Journal of Controlled Release 204 (2015) 70–77

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Evaluation of drug loading, pharmacokinetic behavior, and toxicity of acisplatin-containing hydrogel nanoparticle

Marc P. Kai a,⁎, Amanda W. Keeler b, Jillian L. Perry c, Kevin G. Reuter d, J. Christopher Luft b, Sara K. O'Neal b,William C. Zamboni b, Joseph M. DeSimone a,c,d,⁎a Department of Chemical and Biomolecular Engineering, North Carolina State University, 911 Partners Way, Raleigh, NC 27599, USAb Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, 257 Caudill Lab, Chapel Hill, NC 27599, USAc Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, 257 Caudill Lab, Chapel Hill, NC 27599, USAd Department of Chemistry, University of North Carolina at Chapel Hill, 257 Caudill Lab, Chapel Hill, NC 27599, USA

⁎ Corresponding author at: Department of ChemicalNorth Carolina State University, 911 Partners Way, Raleig

E-mail addresses: mpkai@ncsu.edu (M.P. Kai), desimo

http://dx.doi.org/10.1016/j.jconrel.2015.03.0010168-3659/© 2015 Elsevier B.V. All rights reserved.

a b s t r a c t

a r t i c l e i n f o

Article history:Received 30 October 2014Received in revised form 4 February 2015Accepted 1 March 2015Available online 3 March 2015

Keywords:CisplatinPRINTPEGylationPharmacokineticsCytotoxicity

Cisplatin is a cytotoxic drug used as a first-line therapy for a wide variety of cancers. However, significant renaland neurological toxicities limit its clinical use. It has been documented that drug toxicities can be mitigatedthrough nanoparticle formulation, while simultaneously increasing tumor accumulation through the enhancedpermeation and retention effect. Circulation persistence is a key characteristic for exploiting this effect, and tothat end we have developed long-circulating, PEGylated, polymeric hydrogels using the Particle Replication InNon-wetting Templates (PRINT®) platform and complexed cisplatin into the particles (PRINT-Platin). Sustainedrelease was demonstrated, and drug loading correlated to surface PEG density. A PEG Mushroom conformationshowed the best compromise between particle pharmacokinetic (PK) parameters and drug loading (16 wt.%).While the PK profile of PEG Brush was superior, the loading was poor (2 wt.%). Conversely, the drug loading innon-PEGylatedparticleswas better (20wt.%), but the PKwasnot desirable.We also showed comparable cytotox-icity to cisplatin in several cancer cell lines (non-small cell lung, A549; ovarian, SKOV-3; breast, MDA-MB-468)and a higher MTD in mice (10 mg/kg versus 5 mg/kg). The pharmacokinetic profiles of drug in plasma, tumor,and kidney indicate improved exposure in the blood and tumor accumulation, with concurrent renal protection,when cisplatin was formulated in a nanoparticle. PK parameters were markedly improved: a 16.4-times higherarea-under-the-curve (AUC), a reduction in clearance (CL) by a factor of 11.2, and a 4.20-times increase in thevolume of distribution (Vd). Additionally, non-small cell lung and ovarian tumor AUC was at least twice that ofcisplatin in both models. These findings suggest the potential for PRINT-Platin to improve efficacy and reducetoxicity compared to current cisplatin therapies.

© 2015 Elsevier B.V. All rights reserved.

1. Introduction

Cisplatin has played an important role as a “broad-spectrum” che-motherapeutic in the treatment of many cancers [1]. The side-effectsare not trivial, however, and include significant neurological and renaltoxicities, the latter being dose-limiting [2]. Incorporating the druginto a nanoparticle can increase tumor accumulation while simulta-neously reducing side effects caused by systemic exposure [3].

The key characteristic for this passive targeting of nanoparticles totumors—an important facet of chemotherapeutic delivery—is circula-tionpersistence. The common accepted theory of passive accretion is re-ferred to as the enhanced permeation and retention (EPR) effect. Firstrecognized in 1986, it was attributed to hypervasculature, enhancedpermeability, and little recovery through either venous or lymphatic

and Biomolecular Engineering,h, NC 27599, USA.ne@unc.edu (J.M. DeSimone).

drainage [4]. This theory suggests that, given enough time, circulatingmacromolecules and nanoparticles will eventually deposit in thetumor bed. The first documented definition of EPR involved a studywith proteins, but wide application to nano- and micro-particles hassince been explored [5–10]. In addition to selective accumulation,other advantages of nano-formulations over soluble drug include in-creased exposure through altered pharmacokinetics (PKs), greater solu-bility and biocompatibility, and higher therapeutic index [11–15].

