Recipient Organization
VIRGINIA POLYTECHNIC INSTITUTE
(N/A)
BLACKSBURG,VA 24061
Performing Department
College of Vet Medicine
Non Technical Summary
Brucella spp. causes the most common zoonotic infections in human (500,000 new cases/year) resulting in a chronic debilitating disease. Successful treatment requires a daily dose of combinatorial antibiotic therapy (tetracycline + aminoglycoside or rifampin) for 6 to 8 weeks. Conventional free drugs lack ready access to intracellular bacterium, combined with patient incompliance to complete the therapeutic regime result in failure of complete cure and in some cases relapse (relapse rate of 15%). Additionally, sub-therapeutic doses of free drugs potentiate the development of drug resistant bacteria. Using nanomaterials such as Magnetic Block Ionomer Complexes (MBICs) for drug delivery reduces considerably the dose (mg/kg of body weight) of antibiotics needed for treatment and drug associated toxicity issues. Peptide nucleic acids (PNA) employ anti-sense mechanisms to inhibit transcription and translation of specific bacterial genes that are targeted and are proposed as alternatives for antibiotics. PNAs designed to target genes involved in bacterial DNA, RNA and protein synthesis machinery are shown to possess exceptional growth inhibitory properties. Anti-sense technologies could be used to kill pathogens that are resistant to conventional drugs by targeting essential genes that are required for the pathogen for its survival inside the host. Using liposomes for packaging PNA enhances the therapeutic efficacy by avoiding off-target effects. Additionally, using nanoparticles like MBICs and liposomes allow delivery of therapeutics at the site of action, i.e., the intra-cellular phagosomes of phagocytes where Brucella resides. Here, we propose to use a combination of gentamicin loaded MBICs along with liposome encapsulated PNAs for treating acute brucella infection in mice. The results would potentially help to develop a novel effective therapeutic regime for treating pathogens that cause chronic infections and are difficult to treat (Ex. Brucella spp., Mycobacterium tuberculosis).
Animal Health Component
(N/A)
Research Effort Categories
Basic
(N/A)
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
SPECIFIC AIMS: The overall goal is to develop a novel therapeutic regimen for brucellosis in animals and humans in order to minimize the amount of conventional antibiotics required to cure. The primary objective of this study is to evaluate the therapeutic efficacy of a combinatorial regime of peptide nucleic acid (PNA) and drug-laden nanoparticles (MBICs) - in treating acute Brucella suis infections in a murine model. The working hypothesis is "a combinatorial regime of liposome encapsulated PNA and gentamicin loaded MBICs can be used to effectively treat acute brucellosis with no toxicity". Aim-1. Assess concurrent use of PNA encapsulated in liposomes + drug-laden MBICs ability to significantly reduce the bacterial load in a mouse model of brucellosis. Aim-2. Assess the protection provided by combination of PNA encapsulated in liposomes + drug-laden MBICs and compare them to the conventional combination of free antibiotics (doxycycline + gentamicin).
Project Methods
EXPERIMENTAL DESIGN AND METHODS: Handling and manipulation of Brucella suis culture and infected mice will be done inside our CDC approved BSL-3 and ABSL- 3 facilities respectively. All work involving mice manipulation will be done in compliance with Institutional Animal Care and Use Committee of Virginia Tech guidelines. Aim 1: Procedures: B. suis from our stock culture collection will be grown in tryptic soy broth (TSB) or agar (TSA) at 37C with 5 percent CO2. A dose of 5X104 CFU/mouse in a 100ul inoculum will be administered via intra-peritoneal route. Custom made PNA specific for dnaG gene of B. suis will be acquired commercially from PANAGENE Inc., Korea. Encapsulation of PNA inside liposomes will be done by Encapsula nanosciences LLC., Nashville, TN. Gentamicin loading of MBICs will be done by our on-campus collaborator Dr. Judy Riffle. The drug-laden nanoparticle will be received as a ready to inject suspension and will be used at 20 mg/Kg of body weight per mouse. Commercially available Doxycycline (Sigma) and Gentamicin (Sigma) will be used as controls. A total of 100 female Balb/C mice of 3 - 4 weeks old will be received from a commercial vendor and will be allowed to acclimatize for 2 weeks in our colleges ABSL-3 facility. At the start of the experiment, all the 100 mice will be infected with B. suis via intra-peritoneal route. The mice will be divided into 4 groups (25mice/group) based on the treatment they will receive. First group will receive PBS (control group), second group will receive a combination of Doxycycline (40 mg/kg) and gentamicin (25mg/kg), third group will receive a combination of PNA (5mg/kg) encapsulated in liposomes and Gentamicin loaded MBICs (25 mg/kg) and the fourth group will receive a combination of liposomes (with no PNA encapsulation) and MBICs with no gentamicin as a vehicle control. At one-week (day 7) post infection, 5 mice from each group (total 20 mice) will receive the designated treatments via intra-peritoneal route. At 24h post treatment (day 8), all the four groups of mice will be euthanized and their liver and spleen will be plated on a TSA plate to determine the bacterial load. Likewise, on days 8, 9, 10 and 11 post infection 5 mice from each group will receive their respective treatments and their spleen and liver will be processed as above. The bacterial counts and their statistical significance will be determined using ANOVA and tukeys method. Aim 2: Procedures: The group of mice treated with conventional drugs ((Doxycycline + Gentamicin) in parallel would serve as a control and hallmark for protection. The schedule of closely monitoring the changes in vital organ bacterial load for 5 consecutive days is expected to provide accurate information about the ability of the novel therapeutic regime to restrict bacterial growth in an acute infection. The concurrent quantification of bacterial load after each dose (day 1 group of mice would have received 1 dose, day 2 group of mice would have received 2 doses and so on) would also allow us to determine the effective dose and frequency.