Controlled Production of Poly (3-Hydroxybutyrate-co-3-Hydroxyhexanoate) (PHBHHx) Nanoparticles for Targeted and Sustained Drug Delivery

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 SUN2 1

Guy Hilton Research Centre, Institute of Science and Technology in Medicine, Keele University, Stoke-on-Trent ST4 7QB, UK 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 2

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 systems for drugs, genes, proteins, or as combination products, and compensate the shortcomings of these therapeutic agents to play multiple functions, including effective treatment of various diseases such as destruction of neoplastic tissue,1–4 and mimicking the dynamic biological microenvironment, combining 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) (PHBHHx) consists of random copolymers of 3-hydroxybutyrate and 3-hydroxyhexanoate9 and is one of the few polyhydroxyalkanoates that can be manufactured on a sufficiently large scale at relatively low cost for use in scientific research and the biomedCorrespondence to: Xun Sun (Telephone: +86-28-85502307; Fax: +8628-85501615; E-mail: [email protected]) Correspondence to: Nicholas R. Forsyth (Telephone: +44-1782-555261; Fax: +44-1782-747319; E-mail: [email protected]) 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, biocompatibility, and biodegradability of PHBHHx enable the polymer to meet diverse biomedical requirements and has been used to construct tissue engineering scaffolds11–20 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 polymer employed will have an impact on the polymer physical properties. Nanoparticles have been shown to have an array of applications ranging from nanosensing, drug delivery, gene transfection, and growth factor delivery.26–31 The ability to manipulate 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 tumors40 as well as neurodegenerative diseases. For controlled drug release, a specified drug release profile could be achieved via a controlled manufacturing process. This would lead to improved treatment methods by overcoming drug metabolism and loss via Heathman et al., JOURNAL OF PHARMACEUTICAL SCIENCES

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Table 1.

DOE High, Middle, and Low Settings for Process Variables

Multi Factorial Value High Middle Low

PHBHHx Concentration (mg/mL)

Sonication Power (W)

Number of Sonication Cycles (n)

2.000 1.125 0.250

500 300 100

40 25 10

administration route (i.e., the digestive tract) as the NP would serve to protect the encapsulated drug allowing for reduced drug dosage needed to meet efficacious concentration in body. Nanoparticle drug encapsulation coupled with targeting strategies may reduce circulating free agents, which may reduce harmful side effects currently observed with chemotherapeutic agents used to treat malignant diseases. Thus, an immediate benefit of improved delivery would be in cost reduction during production. More importantly, limiting the administered dose would potentially reduce the risk of systemic toxicity and nonspecific side effects in the patient and could be combined with targeted drug release systems41–45 for advanced drug delivery. The ability to control the size of NPs during production would allow reliable and reproducible manufacture of smaller NPs for applications such as NP-mediated gene transfection. This holds much promise in regenerative medicine as nonviral gene vectors present a significantly reduced safety hazard compared with the viral-based gene vectors.35 The NP-mediated cellular response is crucially important for successful therapies, and particles below 100 nm would actively mediate biological effects such as the binding and activation of membrane receptors and subsequent protein expression,46 a key aspect within a variety of NP applications. The advantages of smaller particles in clinical practice include easier intravenous injection, sterilization by filtration,47,48 and more importantly, the opportunity to avoid clearance from reticuloendothelial system49 as well as the elimination by spleen filtration,50–52 which is critical for managing the in vivo fate of drug for advanced therapeutic performance. Isolating the key production variables affecting the size of NPs would allow for more effective fabrication of smaller NPs and promote effective utilization of their biological and practical benefits. In this work, the controlled production of PHBHHx NPs was investigated via the design of experiment (DOE) method to determine the sensitivity of process variables and the repeatability of producing PHBHHx NPs of a desired size. The DOE approach uses parallel multivariate designed experiments to investigate the action and interaction of variables to improve understanding, and therefore control of complex processes.53 A key aspect of this is identifying controlled and uncontrolled variables, which affect the response from the process, with preliminary studies aimed at highlighting which input variables are affecting the desired process output. Heparzine-Aencapsulated NPs showed conformity to this trend with high process repeatability, combined with proof of principle using paclitaxel and phospholipid–albumin-encapsulated NPs. This study shows that controlled manufacture of PHBHHx NPs via the solvent evaporation method is achievable and has been utilized to produce NPs of below 100 nm. Demonstrating this level of controlled manufacture is essential for developing products in the tightly regulated pharmaceutical industry where precision manufacture is essential for successful commercialization. Heathman et al., JOURNAL OF PHARMACEUTICAL SCIENCES