Non-small cell lung and ovarian are two types of cancer commonlytreated with cisplatin [16]. An orthotopic model of lung cancer has sev-eral advantages over a subcutaneous model. Instilling cells into theorgan of origination provides the appropriate microenvironment,allowing for relevant biological interactions and implications [17,18].Carefully specifying both the type and location of cancer increases theability to correlate potential efficacy with particle distribution andkinetics.

Several other factors, however, also dictate the behavior of a particleonce injected; these include size, elastic modulus, shape, charge, and

71M.P. Kai et al. / Journal of Controlled Release 204 (2015) 70–77

surface chemistry (see Refs [19–22]. for comprehensive reviews).The effect of size on circulation has been well documented, and evi-dence has mounted in favor of particles less than 100 nm in at leastone critical dimension for blood persistence [23–25]. Particle modu-lus is also important for sterile filtration techniques in formulationpreparation and navigating the mechanical barriers of the liver andspleen [26–30]. The role of shape has been elusive due to a shortageof methods for precise shape control. Previous studies have alludedto the effect of particle geometry, most notably studies of high-aspect ratio particles that showed increased circulation time with in-creasing length [31,32]. These studies have been the driving force be-hind utilizing worm-like particles for EPR studies, but calibration-quality fabrication of filamentous geometry and modulus is difficult[33,34]. Surface charge plays a major role in particle stability and bi-ological interactions; cationic particles are more toxic to red bloodcells and cause platelet aggregation compared to neutral or anionicparticles [35]. PEGylation, a polymer surface coating of repeatingethylene glycol units, is a common surface modification utilized toreduce protein binding (opsonization), increase hydrophilicity andstability, and extend circulation half-life [14,36–38]. Finally, the in-terplay between these characteristics complicates studies even fur-ther; synergistic or dysergistic effects may be difficult to observewithout precise control over all variables.

Particle Replication in Non-wetting Templates (PRINT®) is acalibration-quality tool that enables complete, orthogonal control overparticle characteristics [39,40]. The capacity of PRINT to independentlyand systematically vary parameters is a crucial advantage in determin-ing exactly howa given variable affects nanoparticle behavior. Themod-ulus, shape, and surface chemistry of PRINT hydrogels were previouslyoptimized with different PEGylation conformations (non-PEGylated,PEGMushroom, and PEG Brush) [27,41]. The aim of this studywas to in-vestigate the effect of surface PEG density on the loading and release ofcisplatin from nanoparticles, and subsequently determine the in vivobehavior of the optimal formulation. Herein we report a platform andmethod for novel complexation and release of cisplatin from a PRINThydrogel nanoparticle (PRINT-Platin).

2. Materials and methods

2.1. Materials

Commercially available polyethylene glycol diacrylate (PEG700-DA, Mn = 700 Da), 2-aminoethyl methacrylate hydrochloride(AEM), diphenyl(2,4,6-trimethylbenzoyl)-phosphine oxide (TPO),polyvinyl alcohol (PVOH, Mn = 2000 Da), succinic anhydride, cis-diaminedichloroplatinum(II) (CDDP), and sucrose were purchasedfrom Sigma-Aldrich. PTFE syringe filters (13 mm membrane,0.220 μm pore size), dimethylformamide (DMF), triethanolamine(TEA), pyridine, sterile water, borate buffer (pH 8.6), methanol,and trace-metal grade concentrated nitric acid (HNO3) were obtain-ed from Fisher Scientific. Methoxy PEG (5 k)-succinimidyl carboxymethyl ester (mPEG5k-SCM) was purchased from Creative PEGWorks.Tetraethylene glycol monoacrylate (HP4A) was synthesized in-houseas previously described [42]. Conventional filters (2 μm) were pur-chased from Agilent, and polyvinyl alcohol (Mw 2000) (PVOH) waspurchased from Acros Organics. PRINT molds (80 nm × 320 nm) wereobtained from Liquidia Technologies (RTP, NC). Polyethylene tere-phthalate (PET) was purchased in 1000-foot rolls from 3 M. Cisplatinwas acquired from the University of North Carolina Pharmacy. Water,where used, was sterile-grade and 0.2-μm filtered. Cells (A549-luc,SKOV-3, and MDA-MB-468) were purchased from American Type Cul-ture Collection. Fetal bovine serumwas purchased from Atlanta Biolog-icals. RPMI 1640 Mediumwas purchased from Gibco®; Leibovitz's L-15andMcCoy's 5AMedia were purchased fromCorning cellgro®. All com-mercially available materials were used as received.

