Contents lists available at ScienceDirect International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm Alginate lyase immobilized chitosan nanoparticles of ciprofoxacin for the improved antimicrobial activity against the bioflm associated mucoid P. aeruginosa infection in cystic fbrosis Krishna Kumar Patel a , Muktanand Tripathi b , Nidhi Pandey b , Ashish Kumar Agrawal a , Shilpkala Gade a , Md. Meraj Anjum a , Ragini Tilak b , Sanjay Singh a, ⁎ a Department of Pharmaceutical Engineering and Technology, Indian Institute of Technology (IIT-BHU), Varanasi, India b Department of Microbiology, Institute of Medical Sciences, Banaras Hindu University, Varanasi, India ARTICLEINFO Keywords: Bioflm Chitosan Alginate lyase Mucoid P. aeruginosa Cystic fbrosis ABSTRACT Dense colonization of mucoid Pseudomonas aeruginosa within the self-secreted extracellular matrix (mainly al- ginate), called bioflm, is a principal reason for the failure of antimicrobial therapy in cystic fbrotic patients. Alginate is a key component in the bioflm of mucoid P. aeruginosa and responsible for surface adhesion and stabilization of bioflm. To overcome this problem, alginate lyase functionalized chitosan nanoparticles of ci- profoxacin were developed for the efective treatment of P. aeruginosa infection in cystic fbrosis patients. The developed nanoparticles were found to have desired quality attributes and demonstrated sustained release fol- lowing the Higuchi release kinetics. Drug compatibility with the chitosan was confrmed by FTIR while powder X-ray difraction analysis confrmed the entrapment of drug within the nanoparticle matrix. Lactose adsorbed NPs showed promising aerodynamic property. Nanoparticles showed prolonged MIC and signifcant reduction in bioflm aggregation and formation in planktonic bacterial suspension. Nanoparticles exhibited signifcantly higher inhibitory efect against bioflm of P. aeruginosa and reduced the biomass, thickness and density con- frmed by confocal microscopy. Furthermore, developed nanoparticles were haemocompatible and did not ex- hibit any toxicity in vitro MTT assay and in vivo on lungs male Wistar rats. The data in hand collectively suggest the proposed strategy a better alternative for the efective treatment of cystic fbrosis infections. 1. Introduction Cystic fbrosis (CF) is a life threatening disease caused by mutation in the cystic fbrosis transmembrane conductance regulator (CFTR) gene responsible for membrane transport of anions and mucociliary airway clearance. Mutation to CFTR gene leads to compromised mu- cociliary clearance and thereby increases mucus retention, suscept- ibility for persistent microbial infection, exaggerates pulmonary in- fammatory response and progressive airways obstruction leading to complete respiratory collapse. Moreover, it may even may cause exo- crine pancreatic insufciency, biliary disease and metabolic disorder (Flume et al., 2010; Accurso et al., 2005). Among all the bacterium, Pseudomonas aeruginosa is an opportunistic pathogen which afects the majority of CF patients leading to the complication and chronicity of disease by developing the bioflm colonies (Owlia et al., 2014; Hurley et al., 2012). P. aeruginosa invades the lung epithelia and shows the genetic transformation from non-alginate producing (nonmucoid) to alginate producing strain (mucoid). It supports higher bacterial ad- herence in lung mucosa and immune escape (Alipour et al., 2009; Tré- Hardy et al., 2008; Horsman et al., 2012; Sarkisova et al., 2005). Bioflms are mainly comprised of microbial dense colonies protected by the extracellular polymeric matrix. In response to bioflm, most of the antimicrobial therapy fails to eradicate the infection and impose greater tolerance due to impaired penetration in bioflm, protection against the host innate immunity, hydrolytic enzymes, metallic cations, oxidative stress, expression of bioflm-specifc efux pump and also the https://doi.org/10.1016/j.ijpharm.2019.03.051 Received 13 December 2018; Received in revised form 19 March 2019; Accepted 25 March 2019 Abbreviations: CIPR, ciprofoxacin; CH, chitosan; AgLase, alginate lyase; NPs, nanoparticles; CIPR-CH-NPs, ciprofoxacin loaded chitosan nanoparticles; AgLase- CIPR-CH-NPs, alginate lyase functionalized chitosan nanoparticles of ciprofoxacin; PS, particle size; PDI, polydispersity index; ZP, zeta potential; EE, entrapment efciency; MIC, minimum inhibitory concentration; MBIC, minimum bioflm inhibitory concentration; MBEC, minimum bioflm eradication concentration; FT-IR, Fourier transformation infra-red spectroscopy; DSC, diferential scanning calorimetry; XRD, X-ray difraction ⁎ Corresponding author. E-mail address: ssingh.phe@iitbhu.ac.in (S. Singh). International Journal of Pharmaceutics 563 (2019) 30–42 Available online 26 March 2019 0378-5173/ © 2019 Published by Elsevier B.V. T