Figure 1. Cube plot illustration of multifactoral DOE.

MATERIALS AND METHODS Materials Phospholipid, soybean lecithin containing 70%–97% phosphatidylcholine, was supplied by Shanghai Tai-wei Pharmaceutical Company Ltd. (Shanghai, China). Bovine serum albumin (BSA) Fraction V was supplied from Xuzhou Wanbang Bio-Chemical Company Ltd. (Jiangsu, China). Poloxamer188 (F68) was provided by Nanjing Well Chemical Company, Ltd. (Nanjing, China). Sodium deoxycholate (DOC-Na) was supplied by Amresco (Solon, Ohio). PHBHHx (MW 174,000 Da) containing 13.86 mol % (MW 24,116 Da) of R-3-hydroxyhexanoate (HHx) was kindly donated by Professor G. Chen (Tsinghua University, China). All other chemical reagents were of analytical grade or better.

Multifactorial Experimental Design Minitab (16.2.2) was used to design and evaluate DOE utilizing a full factorial with a Yates standard order the number of replicates required was generated using Minitab composed of three factors at two levels yielding eight experimental runs including centerpoint bringing the total number of experimental runs to nine, which produced graphical outputs of the data. Three key factors were assessed, namely, PHBHHx concentration, sonication power, and number of sonication cycles at high and low conditions to enclose the experimental conditions (Table 1). This gave a total of eight experimental conditions (cube plot) with a centerpoint to complete the experimental design (Fig. 1). An N = 3 was taken for each run to account for variability in the measurement system, and a total of three experimental runs were completed to account for batch-to-batch variability, an assessment of common-cause variation within NP production, providing a preliminary indication to the repeatability of the process. R

R

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RESEARCH ARTICLE – Pharmaceutical Nanotechnology

Gel Permeation Chromatography Three milligrams of PHBHHx was dissolved in chromatography grade Chloroform (Avantor, PA, USA) at a ratio of 20% (w/v) and then filtered using 0.22 :m organic solution filters (Membrana, Obernburg, Germany) prior to gel permeation chromatography (GPC) injection. MWs were studied using a GPC (RID-10A; Shimadzu, Koyoto, Japan). The measurement were performed at 40◦ C using a GPC-804C column (Shimadzu, Koyoto, Japan) with all samples referenced against 10% (w/v) chloroform solution of high-MW polystyrene standard (MW 300,000 and 150,000 Da) (Fluka, Milano, Italy, Sigma–Aldrich, St. Louis, MO, USA) and low-MW polystyrene standard (MW 50,000, 30,000, 9000, and 4000 Da) (Shimadzu). Blank PHBHHx Nanoparticle Production PHBHHx–chloroform solutions with a range of concentrations were prepared at a volume of 2 mL (organic phase). 0.5% (w/v) of F68 (150 mg; Nanjing Well Chemical Company) and 0.5% (w/v) of DOC-Na (150 mg; Amresco) were prepared in a volume of 28 mL of 0.22 :m filtered water (aqueous phase). Once both the organic and aqueous phases were dissolved, the aqueous phase was filtered and added dropwise to the organic phase. The mixture was then sonicated (Scientz JY92 – II) for various cycles while on ice (sonication cycle was 1 s sonication at a range of power settings followed by 1 s rest). The NP emulsion was then placed on a rotary evaporator (Buchi Rotavapor R-3) set at 30◦ C until all the organic phase was evaporated (emulsion becomes transparent). Preparation of Phospholipid–Albumin Complexes Albumin-phospholipid complex (ALB–PLC) complexes were prepared using a method adapted from a previous study.54 Albumin and phospholipid at molar ratios of 1:80, 1:100, and 1:120 were dissolved in dimethyl sulfoxide containing 5% (v/v) acetic acid and magnetically stirred for 24 h at 30◦ C. The solution was lyophilized for 24 h to remove the solvent, sealed, and stored at 4◦ C.