2.2. Fabrication, functionalization, and complexation

The PRINT particle fabrication technique has been described previ-ously in detail [41,43]. Briefly, a preparticle solution (PPS)was preparedby dissolving various reactive monomers in methanol. The PPS “solids”were comprised of 69 wt.% HP4A, 20 wt.% AEM, 10 wt.% PEG700DA, and1wt.% TPO. A thin film of the PPS was drawn onto PET, laminated to thepatterned side of the mold, and delaminated at the laminator nip roll.Particles were cured by passing the filled mold through a UV-LED(Phoseon). A PVOH harvesting sheet was hot laminated to the filledmold and cooled to room temperature (r.t.), and particles were re-moved from the mold by splitting the PVOH harvesting sheet from themold. Particles were then harvested by dissolving the PVOH in waterand passed through a 2 μm filter (Agilent) to remove any large particu-lates. To remove the excess PVOH, particles were centrifuged(Eppendorf Centrifuge 5417R) at ca. 21,000×g for 15min at 4 °C, the su-pernatant was removed, and the particles were re-suspended in sterilewater. This purification process was repeated two times in water,followed by three times in DMF, to prepare particles for PEGylationand succinylation.

The particles (1mg/mL) were reacted with excess TEA for 10min atr.t. on a shaker plate (Eppendorf, 1400 rpm). The mPEG5k-SCMwas dis-solved in DMF (14 mg for PEG Brush; 2.05 mg for PEG Mushroom, pre-viously optimized [41]; none for non-PEGylated and non-succinylated)and added to the reaction mixture. The reaction mixture was shakenovernight and quenched with borate buffer (100 μL). The nanoparticlesuspension was then washed three times with DMF via centrifugation.Particles were succinylated by reaction with an excess of pyridine andsuccinic anhydride (100× molar excess with respect to amine groups;no succinic anhydridewas added to non-succinylated particles). The re-action was carried out in a sonicator bath (Branson Ultrasonic Cleaner1.4 A, 160 W) for 30 min. Following succinylation, the particles werewashed by centrifugation once in DMF, followed by a borate bufferwash to neutralize any succinic acid side product, and then threewashes with sterile water.

Cisplatin complexationwas achieved by incubating the particles in asolution of CDDP (2× molar excess with respect to carboxyl groups) inwater at r.t. for N24 h under constant agitation (Eppendorf, 1400 rpm).After incubation in the complexation solution, particles were washedwith sterile water by centrifugation and resuspended in 9.25 wt.% su-crose (aq) at the appropriate dose concentration. Aliquots were flashfrozen in liquid nitrogen and stored at−20 °C until needed.

2.3. Characterization

Particle concentrations were determined by thermogravimetricanalysis (TGA) using a TA Instruments Q5000 analyzer. Particles werevisualized via scanning electron microscopy (SEM) using a Hitachi S-4700 microscope. Prior to imaging, SEM samples were coated with1.5 nm of gold–palladium alloy using a Cressington 108 auto sputtercoater. Particle size and zeta potential were measured by dynamiclight scattering (DLS) on a Zetasizer Nano ZS (Malvern Instruments,Ltd.) at 37 °C.

For release studies, PRINT-Platin was incubated at 1 mg/mL in PBSbuffer on a shaker at 1400 rpm, 37 °C (n = 3 per particle type). An ali-quot was taken from each sample at 24, 48, 72, and 168 h. The samplealiquot was centrifuged (ca. 21,000 ×g, 15 min, 4 °C) to isolate cisplatinin the supernatant released from the particle pellet. The supernatantsamples were stored before analysis.

