Development of a novel anti-infectivity platform for the treatment of neglected tropical and infectious diseases

This dissertation focuses on the design of novel strategies for the prevention and treatment of neglected tropical and infectious diseases using polyanhydride nanoparticles as a drug delivery platform. The overall goal of this research is to design efficacious drug delivery vehicles that are effective against the filarial nematode Brugia malayi and B. pahangi and intracellular bacteria Mycobacterium tuberculosis, M. paratuberculosis, and M. marinum. The primary goal is to use the polyanhydride nanoparticles to deliver high enough microenvironmental concentrations to cause the death of the organism, thereby reducing the mortality, morbidity, and transmission of the disease. While a wide array of vaccine delivery vehicles have been investigated the majority of the research has focused on the use of polyesters and polyanhydride. This research presented shows the benefits of biodegradable polyanhydride as an effective drug delivery platform using the polymers and copolymers of various chemistries. We demonstrate the use of a polyanhydride nanoparticle-based platform for the co-delivery of the antibiotic doxycycline with multiple antiparasitic drugs, (ivermectin, diethylcarbamazine, and albendazole) to reduce microfilariae burden and to rapidly kill adult worms. By encapsulating two of these drugs, ivermectin, and doxycycline, into biodegradable polyanhydride nanoparticles, we report the ability to effectively kill adult B. malayi and B. pahangi worms with up to a 4,000-fold reduction in the amount of drug used. Additionally, we significantly reduced the average time to death of the macrofilaria (five-fold) when the anti-filarial drug cocktail was delivered within polyanhydride nanoparticles. Knowledge gained from filarial studies was then applied to kill the intracellular bacteria Mycobacterium, we sought to examine the effectiveness of the first line antituberculous rifampicin, isoniazid, pyrazinamide, and ethambutol, encapsulated into various polymer and copolymer combinations in vitro. Initial experiments focused on particle internalization, co-localization, and reduced macrophage bacterial population. Subsequently, gained knowledge from the in vitro effectiveness was then applied to systemic acute M. tuberculosis in vivo infection focusing on multi-drug resistance and extensively drug-resistant strains. We observed through short course chemotherapy utilizing the nanoparticle-based drug delivery platform 20:80 CPTEG:CPH, we were able to reduce the prevalence of a persister bacterial population significantly. In summary, the studies described herein support the rational design and use of a polyanhydride nanoparticle drug delivery platform to reduce both the course of treatment and the amount of drug needed to treat neglected infectious disease effectively

A data analytics approach for rational design of nanomedicines with programmable drug release

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 between) polymer, drug, and nanoparticle properties gives rise to nonlinear behavioral relationships and a large data space. These factors complicate both first-principles modeling and screening of nanomedicine formulations. Predictive analytics may offer a more efficient approach toward 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. Non-linear 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 training data set. The models were shown to be robust after inclusion of these new formulations in that the new inclusions did not significantly change model regression. This approach provides the first steps towards 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

Development of a novel anti-infectivity platform for the treatment of neglected tropical and infectious diseases

This dissertation focuses on the design of novel strategies for the prevention and treatment of neglected tropical and infectious diseases using polyanhydride nanoparticles as a drug delivery platform. The overall goal of this research is to design efficacious drug delivery vehicles that are effective against the filarial nematode Brugia malayi and B. pahangi and intracellular bacteria Mycobacterium tuberculosis, M. paratuberculosis, and M. marinum. The primary goal is to use the polyanhydride nanoparticles to deliver high enough microenvironmental concentrations to cause the death of the organism, thereby reducing the mortality, morbidity, and transmission of the disease. While a wide array of vaccine delivery vehicles have been investigated the majority of the research has focused on the use of polyesters and polyanhydride. This research presented shows the benefits of biodegradable polyanhydride as an effective drug delivery platform using the polymers and copolymers of various chemistries. We demonstrate the use of a polyanhydride nanoparticle-based platform for the co-delivery of the antibiotic doxycycline with multiple antiparasitic drugs, (ivermectin, diethylcarbamazine, and albendazole) to reduce microfilariae burden and to rapidly kill adult worms. By encapsulating two of these drugs, ivermectin, and doxycycline, into biodegradable polyanhydride nanoparticles, we report the ability to effectively kill adult B. malayi and B. pahangi worms with up to a 4,000-fold reduction in the amount of drug used. Additionally, we significantly reduced the average time to death of the macrofilaria (five-fold) when the anti-filarial drug cocktail was delivered within polyanhydride nanoparticles. Knowledge gained from filarial studies was then applied to kill the intracellular bacteria Mycobacterium, we sought to examine the effectiveness of the first line antituberculous rifampicin, isoniazid, pyrazinamide, and ethambutol, encapsulated into various polymer and copolymer combinations in vitro. Initial experiments focused on particle internalization, co-localization, and reduced macrophage bacterial population. Subsequently, gained knowledge from the in vitro effectiveness was then applied to systemic acute M. tuberculosis in vivo infection focusing on multi-drug resistance and extensively drug-resistant strains. We observed through short course chemotherapy utilizing the nanoparticle-based drug delivery platform 20:80 CPTEG:CPH, we were able to reduce the prevalence of a persister bacterial population significantly. In summary, the studies described herein support the rational design and use of a polyanhydride nanoparticle drug delivery platform to reduce both the course of treatment and the amount of drug needed to treat neglected infectious disease effectively.</p