then placed on a rotary evaporator (Buchi Rotavapor R-3) set at 30◦ C until all the organic phase was evaporated (emulsion becomes transparent). Nanoparticle Characterization of Size, Zeta Potential, and Polydispersity Index The mean particle size, polydispersity index (PDI), and zeta potential of the resulting PHBHHx NPs were characterized using dynamic light scattering and electrophoretic light scattering technology, respectively, with a Zetasizer Nano ZS90 instrument (Malvern Instruments Ltd., Malvern, U.K.), using water (dH2 O) as a dispersant at 25◦ C with each cycle of the measurement automatically determined by the instrument system. The particle size was displayed by intensity distribution, and the size distribution was evaluated by PDI. In Vitro Stability Study Nanoparticles and ALB–PLC NPs were produced as described above, and the size as well as the zeta-potential were measured at the indicated time points (0, 12, 24, 48, 96, and 168 h) from samples stored at 4◦ C or room temperature, respectively. Entrapment Efficiency The entrapment efficiency (EE) of Heparzine-A-loaded NPs was measured based on a modified centrifugation method.55 Drug was extracted with 5% (v/v) of Triton X-100 under sonication, passed through a 0.22 :m filter and measured by mass spectroscopy to determine the total amount of drug (Xt ). In addition, the same volume of drug-encapsulated NPs was taken and 10% acetic acid in dH2 O (v/v) was added dropwise to neutralize the surface charge of the NPs detected via zeta potential. The resulting solution was placed into a centrifuge filter (100,000 MW cutoff; Millipore) and centrifuged until all of the aqueous solution containing the free drug of interest had passed through the filter (Xf ). The EE was then calculated using the following equation:  EE =

Preparation of Drug-Loaded PHBHHx Nanoparticles PHBHHx NPs were prepared with adjustments from a previous study.34 0.5% (1 mg) of ALB–PLC or drug and 3% (7.5 mg) PHBHHx (w/v) chloroform solution were prepared at a volume of 2 mL (organic phase); 150 mg F68 (Nanjing Well Chemical Company) and 150 mg DOC-Na (Amresco) were prepared at a volume of 28 mL of 0.22 :m filtered water (aqueous phase). Once both the organic and aqueous phases were dissolved, the aqueous phase was filtered and added dropwise to the organic phase. The mixture were then sonicated (Scientz JY92 – II) for various cycles while on ice (sonication cycle was 1 s sonication at a range of power settings and 1 s rest). The NP emulsion was

3

Xt − Xf Xt

 × 100

Haperzine-A Release Profile Release profile analysis was undertaken on four samples produced utilizing the parameters shown in Table 2. Briefly, 2 mL of Haperzine-A-loaded NP was placed into a dialysis membrane with a MW cutoff of between 8000 and 14,000 Da (Sigma). The dialysis membrane was then immersed in 10 mL of PBS and mechanically stirred (100 rpm) at a temperature of 37◦ C.34 At predetermined time points over a 14-day period, 200 :L of PBS was removed and replaced by fresh PBS. The 200 :L of removed PBS containing released Haperzine-A

Table 2. Haperzine-A PHBHHx Nanoparticle Properties Utilizing two Different Sets of Parameters (PHBHHx Concentration, Sonication Power, and Sonication Cycles) Sample

PHBHHx (mg/mL), Power(W) Cycles (C)