Cisplatin loading and release were assessed using an Agilent 1200series high-performance liquid chromatography (HPLC) system withan ultraviolet detector. The mobile phase consisted of 90% 0.9-wt.%NaCl (aq) and 10%methanol, by volume. A five minute isocratic elutionprotocol was used with a ZORBAX Eclipse Plus C18 column (AgilentTechnologies). The product was eluted at a flow rate of 1 mL/min andmonitored at a wavelength of 210 nm. Drug loading was determined

72 M.P. Kai et al. / Journal of Controlled Release 204 (2015) 70–77

by analysis of the complexation solution pre- and post-incubation. Thenet difference in cisplatin concentration was calculated as weight per-cent in the particle.

2.4. Cytotoxicity

Cells were seeded in 200 μL of media [RMPI 1640Medium for A549;McCoy's 5A Medium for SKOV-3; Leibovitz's L-15 Medium for MDA-MB-468; all media was supplemented with 10% fetal bovine serum] ata density of 5000 cells per cm2 into a 96-well microtiter plate. Cellswere allowed to adhere for 24 h and subsequently incubatedwith eitherPRINT-Platin or cisplatin at concentrations ranging from 9.8 nM to80 μM (drug concentration) for 72 h at 37 °C in a humidified 5% CO2 at-mosphere. After the incubation period, all media/particles were aspirat-ed off cells. 100 μL fresh medium was added back to cells, followed bythe addition of 100 μL CellTiter-Glo® Luminescent Cell Viability Assayreagent (Promega, Madison, WI). Plates were placed on a microplateshaker for 2 min, then incubated at r.t. for 10 min to stabilize lumines-cent signal. The luminescent signal was recorded on a Molecular Dy-namics SpectraMax M5 plate reader. The viability of the cells wasexpressed as a percentage of the viability of cells grown in the absenceof particles or drug.

2.5. Animals

All experiments involving mice were performed in accordance withtheNational Research Council's Guide to Care and Use of Laboratory An-imals (1996), under an animal use protocol approved by the Universityof North Carolina Animal Care and Use Committee. All studies used fe-male Foxn1nu (athymic nude, C57BL/6 J background) mice (5 weeksold, 17–27 g, Jackson Laboratory). For the tumor accumulation PKstudy, an orthotopic non-small cell lung (A549-luc)model and a subcu-taneous ovarian (SKOV-3) model were used. Cell cultures were pre-pared and maintained per vendor specifications. For the orthotopiclung model, a 40-μL suspension of luciferase-expressing A549 cells(5 × 106 cells per mouse in a 50:50 Matrigel:PBS blend) was implantedinto the lung via intra-thoracic inoculations, as previously described[18]. For the subcutaneous ovarian model, a 200-μL suspension ofSKOV-3 cells (5 × 106 cells per mouse in a 50:50 Matrigel:PBS blend)was injected into the left flank of the mice. Tumor growth was moni-tored by bioluminescence (A549) or volume (SKOV3) via caliper mea-surements (L2 × W/2).

2.6. Pharmacokinetic study in healthy mice

PRINT-Platin or cisplatin was dosed via tail vein injections at 3 mgdrug per kg of mouse weight in naïve animals based on drug amount.Nanoparticles were injected as a suspension in an isotonic sucrose solu-tion (9.25wt.%), and cisplatinwas dosed in prescription form (pH-adjust-ed 0.9 wt.% NaCl solution). Liver, spleen, and kidney were harvested andflash-frozen in liquid nitrogen at 0.083, 0.5, 1, 6, 24, and 72 h post-injection. Four mice per arm were examined at each time point. Bloodwas collected in EDTA by cardiac puncture at the same time points andcentrifuged (300 ×g, 5 min, 4 °C) to isolate plasma from the cell fraction.A portion of the plasma samples from the soluble drug armwas set asideto separate protein-bound cisplatin from unbound cisplatin. For the sepa-ration of protein-bound cisplatin from unbound cisplatin, plasma was pi-petted into a Centrifree ultracentrifugation device (EMD Millipore Co.)and centrifuged at 2500 ×g for 30 min at 25 °C.

Pharmacokinetic analysis of the measured plasma concentrationswas performed using PKSolver [44]. Data was fit to either a one- ortwo-compartment model, and the Akaike information criterion (AIC)was used to compare goodness of fit for each nanoparticle type [45].

