RESEARCH ARTICLE – Pharmaceutical Nanotechnology Controlled Production of Poly (3-Hydroxybutyrate-co-3-Hydroxyhexanoate) (PHBHHx) Nanoparticles for Targeted and Sustained Drug Delivery THOMAS R. J. HEATHMAN, 1 WILLIAM R. WEBB, 1 JIANFENG HAN, 2 ZHENG DAN, 2 GUO QIANG CHEN, 3 NICHOLAS R. FORSYTH, 1 ALICIA J. EL HAJ, 1 ZHIRONG R. ZHANG, 2 XUN SUN 2 1 Guy Hilton Research Centre, Institute of Science and Technology in Medicine, Keele University, Stoke-on-Trent ST4 7QB, UK 2 Key Laboratory of Drug Targeting and Drug Delivery Systems, Ministry of Education, West China School of Pharmacy, Sichuan University, Chengdu 610041, China 3 Department of Biological Sciences and Biotechnology, School of Life Sciences, Tsinghua University, Beijing 100084, China Received 19 August 2013; revised 25 March 2014; accepted 7 May 2014 Published online in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.24035 ABSTRACT: The ability to control the size and quality of nanoparticles (NPs) during production is critical for their success as a commercial product for clinical applications. Here, we employed a statistical design of experiment approach to identify the key process variables affecting the size of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) NPs during production via the solvent evaporation method. The number of sonication cycles had a standardzed effect on NP size of 55, with sonication power at 25, and PHBHHx concentration at 27 with a combination of these variables having a lower yet significant effect on NP size (p < 0.05). The PHBHHx NPs were stable for at least 7 days with an average polydispersity index of 0.18, a zeta potential of −10 to −40 mV, and an encapsulation efficiency of 63.5 ± 2%. These data were utilized to produce a prediction graph whereby particles could be produced with sizes ranging from 90 to 205 nm with a low mean curve prediction error of 1.96% for Haperzine-A-loaded NPs. Furthermore, a range of drug encapsulates NPs were produced and showed a sustained release of the encapsulated drug. This study demonstrates the ability to control the size of drug-loaded particles by manipulation of the production variables, which will allow targeted and controlled drug release to fit a variety of applications. C  2014 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci Keywords: nanoparticle; biomaterials; drug delivery systems; nanotechnology; polymeric drug carrier; DOE; polyhydroxyalkanoates; drug release; controlled production INTRODUCTION Nanotechnology has quickly evolved because of its ability to develop structures that can serve as controlled delivery sys- tems for drugs, genes, proteins, or as combination products, and compensate the shortcomings of these therapeutic agents to play multiple functions, including effective treatment of var- ious diseases such as destruction of neoplastic tissue, 1–4 and mimicking the dynamic biological microenvironment, combin- ing cells, scaffolds, and drug releasing nanoparticles (NPs) to efficiently regenerate functional tissue. 5–7 Biocompatibility is a key factor in the use of biomaterials for clinical products, where the coexistence of biomaterials and tissues can occur without causing unacceptable harm to the body. 8 Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHB- HHx) consists of random copolymers of 3-hydroxybutyrate and 3-hydroxyhexanoate 9 and is one of the few polyhydroxyalka- noates that can be manufactured on a sufficiently large scale at relatively low cost for use in scientific research and the biomed- Correspondence to: Xun Sun (Telephone: +86-28-85502307; Fax: +86- 28-85501615; E-mail: xunsun22@gmail.com) Correspondence to: Nicholas R. Forsyth (Telephone: +44-1782-555261; Fax: +44-1782-747319; E-mail: n.r.forsyth@pmed.keele.ac.uk) Thomas R. J. Heathman and William R. Webb contributed equally to this work. Journal of Pharmaceutical Sciences C  2014 Wiley Periodicals, Inc. and the American Pharmacists Association ical industry. 10 The adaptable mechanical properties, biocom- patibility, and biodegradability of PHBHHx enable the poly- mer to meet diverse biomedical requirements and has been used to construct tissue engineering scaffolds 11–20 as well as combining with different mesenchyme-derived cell types and as a drug delivery device. 21–24 Research conducted by Wang et al. 25 has previously shown PHBHHx containing between 10% and 15% HHx component to be less brittle than those PHBHHx polymers HHx component of less than 10% HHx. However, the reduction in molecular weight (MW) of the poly- mer employed will have an impact on the polymer physical properties. Nanoparticles have been shown to have an array of appli- cations ranging from nanosensing, drug delivery, gene trans- fection, and growth factor delivery. 26–31 The ability to manipu- late NP size in order to fit the needs of a desired application would provide increased utility to treat a range of diseases, including cardiovascular disease, 32 osteoarthritis, 33 diabetes, 34 cancer, 32,35–38 and neurodegenerative disease. 39 Recently, NPs have been shown to pass across the blood–brain barrier, which is a major obstacle when delivering chemotherapeutical drugs for the treatment of nervous system tumors 40 as well as neu- rodegenerative diseases. For controlled drug release, a spec- ified drug release profile could be achieved via a controlled manufacturing process. This would lead to improved treat- ment methods by overcoming drug metabolism and loss via Heathman et al., JOURNAL OF PHARMACEUTICAL SCIENCES 1