1 2 3 4

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0.25, 500, 30 0.25, 100, 10 2, 500, 30 2, 100, 10

Size (nm)

PDI

Zeta Potential (mV)

Encapsulation Efficiency (%)

± ± ± ±

0.11 ± 0.03 0.17 ± 0.04 0.07 ± 0.02 0.44 ± 0.06

−17.83 ± 1.39 −21.18 ± 3.08 −23.37 ± 1.14 −32.10 ± 2.38

60.9 ± 4.9 75.5 ± 4.5 22.8 ± 5.4 21.4 ± 7.3

108.33 119.93 134.77 183.27

3.69 13.02 7.56 5.85

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was then analyzed using liquid chromatography–tandem mass spectrometry (LC–MS/MS). LC–MS/MS Detection of Paclitaxel and Haperzine-A The LC–MS/MS system consisted of an Agilent 1200 series RRLC, which includes an SL auto-sampler, degasser, SL binary pump and an Agilent triple-quadrupole MS (Agilent). The system was controlled with B01.03 software for qualitative analysis and B01.04 software for quantification. Separation was performed in a Diamonsil ODS column (50 × 3.5 mm2 , 3.4 :m) with a corresponding guard column (ODS, 5 :m). The column was maintained at 30◦ C and the injection volume was 1 :L. The mass spectrometer was operated using an electrospray source configured to positive ion mode and the quantification analysis was performed using multiple reaction monitoring. [M+H] of each analyte was selected as the precursor ion. Instrumental

Figure 2. Pareto chart of standardized effects for the size production of PHBHHx NPs.

parameters were as follow: gas temperature: 350◦ C; with a gas flow: 8 mL/min; nebulizer: 30 psi; capillary: 4000 V. Gas chromatographic separation of Haperzine-A was performed using a mobile phase 83%:17% H2 O and MeOH containing 0.1% (v/v) formic acid with a flow rate of 0.4 mL/min. Mass spectrometry detection using MW from the positive molecular ion mode [M+H]+ m/z 243–226; Dwell: 300; fragmentor: 185; collision energy: 4; polarity: positive. Statistical Analysis Results were deemed to be significant if p < 0.05 using a twotailed Students t-test.

RESULTS Statistical DOE Confirmation of PHBHHx and the HHx MW was undertaken prior to the undertaking of the DOE. The MW of PHBHHx was confirmed to be 174,000 Da with the HHx component having a MW of 24,116 Da by GPC analysis. Design of experiment was utilized to find the production parameters (PHBHHx concentration, sonication power, and number of sonication cycles) with the greatest effect on NP size, PDI, and zeta potential to control the size of NPs during production. Lower and upper limits for the DOE were established in an earlier study conducted within the laboratory utilizing individual parameter analysis (data not shown). The effect of the number of sonication cycles on NP size was found to be at least twofold over the other production parameters (Fig. 2), although all production variables had a significant effect (p < 0.05) on NP size. PHBHHx concentration and sonication power had a standardized effect of 25–30 (p < 0.05) on the size of NPs. To produce the smallest NP diameter (90–100 nm), 40 sonication cycles, a sonication power of 500 W, and 0.25 mg/mL PHBHHx concentration (Fig. 3a) was required. The number of sonication cycles had the greatest effect on PDI (0.14–0.18), with the lowest PDI of 0.14 produced at 40 sonication cycles (Fig. 3b). It

Figure 3. Interaction plots. (a) Mean PHBHHx NP size and (b) mean PDI. Heathman et al., JOURNAL OF PHARMACEUTICAL SCIENCES

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RESEARCH ARTICLE – Pharmaceutical Nanotechnology

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Figure 4. Blank PHBHHx NP size prediction curves.

