insinuate that salicylate-based polymer prevents biofilm accumulation by inhibiting the las quorum sensing system. Furthermore, unlike control polymer, salicylate-based polymer implanted subcutaneously for a period of 4 weeks-resisted cell-mediated degradation and remained intact. Histological and immunohistochemical analysis indicated a reduction in overall encapsulation and paucity of macrophages in the area of the salicylate-based polymer implant.