Effects of a postbiotic, with and without a saponin-based product, on turkey performance

ABSTRACT: Modern poultry production relies on an ability to prevent and mitigate challenges to bird health, while maintaining a productive bird. A number of different classes of biologics-based feed additives exist, and many have been tested individually for their impacts on poultry health and performance. Fewer studies have examined the combinations of different classes of products. In this study, we examined the use of a well-established postbiotic feed additive (Original XPC, Diamond V) on turkey performance, with and without the addition of a proprietary saponin-based feed additive. This was accomplished in an 18-wk pen trial utilizing 22 pen replicates per treatment across 3 treatments (control, postbiotic, and postbiotic plus saponin). Significant differences in body weight were identified at wk 12 and 15 of age, with the postbiotic plus saponin treatment group resulting in heavier birds at both timepoints. Significant differences in feed conversion ratio were observed from 0 to 18 wk of age, with the postbiotic alone having improved FCR compared with the control group. No significant differences were observed for livability or feed intake. This study demonstrates that a combination of a postbiotic plus saponin may exert additive effects on the growth of the turkey

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

A data analytics approach for rational design of nanomedicines with programmable drug release

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 between) polymer, drug, and nanoparticle properties gives rise to nonlinear behavioral relationships and a large data space. These factors complicate both first-principles modeling and screening of nanomedicine formulations. Predictive analytics may offer a more efficient approach toward 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. Non-linear 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 training data set. The models were shown to be robust after inclusion of these new formulations in that the new inclusions did not significantly change model regression. This approach provides the first steps towards 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.This document is the unedited Authors version of a Submitted Work that was subsequently accepted for publication in Molecular Pharmaceutics, copyright American Chemical Society after peer review. To access the final edited and published work see DOI: 10.1021/acs.molpharmaceut.8b01272. Posted with permission.</p

Panel A: Scanning electron photomicrograph of 20:80 CPTEG:CPH nanoparticles loaded with 5% IVM, 5% DOX, and 2% rhodamine.

<p>Scale bar: 500 nm. Panel B: Release kinetics of ivermectin (IVM) and doxycycline (DOX) from 20:80 CPTEG:CPH nanoparticles quantified using HPLC.</p

The motility of <i>B</i>. <i>malayi</i> MF treated with decreasing doses of either soluble or encapsulated ivermectin (IVM)/doxycycline (DOX) was recorded for 14 days post treatment.

<p>The recorded motility scores (Panel B) were used to calculate an average time to death for each treatment group and dose (Panel A). To calculate the average motility score for each time, dose and treatment group, triplicate wells containing a minimum of 200 MF in each well were treated as indicated. A motility score of 0 was equated with death and the average time to death was plotted along with standard error. Data presented are from one of two experiments with similar results. Significance was determined at p<0.05, 0.01, or 0.001 as noted using a Student’s T test.</p

Confocal microscopy of female <i>B</i>. <i>malayi</i> with nanoparticles.

<p>Worms were incubated for 96 h with either soluble controls (panel A) or 20:80 CPTEG:CPH nanoparticles containing ivermectin (IVM), doxycycline (DOX), and rhodamine B (panel B), fixed and imaged by LSCM. Controls contained 195 μM each of IVM and DOX, and 3.9 μM rhodamine B, while the nanoparticles contained 5 μM of each drug and 0.1 μM of rhodamine B. Left panels are DNA (blue), rhodamine (red) and bright field image overlays and right panels are the respective individual rhodamine images collected using identical image acquisition settings. Inset box within the nanoparticle-treated worm outlines the area selected for side view rendering (C). Representative images shown demonstrate the accumulation of nanoparticles within tissues throughout the worm (B) as compared to the higher amount of soluble rhodamine that was not detected within the body of the worms.</p

Panel A. Table outlining survival of <i>B</i>. <i>malayi</i> females after treatment with 195, 49, 10, 5, 1.95, or 0.049 μM concentrations of ivermectin (IVM) and doxycycline (DOX) delivered solubly or encapsulated into 20:80 CPTEG:CPH nanoparticles.

<p>Panel B. Average number of days to death of <i>B</i>. <i>malayi</i> females after administration of soluble or encapsulated IVM/DOX treatments and comparison to control worms. Significance was determined at p<0.05, 0.01, or 0.001 as noted using a Student’s T test. Panel C. Average motility scores of <i>B</i>. <i>malayi</i> females after IVM/DOX treatments scored using a 2X objective on a Nikon Microscope following a 0–5 scoring system, as described in the Methods. Panel D. Average number of microfilaria shed by <i>B</i>. <i>malayi</i> females after administration of soluble or encapsulated IVM/DOX treatments and comparison to control worms. The NP only group contains comparable amount of rhodamine and the total amount of particle in this group corresponds to that of the highest drug concentration of 195 μM. At 14 days, all worms treated with NP only with a motility score of 0 remained viable based on the MTT assay and recovery of motility upon transferring to fresh medium^†. Significance was determined at p<0.05, 0.01, or 0.001 as noted using a Student’s T test.</p