is important to note the curved response in Figure 3, where the centerpoint does not lie directly between the high and low value. This gives a clear indication that the values chosen are converging on optimum for the given response with the limited number of repeat runs, which is critical for a high level of process understanding.56 Controlled Nanoparticle Size Production A size range of NPs was produced by varying the number of sonication cycles whilst maintaining the same PHBHHx concentration at high and low sonication power. This process was first completed with blank NPs in order to obtain a base curve for NP size production over two batches (to account for the associated common cause variability). From Figure 4, it can be seen that this process produced a NP range of 90–215 nm with zeta potential (−35.2 ± 7.79) and PDI (0.168 ± 0.029) within accepted limits.57 This produced two curves at sonication power of 100 and 500 W that can be used to produce NPs of a desired size with R2 = 0.9948 and 0.9955, respectively (Fig. 4). The data obtained from the three independent runs were then analyzed using Minitab and utilizing the capability sixpack analysis (Fig. 5). This analysis showed a CpK index of 4.13, which is well above 1 and the capability histogram showing normal distribution between the lower and upper limits and the normal probability plot following a straight line (Fig. 5). These combined observations show the DOE can be utilized to enable reliable production of NPs of a given size as the process variables in this preliminary study are far narrower than the specification tolerance. Following the evaluation of blank NPs, the process was repeated with Heparzine-A-encapsulated NPs to see if the same rule applied to this complex enabling size evaluation with blank NPs and the results extrapolated for drug-encapsulated NPs. Paclitaxel and ALB–PLC-encapsulated NPs were also compared to determine the proof of concept for other encapsulated drugs using this process method (Table 3). From Figure 6, it can be seen that these encapsulated drugs conformed to the same production size curve as the blank NPs, meaning that the original curve can be used to select a desired NP size, for a specified application. The accuracy of this relationship has also been evaluated in Table 2, which shows that the overall (mean) prediction error from blank to Heparzine-A-encapsulated NPs R

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Figure 5. Capability six-pack output from Minitab 14R showing capability histogram, normal probability plot, and capability plot showing Cpk = 4.13.

as 2.70 ± 2.84 nm, which equates to 1.96 ± 2.06%, with a prediction error range of <1.0–7.88 nm or <1.0%–6.35% for the three drugs, compared with blank NPs. Haperzine-A Release Profile for Sustained Release A range of production settings was utilized to undertake the release profile study (Table 4). The release profile study showed a sustained release of Haperzine-A over 14 days, when compared with the control (free-drug comparison) (Table 5 and Fig. 7). Loading concentrations for the Samples 1 and 2 were calculated to be 412 ng/mL with Samples 3 and 4 having a loading concentration of 3333 ng/mL, which gave Samples 3 and 4 and eightfold larger burst release. This clearly shows that controlling the size of NP can have a profound effect on not only release profiles but on providing a desired burst release. Particular attention in future studies should undertake further release studies should be conducted over a longer period of time. Characterization of Drug-Loaded Nanoparticles A blank NP stability study was conducted over 168 h (7 days) to assess the change in size, PDI, and zeta potential of the NPs (Figs. 8a–8c). The NP size was stable for the full 168 h at both high and low sonication settings over the range of NP sizes. There was also no difference to NP size stability when stored at room temperature compared with 4◦ C. The PDI of the blank NPs showed a slight fluctuation of ±0.1 over the 168-h period, resulting in a slightly elevated PDI to a maximum of 0.25 after 168 h (Fig. 8b), but was still below the 0.3 benchmark58,59 Heathman et al., JOURNAL OF PHARMACEUTICAL SCIENCES

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Table 3.

