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Nanoparticles Targeted Against Cryptococcal Pneumonia by Interactions Between Chitosan and Its Peptide Ligand Yixuan Tang, Shuang Wu, Jiaqi Lin, Liting Cheng, Jing Zhou, Jing Xie, Kexin Huang, Xiaoyou Wang, Yang Yu, Zhangbao Chen, guojian Liao, and Chong Li Nano Lett., Just Accepted Manuscript • DOI: 10.1021/acs.nanolett.8b02229 • Publication Date (Web): 27 Sep 2018 Downloaded from http://pubs.acs.org on September 27, 2018
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Nanoparticles Targeted Against Cryptococcal Pneumonia by Interactions Between Chitosan and Its Peptide Ligand Yixuan Tang,† Shuang Wu, † Jiaqi Lin,‡ Liting Cheng,† Jing Zhou,† Jing Xie,† Kexin Huang, † Xiaoyou Wang,† Yang Yu,† Zhangbao Chen,† Guojian Liao,† and Chong Li†*
†
Key Laboratory of Luminescent and Real-Time Analytical Chemistry (Southwest
University), Ministry of Education, College of Pharmaceutical Sciences, Southwest University, Chongqing 400715, P.R. China. ‡ David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA.
ABSTRACT: Inspired by the fact that chitosan is a representative constituent of the ectocellular structure of Cryptococcus neoformans, and a typical biomaterial for improving drug oral absorption, we designed an elegant and efficient C. neoformans-targeted drug delivery system via oral administration. A chitosan-binding peptide screened by phage display was used as the targeting moiety, followed by conjugation to the surface of poly(lactic-co-glycolic acid) nanoparticles as the drug carrier, which was then incubated with free chitosan. The non-covalently bound chitosan adheres to mucus layers and significantly enhances penetration of nanoparticles through the oral absorption barrier into circulation, and then, re-exposed the targeting ligand for 1
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later recognition of the fungal pathogen at the site of infection. After loading itraconazole as a model drug, our drug delivery system remarkably cleared lung infections of C. neoformans and increased survival of model mice. Currently, targeted drug delivery is mainly performed intravenously; however, the system described in our study may provide a universal means to facilitate drug targeting to specific tissues and disease sites by oral administration, and may be especially powerful in the fight against increasingly severe fungal infections. KEYWORDS: chitosan, targeted drug delivery, fungal infection, oral route, peptide ligand
Oral delivery is the most preferred method for drug administration because of various advantages over many other routes of drug delivery, including patient convenience and compliance, relatively low cost in manufacturing, and preserving process.1 However, oral administration of many existing drugs is beset by constraints such as poor physical/chemical characteristics of drug molecules and physiological barriers of the gastrointestinal tract with a harsh environment.2-6 To solve these challenges, functional biomaterials, as formulation ingredients, have played an increasingly important role in recent decades, among which chitosan has been widely explored.7-11 Chitosan, a naturally occurring polysaccharide mainly from crustaceans, can work as a carrier material to prevent drug inactivation and degradation caused by low pH and enzymes of the gastrointestinal tract, and more importantly, can regulate the intestinal barrier as a permeation enhancer.12-17 Chitosans have a special feature being capable of transiently 2
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opening tight junctions between epithelial cells while remaining mainly restricted in the mucosal surface for subsequent elimination, thereby providing a very promising material for efficient oral drug delivery.18-19 Currently, the rapidly increasing incidence of invasive fungal diseases poses a great challenge for human health, especially in developing countries. As an important example of human pathogenic fungi, Cryptococcus neoformans causes an estimated million cases of lethal cryptococcal pneumonia and/or central nervous system cryptococcosis, leading to an estimated annual mortality of 700 000 deaths worldwide.20-21 Even worse is that the very limited categories of existing corresponding therapeutics may not adapt to the rapid development of the disease, further confounded by a vicious circle in that a severely affected area often has limited access to advanced medications and hospitalization.22 Therefore, it is critically necessary not only to continuously develop new antifungal medications, but also pursue ongoing exploration to develop effective approaches or vehicles for drug delivery, thereby providing enhanced therapeutic efficacy, decreased side and toxic effects, improved compliance, as well as simple yet efficient therapy. As an infectious microorganism, a fungal cell is fundamentally different from a host human cell. Thus, the unique ectocellular structure of fungi may provide possible recognition sites for drug targeting. Interestingly, many fungi have chitosan or chitosan-like molecules in the cell wall as a dynamic component, reflecting the pathogenic state of the fungal cell.23-24 Moreover, for some fungi like C. neoformans, which possess capsules surrounding the cell body, chitosan is also intercalated within the elastic matrix network with variable size and porosity, suggesting chitosan is a promising general target for pathogenic fungal tracking and elimination.23, 25-27
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In the current study, we constructed and evaluated an efficient fungal-targeted drug delivery
system
against
cryptococcal
pneumonia
through
conjugation
of
a
chitosan-binding peptide (CP) screened by phage display as a targeting moiety to the surface of poly (lactic-co-glycolic acid) (PLGA) nanoparticles (CP-NPs) (Scheme 1A). We hypothesized that if CP-NPs were pre-incubated with free chitosan in vitro, the formed chitosan-bound nanoparticles (C-CP-NPs) will have a two-step targeting procedure via the oral route (Scheme 1B) specifically: (i) the non-covalently bound chitosan will significantly enhance NP permeability across the oral absorption barrier, and (ii) the bound chitosan will mainly adhere to the mucosal layer, releasing the CP-NPs into circulation for further accumulation at the infection site.
The 12-mer peptide (ADGVGDAESRTR) was obtained by screening random peptide libraries, and binding affinity between CP and chitosan was determined by surface plasmon resonance (SPR). Our findings show that CP is a specific, avid ligand of chitosan with a binding constant at 5.27 × 10-8 M (KD) (Figure 1A), while a scrambled peptide served as a negative control (Figure S1). Further investigation through molecular dynamic simulations indicates that the peptide favorably interacts with chitosan. There is a significant hydrogen bond originating from an attraction between the amine group on the chitosan and the carboxylate group on the peptide; hydrogen bonding also occurs between the amide group on the peptide backbone and the hydroxyl group on the chitosan as well as between two hydroxyl groups on both molecules (Figure 1B and 1C and Figure S2). Radial distribution functions (RDFs) further indicate the distance between the two molecules in terms of peptide residue specificity (Figure S3). Positively charged
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residues (Arg) are relatively distant from the chitosan chain, while negatively charged residues (Asp and Glu) are mostly in the vicinity of the chitosan (
