Delivery of LLKKK18 loaded into self-assembling hyaluronic acid nanogel for tuberculosis treatment João P. Silva a, ⁎, Carine Gonçalves b,c , César Costa a , Jeremy Sousa b,c , Rita Silva-Gomes b,c , António G. Castro b,c , Jorge Pedrosa b,c , Rui Appelberg d , F. Miguel Gama a, ⁎ a CEB - Centre of Biological Engineering, University of Minho, Campus de Gualtar, Braga 4710-057, Portugal b Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, Braga, Portugal c ICVS/3B's-PT Government Associate Laboratory, Braga/Guimarães, Portugal d Department of Immunophysiology, University of Porto, 4050-313 Porto, Portugal abstract article info Article history: Received 2 March 2016 Received in revised form 24 May 2016 Accepted 30 May 2016 Available online 01 June 2016 Tuberculosis (TB), a disease caused by the human pathogen Mycobacterium tuberculosis, recently joined HIV/AIDS on the top rank of deadliest infectious diseases. Low patient compliance due to the expensive, long-lasting and multi-drug standard therapies often results in treatment failure and emergence of multi-drug resistant strains. In this scope, antimicrobial peptides (AMPs) arise as promising candidates for TB treatment. Here we describe the ability of the exogenous AMP LLKKK18 to efficiently kill mycobacteria. The peptide's poten- tial was boosted by loading into self-assembling Hyaluronic Acid (HA) nanogels. These provide increased stabil- ity, reduced cytotoxicity and degradability, while potentiating peptide targeting to main sites of infection. The nanogels were effectively internalized by macrophages and the peptide presence and co-localization with mycobacteria within host cells was confirmed. This resulted in a significant reduction of the mycobacterial load in macrophages infected in vitro with the opportunistic M. avium or the pathogenic M. tuberculosis, an effect accompanied by lowered pro-inflammatory cytokine levels (IL-6 and TNF-α). Remarkably, intra-tracheal admin- istration of peptide-loaded nanogels significantly reduced infection levels in mice infected with M. avium or M. tuberculosis, after just 5 or 10 every other day administrations. Considering the reported low probability of resis- tance acquisition, these findings suggest a great potential of LLKKK18-loaded nanogels for TB therapeutics. © 2016 Elsevier B.V. All rights reserved. Keywords: Antimicrobial peptide Macrophages Infectious diseases Cathelicidin Mycobacteria 1. Introduction Tuberculosis (TB) is a life-threatening disease caused by Mycobacte- rium tuberculosis. The World Health Organization recently reported about 9.6 million TB cases in 2014, having its death toll increased to around 1.5 million people, which places TB alongside HIV/AIDS as the deadliest infectious diseases [1]. The emergence of multidrug-resistant TB (MDR-TB) strains at an es- timated rate of 3.3% is a further concern. MDR-TB may be mostly attrib- uted to the selection of drug resistance mutations resulting from overuse and/or misuse of antibiotics. In addition, it has been demon- strated that primary transmission of MDR strains plays an important role in MDR-TB epidemics [2]. Tuberculosis transmission occurs mostly via the respiratory tract [3]. Mycobacteria are then phagocytized by alveolar macrophages and quickly spread across the lungs by lymphatic circulation. Weeks later, bacilli may disseminate from the lungs to highly irrigated organs such as liver and kidneys [4,5]. Inside macrophages, mycobacteria are able to multiply within phagosomes, provided they arrest the phagosome maturation and its fusion with lysosomes, thereby avoiding contact with potent hydrolytic enzymes and antigen-presenting organelles present in the host macrophage [6,7]. TB control represents a major challenge. The only vaccine available, Bacille Calmette-Guérin (BCG), successfully prevents childhood TB but fails in protecting adults already infected or sensitized to mycobacteria [8]. Standard chemotherapy for drug-susceptible TB consists in the ad- ministration of a four antibiotic cocktail comprising isoniazid, rifampicin, pyrazinamide and ethambutol over a 6-month period. The presence of mycobacteria in stationary or low proliferating phases, as well as of mul- tidrug-resistant strains, requires a more prolonged treatment regimen, in- volving the administration of pyrazinamide in combination with second- line drugs (e.g. fluoroquinolones, ethionamide, cycloserine, capreomycin or prothionamide) that can last up to 24 months [9,10]. However, toxicity issues and elevated costs associated with these drugs, as well as low patient compliance to the long-lasting regimens, represent crucial drawbacks to the pharmacotherapy [11,12]. Journal of Controlled Release 235 (2016) 112–124 ⁎ Corresponding authors. E-mail addresses: jpsilva@deb.uminho.pt (J.P. Silva), fmgama@deb.uminho.pt (F.M. Gama). http://dx.doi.org/10.1016/j.jconrel.2016.05.064 0168-3659/© 2016 Elsevier B.V. All rights reserved. Contents lists available at ScienceDirect Journal of Controlled Release journal homepage: www.elsevier.com/locate/jconrel