Haperzine-A Nanoparticle Properties

Drug-Loaded NPs Haperzine-A (1:60) Haperzine-A (1:90) Haperzine-A (1:120)

Size (nm)

PDI

Zeta Potential (mV)

EE (%)

95.2 ± 0.73 94.2 ± 1.46 93.3 ± 1.46

0.128 ± 0.007 0.146 ± 0.012 0.137 ± 0.013

− 12.2 ± 0.35 − 13.8 ± 1.85 − 15.33 ± 1.10

49.98 ± 1.50 48.81 ± 1.69 63.50 ± 1.57

figure for in vivo drug delivery applications. There were minor differences between the NPs PDI after 168-h storage at room temperature in comparison with 4◦ C. The zeta potential of the blank NPs remained stable over the 168-h period albeit with minor fluctuations at the measurement intervals (Fig. 8c). This considered, the zeta potential for the NPs remained in the accepted range of −10 to −40 mv for the duration of the study and there was again no observed difference between those stored at 4◦ C and those stored at room temperature. The encapsulation efficiency of Heparzine-A (Table 3) was found to be highest at a drug to polymer ratio of 1:120 with 63.50 ± 1.57% with lower entrapment of around 50% for ratios of 1:60 and 1:90. Transmission electron microscopy (Figs. 9a and 9b) showed that the Heparzine-A-loaded NPs were spherical in shape and that the size distribution of the NPs was low.

DISCUSSION

Figure 6. PHBHHx-encapsulated product size curves for (a) paclitaxel, (b) Heparzine-A, and (c) ALB–PLC NP.

Heathman et al., JOURNAL OF PHARMACEUTICAL SCIENCES

Experimental design is a vital aspect of any research that is often overlooked, but when well executed, has the potential to deliver high impact results with precise planning and reduced cost. A factorial experiment consists of two or more factors, taking into account the effect of each variable on the process as well as the interaction of multiple variables in order to optimize production. Within a manufacturing process such as NP production, the control of these variables is critical and the knowledge of how they interact as well as which variables have the largest effect on controlling the desired process output will lead to more tightly controlled production. This interaction of variables is often ignored but is critical for a full understanding of the process.60 Lamprecht et al.61,62 achieved variation in poly(lactic-coglycolic acid) (PLGA) and BSA NP size by altering production variables (homogenization time, polymer concentration, and surfactant concentration) using a “one factor at a time” (OFAT) approach. The absence of consideration of the interaction of competing variables within their process resulted in the production of NPs with a minimum size of 230 nm with no indication of repeatability. A separate study by Gutierro et al.63 also varied PLGA–BSA NP size by altering the production process and managed to reduce the NPs from 1000 to 200 nm using an OFAT approach, which led to a reduced immune response. Gryparis et al.64 employed a sequential-multivariate batch method without testing combinational parameter effects to vary the size of PLGA NPs and were able to create drug-encapsulated NPs with a minimum size of 150 nm and a PDI of close to 0.3, the accepted limit for clinical applications.65 Perhaps the most rigorous study to date on size control of NPs employed a five-factor three-level experimental design for the production of PLGA–BSA NPs.66 Detailed computer analysis of this factorial design revealed a theoretic minimum DOI 10.1002/jps.24035

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Table 4. Sonication Power (W)

Comparison Between Prediction Curve and Drug-Loaded PHBHHx Nanoparticles Curve Prediction (nm)

100

204.5 167.7 141.7 124.2 500 165.7 118.3 99.8 93.2 Overall average prediction error

Table 5.

Paclitaxel

Haperzine-A

ALB–PLC NP

Prediction Error (nm)

Prediction Error (%)

Prediction Error (nm)

Prediction Error (%)

Prediction Error (nm)

Prediction Error (%)

5.47 4.26 1.96 0.07 3.98 1.54 0.30 0.85 2.30 ± 2.02

2.67 2.54 1.38 0.06 2.40 1.31 0.30 0.91 1.45 ± 1.01

5.65 0.26 4.08 7.88 0.48 0.54 1.65 1.03 2.70 ± 2.84

2.76 0.15 2.86 6.35 0.29 0.46 1.66 1.10 1.96 ± 2.06

0.44 3.69 6.52 7.75 4.28 2.68 1.21 0.16 3.34 ± 2.78

0.21 2.20 4.60 6.24 2.58 2.26 1.21 0.17 2.44 ± 2.10

Accumulate Release Profile Samples 1 and 2 were Compared with the Encapsulated Drug Concentration of 412 ng/mL Time (h)

