Communications Fabrication of Elastin-Like Polypeptide Nanoparticles for Drug Delivery by Electrospraying Yiquan Wu,���,��� J. Andrew MacKay,�� Jonathan R. McDaniel,�� Ashutosh Chilkoti,*,���,�� and Robert L. Clark*,���,��� Center for Biologically Inspired Materials and Material Systems, Duke University, Durham, North Carolina 27708, Department of Mechanical Engineering and Materials Science, Pratt School of Engineering, Duke University, Durham, North Carolina 27708, and Department of Biomedical Engineering, Duke University, 136 Hudson Hall, Box 90281, Durham, North Carolina 27708 Received September 15, 2008 Revised Manuscript Received November 19, 2008 The development of environmentally responsive drug carriers requires new methods for assembling stimuli- responsive nanoparticulates. This communication describes a novel application of electrospray to construct bioresponsive peptide-based particulates, which can encapsulate drugs. These particles are composed from genetically engineered elastin-like polypeptides (ELPs), a biodegradable, biocompatible, and bioresponsive polymer. To generate nanoparticles (300-400 nm in diameter), ELPs and drugs are codissolved in organic solvent, accelerated across a voltage gradient, dried by evaporation during transit, and collected from a target surface. These findings indicate that particle diameter, polydispersity, and morphology are strong functions of the solvent concentration, spraying voltage, and polymer molecular weight. Surprisingly, the loading of drug at 20 w/w% did not influence particle morphology furthermore, drug release from these particles correlated with the pH-dependent solubility of the parent ELPs. These studies suggest that electrospray is an efficient and flexible method for generating stimuli-responsive drug particles. 1. Introduction Electrospraying can create particles by applying a uniform electrohydrodynamic force to break up liquids into fine jets1,2 and is an emerging method for the rapid and high throughput production of nanomeso scale particles of controlled morphology for controlled release drug delivery applications. A major rationale driving nanoparticle drug formulation is to adjust the kinetics of drug distribution in the body, thus improving the efficacy of existing drugs. At a minimum, these nanoparticles must display (1) reliable drug entrapment, (2) uniform particle dimensions, (3) sufficient stability under physiological condi- tions, (4) adequate release either over time or in response to a stimuli, and (5) biocompatibility. To this end, electrospray encapsulation of drugs into an environmentally responsive peptide matrix offers a unique opportunity to address the requirements of nanoparticle drug delivery. The morphology and size of electrosprayed polymer nanoparticles is strongly influenced by a host of variables that include processing parameters and the material properties of the solution such as polymer composition, molecular weight (MW), the solvent used for polymer dissolution, and the presence and concentration of other cosolutes (such as a drug).3 In this communication, the effect of electrospray processing conditions (i.e., flow rate, spraying voltage), the molecular weight of polymer and its concentration were investigated on the morphology and size of electrosprayed particles using a well-defined set of monodisperse recombinant biopolymers and doxorubicin (Dox), a cancer chemotherapeutic.4,5 We demonstrate that electrospraying is a viable method to generate nanomeso scale biopolymer particles of defined morphology, and the release of the drug is controlled by the dissolution of the particles. We chose recombinant elastin-like polypeptides (ELPs) as the electrosprayed polymer. ELPs are derived from a pentapep- tide Val-Pro-Gly-Xaa-Gly (VPGXG) repeat in the amino acid sequence of human tropoelastin, where the ���guest residue,��� Xaa, can be any combination of natural amino acids except proline.6,7 We selected ELPs for the following reasons: first, ELPs undergo a thermodynamically driven inverse phase transition at a characteristic temperature, above which they phase separate from bulk water.8 The ELP transition temperature is similar to a lower critical solution temperature (LCST), above which the system separates into a liquid phase and an insoluble coacervate phase. During electrospraying the solvent, water in this case, is rapidly evaporated leading to the formation of a dehydrated particle. We speculated that upon exposure to an aqueous environment in the body, the ability to undergo coacervation would hinder release of Dox if the solution conditions promote the existence of the insoluble phase.9,10 Second, ELPs are of interest for biomedical applications,11-13 including drug delivery.14 A previous report has demonstrated possible applications of ELP particulates, but to date, ELP particles have not been developed for sustained release of an encapsulated therapeutic,9 exposing a need for efficient processes capable of converting ELPs into micrometer to nanometer diameter particles. Third, ELPs can be produced recombinantly, which allows precise control over * To whom correspondence should be addressed. E-mail: rclark@ duke.edu (R.L.C.) chilkoti@duke.edu (A.C.). ��� Center for Biologically Inspired Materials and Material Systems. ��� Pratt School of Engineering. �� Department of Biomedical Engineering. Biomacromolecules 2009, 10, 19���24 19 10.1021/bm801033f CCC: $40.75 ��� 2009 American Chemical Society Published on Web 12/10/2008