2.7. Tumor accumulation

Once tumors had reached sufficient size post-tumor inoculation,mice were randomized into dosing groups. PRINT-Platin or cisplatinwas dosed via tail vein injections at the maximum tolerated dose(MTD), based on drug amount. Tumors were harvested and flash-frozen in liquid nitrogen at 0.083, 0.5, 1, 6, 24, and 72 h post-injection.Four mice per arm were examined at each time point.

2.8. Maximum tolerated dose (MTD)

Cisplatin or PRINT-Platin (n = 3) was dosed intravenously in naïvemice via tail-vein once per week for six weeks. Acceptable body weightloss was specified as ≤20%, per protocol guidelines. Injections and ani-mal welfare monitoring were performed with the assistance of theAnimal Studies Core (UNC-CH).

2.9. ICP-MS validation, quantification, and analysis

Tissue sample preparation was performed as previously described[46]. Briefly, tissue (kidney, tumor, liver, spleen) and plasma sampleswere digested in concentrated HNO3 spiked with 200 ng/mL Iridium(Ir; analytical internal standard, Inorganic Ventures, Christiansburg,VA) for 60–90min at 90 °C. Deionizedwater was added to bring sampleto volumeandHNO3 concentration of 3.5%, and the sampleswere storedat 4 °C until platinum (Pt) analysis was completed. Inductively-coupledplasmamass spectroscopy (ICP-MS) analysis (Agilent 7500cx)was per-formed and validated as previously described [46,47].

3. Results and discussion

3.1. Characterization and drug release of calibration-quality nanoparticles

Incorporation of cisplatin into PRINT hydrogels was achieved via li-gand exchange of a chloride atom on the drug with a carboxyl groupwithin the particle matrix (Fig. 1). Complexation of cisplatin into a ma-trix was previously shown in micelles containing aspartic acid and wasdemonstrated by this group using a PEG-based hydrogel [48].

Since hydrogels swell based upon composition and conditions, itwas important to monitor the particles for any changes during produc-tion. Demonstration of stability in the solid-state form was also impor-tant for long-term storage potential. Fig. 2 shows a representativecomparison of SEM images andDLSmeasurements for purified particlesafter harvest and cisplatin-loaded particles that were flash frozen in su-crose. These measurements indicate that particle integrity was main-tained throughout the functionalization and complexation process,with no apparent change in particle characteristics. This validation ofthemorphology, size, and stability provides confirmation of an effectiveand suitable process for formulation and storage of cisplatin-loaded,PEGylated PRINT hydrogels.

The loading and release of cisplatin as a function of surface PEG con-formation was then investigated via HPLC. An inverse correlation be-tween PEG density and drug loading was observed; cisplatin contentincreased as PEGylation decreased (Fig. 3A). Although non-PEGylatedparticles afforded the highest drug loading, previous studies revealedthat the in vivo PK was quite poor compared to PEG Brush and PEGMushroom particles [41]. These studies showed an increase of 200-times and 136-times the clearance of PEG Brush and PEG Mushroomparticles, respectively. Furthermore, the exposure (area under the curve[AUC]) was reduced by a factor of 127 and 86 over Brush andMushroomconformations, respectively. Conversely, although the PEG Brush confor-mation had the best PK parameters, it had the lowest cisplatin loading.It was hypothesized that steric hindrance from the thicker PEG coatingon the surface prohibited cisplatin from diffusing into the interior of theparticle. The PEG Mushroom conformation offered a moderate improve-ment in PK parameters compared to PEG Brush and only a small decrease

Fig. 1.Nanoparticle-cisplatin complexation schematic. The particle (red) was PEGylated and contained excess amines (A). The amineswere converted to carboxyl groups by succinylation(B), which complexwith cisplatin via ligand exchangewith a chlorine atom (C). (For interpretation of the references to color in this figure legend, the reader is referred to theweb versionof this article.)

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in cisplatin loading compared to non-PEGylated. Thus, PEG Mushroomwas considered the optimal particle—hereafter referred to as PRINT-Platin—since it offered the best compromise between drug loading andcirculation behavior. The release profile of cisplatin from the particlesalso supported this conclusion (Fig. 3B). The majority of encapsulateddrug was released from each particle type; demonstration of releasefrom the carrier is vital for effective delivery. An additional negative con-trol (non-succinylated particles) presented minimal release, which wasexpected since there were no carboxyl groups for cisplatin complexation.