Sample 1

Accumulative release (%) SD Sample 2 Accumulative release (%) SD Sample 3 Accumulative release (%) SD Sample 4 Accumulative release (%) SD Free drug (412 ng/mL) Accumulative release (%) SD Free drug (3333 ng/mL) Accumulative release (%) SD

1/6

1/2

1

2

4

12

24

48

72

96

144

192

240

336

9.05 1.79 10.41 1.25 12.31 0.89 11.67 2.79 37.36 5.53 27.76 3.77

14.04 2.08 18.04 0.45 14.90 3.33 16.53 0.86 85.21 7.90 52.73 3.44

19.18 2.85 20.55 0.71 19.92 2.74 20.37 3.04 96.89 7.94 69.81 7.17

21.13 1.51 21.74 0.24 20.99 2.65 21.89 2.08 97.53 6.08 80.35 4.75

24.16 2.44 21.49 0.85 22.25 2.29 22.01 0.75 97.21 3.75 88.44 3.97

25.20 2.76 22.76 1.96 22.41 0.70 22.55 4.09 97.53 11.72 89.67 5.49

25.76 0.96 24.00 1.68 23.29 3.42 22.11 2.79 97.61 7.56 89.30 7.02

28.91 2.60 24.04 0.91 22.17 0.63 21.83 3.14

28.57 3.10 25.35 1.27 23.93 3.22 23.72 2.75

28.50 1.98 25.38 0.83 23.28 4.13 23.36 3.25

28.58 0.30 25.43 0.70 24.33 6.84 22.99 2.99

28.41 0.44 26.19 0.81 24.26 6.69 22.53 3.73

28.87 1.13 26.53 0.32 24.54 6.40 22.53 3.30

28.66 0.54 26.11 0.47 24.51 3.32 22.45 3.53

Samples 3 and 4 were compared with the encapsulated drug concentration of 3333 ng/mL.

Figure 7. PHBHHx Haperzine-A drug release profile over 14 days.

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NP diameter for their double emulsion production method of 122.4 nm. As described previously, NPs with a mean diameter of less than 100 nm would be highly desirable to mediate cellular response, for sterilization via filtration, for use in gene transfection and to actively cross the blood–brain barrier. By producing a NP size range of 90–205 nm, we demonstrate the advantage of using PHBHHx for NP production to fit a variety of applications. In this study, blank NPs showed the same size trend as encapsulated NPs meaning that various biocompatible polymers can be characterized by this method in the knowledge that the introduction of drugs to the NPs may not affect the production size. From the data in Table 2, it is evident that the process described is highly repeatable and can accurately produce NPs of a desired size based on the number of replicates. This is a critical component of any production system because if a process is not reproducible or repeatable it cannot be used for reliable or commercial production. If batch to batch production is not considered within an experimental process, then common cause variability is not accounted for and the process cannot be reliably evaluated. A large number of experimental methods only account for interbatch variability, which is an assessment of the accuracy of the measurement system in use, that is, an N of three on one occasion, but gives no indication of the repeatability of the experimental methodology. Bridging the gap between industry and research by implementing a DOE approach in research laboratories is an invaluable way to attract industry and produce high quality research. This is a highly critical consideration for the successful commercialization of pharmaceutical products, which should be the aspiration of all research groups not only in the field of drug delivery and pharmaceutical research but also biomedicine. One consideration that must be noted in this process is the effect of sonication on the encapsulated protein or drug as high sonication power may have a destructive effect on the encapsulated protein. In this study, the total drug concentration was calculated from the amount of drug input at the start of the

Figure 8. PHBHHx NP stability testing, (a) PHBHHx NP size, (b) PDI, and (c) zeta potential.