the composition and MW,6,13 variables that are important in controlling not only the phase transition behavior of ELPs but also the electrospraying, drug encapsulation, and drug release processes. 2. Experimental Section Synthesis of ELPs Materials. Two ELPs with a MW of 17.8 and 70.2 kD, with a guest residue composition of Val/Ile/Glu [1:3:1], were synthesized for this study to examine the effect of polymer MW on the morphology of the electrosprayed particles. The low molecular weight ELPs had the peptide sequence SKGPG(VGVPGIGVPGI- GVPGEGVPGIGVPG)8WPC. The high molecular weight ELP had the peptide sequence SKGPG(VGVPGIGVPGIGVPGEGVPGIGVPG)32- WPC(GGC)7. We selected ELPs containing glutamic acid residues because (1) we speculated that the inclusion of charges would promote formation of nanoparticles in electrospraying via Coulombic repulsion based on our previous observation that highly charged ELP fusion proteins tend to form nanoparticles (as opposed to microparticles during coacervation), and (2) Glu residues convey a strong pH dependence to the transition temperature of the ELP, allowing us to conveniently and systematically examine the effect of LCST under isothermal conditions by modulation of the pH on the release of the encapsulated drug without the need to synthesize a large set of ELPs with different LCSTs. The ELPs were synthesized by heterologous expression of a plasmid- borne synthetic gene of the ELP in E. coli, as described previously.13 Genes encoding the ELPs were constructed using recursive directional ligation in a modified pUC19 plasmid in TOP10 cells (Invitrogen Corporation, Carlsbad, CA),13 ligated into a modified pET25b+ vector (Novagen, Madison, WI), expressed in BLR(DE3) cells (Novagen, Madison, WI) and purified by inverse transition cycling.15,16 Cysteine residues in these ELPs were reduced during purification by ���10 mM tris carboxyethyl phosphine hydrochloride (TCEP Pierce Biotechnol- ogy, Inc., Rockford, IL). The purified polymers used for this study were obtained from multiple batches at yields greater than 100 mg/L bacterial culture. After purification, ELP molecular weight and purity were determined by capillary electrophoresis using an Experion instrument (Bio-Rad, Hercules, CA). Electrophoresis confirmed a single 26 kD band (98% purity) for the 17.8 kD ELP and a 105 kD band (100% purity) for the 70.2 kD ELP. The observation of increased MW of ELPs is consistent with previous observations that ELPs under electrophoresis run at ���20-40% higher molecular weight than do globular protein standards.13 Fabrication of Nanoparticles. Figure 1 shows a schematic illustra- tion of the electrospraying setup. A 5 mL syringe was filled with ELP solution and a syringe pump was used to dispense the solution. The solution was dispensed at set flow rates of 0.05 mL/h and 0.1 mL/h in separate experiments. A high-voltage power supply with a range of 0-30 kV was used to generate an electric field between the nozzle and the collector. ELP particles were electrosprayed onto a collector located at a set distance of 40 cm away from the nozzle tip. A stable cone-jet mode can not always be guaranteed during electrospraying water solution, but several methods have been developed to deal with this challenge, such as using deionized water as solvent,17 using a sheath of inert gas or vacuum chamber to prevent electrical discharge,18,19 using a novel electrospraying nozzle with a nonconductive fiber,20 using a tine silica nozzle with a 20 ��m inner diameter,21 and using AC voltage superimposed on the DC voltage.22 In this experiment, a ring electrode slightly below the electrospraying nozzle was incorporated to produce a uniform spraying pattern by creating a cylindrical electric field to help atomization.2 ELPs Particle Characterization. The morphologies of electro- sprayed ELP particles were characterized by a field emission scanning electron microscope (FESEM, FEI XL 30 SEM-FEG) operated at an accelerating voltage of 5 kV and a working distance of 5 mm. The diameters of particles and size distribution were measured using Image J analysis software (NIH). Prior to SEM analysis, the ELP particles were collected on an aluminum disk and sputter-coated with gold. ELP Thermal Characterization. ELP transition temperatures were characterized over a range of concentrations (5, 10, 25, 50, and 100 ��M ELP in phosphate buffer) by raising the temperature at 1 ��C/min on a CARY UV-vis spectrophotometer (Varian, Palo Alto, CA). The transition temperature was defined at the maximum first derivative of the optical density at 350 nm. DOX Release. Each piece of silica was massed and incubated at 37 ��C in 200 ��L of buffer of a given pH (pH 2.5: 10 mM succinate, 140 mM NaCl pH 5.5: 10 mM sodium succinate, 140 mM NaCl pH 7.5: 10 mM Na2PO4, 140 mM NaCl). For each time-point, 200 ��L was gently removed and replaced with fresh solution. Dox concentration was determined by fluorescence (excitation: 485 nm emission: 590 nm) using a Victor-3 96-well plate reader (Perkin-Elmer, Waltham, MA) and computed from a standard curve. Standards and samples were quenched in 1:1 v/v 0.1 M sodium phosphate buffer, pH 7.5 prior to measurement. At the end of the study, each piece of silica was washed in pH 7.5 buffer to recover any remaining ELP and Dox. 3. Results and Discussion Figure 2 shows SEM images of electrosprayed particles generated from ELPs with a molecular weight of 70.2 kD and a solution concentration of 1 w/v% using different spraying voltages and flow rates. Figure 2a-c show the ELP particles produced at a flow rate of 0.05 mL/h and a spraying voltage of 7, 8, and 9 kV, respectively. At a spraying voltage of 7 kV, both particulate and fibrous ELP structures were produced, and the particles were connected by the fibers. As the spraying voltage increased to 8 kV and greater, particles with fibrous tails were formed. The particle sizes were distributed over a range of 115-680 nm. Figure 2d-f show that when the flow rate of solution was increased to 0.1 mL/h, more fibrous ELP structures were produced. Using a spraying voltage of 7 kV, a predominantly fibrous microstructure was obtained, as shown in Figure 2d. Increasing the spraying voltage from 7 to 9 kV generated more ELP particles and fewer fibers, as shown in Figure 2f. This trend was a result of a shift in the balance of forces on the surface of the charged droplets of ELP solution. As the applied voltage was increased, the electrostatic force more easily overcame the surface tension on the droplets and atomized the charged droplets into particles. Increasing the flow rate of solution drove more precursor materials to be electro- sprayed and thus made it easier to generate fiber or tail structures when using high molecular weight polymers.23,24 Figure 1. Schematic diagram of an electrospraying setup for preparing ELP drug delivery particles. 20 Biomacromolecules, Vol. 10, No. 1, 2009 Communications