Figure 9. SEM of PHBHHx Haperzine-A-encapsulated NPs (a) low magnification and (b) high magnification. Heathman et al., JOURNAL OF PHARMACEUTICAL SCIENCES

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process, taking into account the overall efficiency. In contrast, traditional encapsulation efficiency calculations use the total drug concentration after breakdown of the NPs via sonication and therefore do not account for the destruction of drug due to sonication. It can be noted from a previous study that a sonication power of 400 W had no apparent effect on the encapsulation of insulin,34 however repeat and high power sonication on the formed NP to release the entrapped drug or protein may cause destruction of the analyte of interest with future studies paying particular attention to the methods employed to evaluate encapsulation efficiency. At higher sonication power and duration, there was a tighter distribution of particle sizes and a reduction in PDI compared with the lower settings, which is consistent with previous studies.67 This is because of the increased exposure to ultrasonic force, which acts to push the size distribution curve down toward the lower size limit, reducing the overall range and creating a tighter size distribution. This is advantageous from a process perceptive because creating a tighter size distribution means that the process is inherently more reproducible when creating smaller NPs. The NPs in this study were assessed over 7 days and showed no size deviation for this period as well as small and acceptable changes to PDI and zeta potential, which is critical for the viability of the NPs for clinical applications.68 This was found to be the case for both 4◦ C and room temperature, showing the viability of this method in terms of stability. Clearly, storage will need to be considered in relation to the encapsulated drug as well as most proteins will need to be stored at 4◦ C to retain their integrity. In this study, we created NPs as small as 90 nm with a PDI of well below 0.3, which is far smaller than the theoretical minimum of 122.4 nm using PLGA–BSA as described by Feczko et al.66 This shows that the use of PHBHHx as a drug encapsulating polymer has greater potential compared with PLGA, producing smaller NPs as required for intravenous injection and transport across the blood–brain barrier as well as opening up new avenues in the use of NPs in gene transfection. Combining this key attribute with the highly favorable biodegradability of PHBHHx and the potential for cost-effective large-scale polymer production means that this system has remarkable and unique commercial potential as a drug delivery device to suit a multitude of applications within the field of nanomedicine. The CpK index of 4.13 shows the utilization of the DOE methodology for NP production has the potential to produce high quality research and has the potential to draw pharmaceutical industry attention.

CONCLUSIONS It has been shown that the DOE approach can be employed to control the production of NPs to fit a variety of applications within nanomedicine, whereby a size prediction curve can be produced for each polymer, which can then be applied to any encapsulated drug to achieve a desired NP size. Preliminary release profile analysis has shown steady and sustained release of NP entrapped drug over a 14-day period and the manipulation of NP size can be controlled not only to deliver a sustained release but also a tailored burst release. The knowledge gained from this preliminary optimization is important to pave the way for more in depth analysis to obtain a full understandDOI 10.1002/jps.24035

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ing of the process. This study highlights the most sensitive aspects of NP production via the solvent evaporation method, which can be used to reduce variability in the production process leading to increased precision within the manufacture of NPs. Reliable production of drug-encapsulated NPs provides key evidence that PHBHHx can be used to investigate new applications in the field that require the production of NPs less than 100 nm. Investigating the effect of different surfactants during production using the solvent evaporation method would potentially lead to the creation of smaller NPs and is a key area for further investigation. We have shown reasonable PHBHHx NP EE of the hydrophobic drug Heparzine-A, which can be combined with this controlled manufacture process for targeted and disease specific treatments. Aligning research practices with those of current commercial research institutions has been highlighted as an invaluable way to attract commercial interest and to allow research to evolve alongside industry, which is critical for the success of nanotechnology drug, controlled and targeted drug delivery, and biomedicine in general.

ACKNOWLEDGMENTS The authors would like to acknowledge the EPSRC Doctoral Training Centre in Regenerative Medicine, Keele University Acorn Funding, the Medical Research Council, the North Staffordshire Medical Institute, and the HYANJI Scaffold Project (European Commission Framework 7 program) for funding this research. The authors also acknowledge all staff and students in the Department of Pharmacy at Sichuan University and Antonella Lisella, Pisa University, Italy.

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