3.2. Cytotoxicity of drug and particles on A549, SKOV-3, and MDA-MB-468

To evaluate the in vitro toxicity of the new formulation, we investi-gated the effects of PRINT-Platin on several cancer cell lines as comparedto cisplatin. As discussed previously, the PEG Mushroom particle wasused as it appeared to possess the appropriate balance between cisplat-in content and pharmacokinetic parameters. Non-small cell lung, ovar-ian, and breast cancers were screened to demonstrate that particleeffectiveness was independent of tumor selection. The former two dis-eases are approved indications of cisplatin, and the latter breast line rep-resented potential future targets. The nanoparticle complex showedcytotoxicity in all cell lines tested, but was less toxic compared to thesoluble drug (Fig. 4). This was an expected shift due to the release pro-file of the drug from the particle matrix. Cells were exposed to particlesfor 72 h, at which point approximately 60% of the encapsulated drugwould have released (Fig. 3B). Blank particles showedno toxicity, agree-ing with previous studies on PEG-based PRINT hydrogels [49–51].

Verification that active drug is recovered in a biological setting isparamount to continued investigations of the formulation, as severalother cisplatin-nanoparticle platforms failed to achieve results due to

Fig. 2. SEMmicrograph and dynamic light scattering data for (A) bare (not functionalized) 80 nPRINT hydrogels.

a lack of drug release [52,53]. Coupled with HPLC analysis, the cytotox-icity assays confirmed in vitro release of cisplatin from PRINT-Platin.

3.3. Pharmacokinetic study in healthy mice

In order to determine the benefits of formulating cisplatin in a nano-particle, quantitative analysis of the drug was performed using ICP-MS.An equimolar amount of drug was injected for accurate comparison ofcisplatin distribution. Fig. 5 portrays the plasma kinetics and parametersof PRINT-Platin and cisplatin. Given at the same concentration, PRINT-Platin was retained in the blood at a higher concentration at all timepoints (Fig. 5A). The pharmacokinetic (PK) parameters revealed amarked improvement in the circulation behavior of PRINT-Platin ascompared to cisplatin (Fig. 5B). Therewas a 16.4-times higher exposure,expressed as area-under-the-curve (AUC), representing a higher totalamount of drug reaching circulation. A decrease in the clearance (CL)by a factor of 11.2 indicated slower removal of the drug from thebody. Finally, the volume of distribution (Vd) increased by 4.20-times,which implied an apparent decrease in the amount of drug that wastissue-bound for the particulate form. Overall, the PK of cisplatin wasvastly improved by the nanoparticle formulation.

Encapsulation within a vehicle is also important and affords protec-tion of cisplatin from protein binding and inactivation. Fig. 5C shows themajority of drug is retained within the particle while in circulation, po-tentiallyminimizing toxicity in non-target, healthy tissues. Finally, renalprotection was demonstrated in the particle formulation. Fig. 5D re-vealed lower kidney accumulation of PRINT-Platin compared to cisplat-in. This suggests the potential for the nano-formulation to mitigatenephrotoxicity, increasing the therapeutic index to improve a patient'squality of life and increase the tolerable drug dose, with implications

m× 320 nm PRINT hydrogels and (B) cisplatin-loaded, PEG Mushroom 80 nm × 320 nm

Fig. 3. Loading (A) and release profile (B) of cisplatin from 80 nm × 320 nm PRINThydrogels as a function of surface PEG conformation in PBS at 37 °C.

MDA-MB-468

A549

SKOV-3

74 M.P. Kai et al. / Journal of Controlled Release 204 (2015) 70–77

for better efficacy [2,54]. The pharmacokinetic profile in both liver andspleen displayed higher accumulation of PRINT-Platin compared to cis-platin (Supplemental Fig. 1) as expecteddue to the enhanced sequestra-tion of particles by these organs, however, no adverse toxicity wasobserved.

Fig. 4. Cytotoxicity of cisplatin, cisplatin-loaded 80 nm × 320 nm PEG Mushroomhydrogels, and blank 80 nm × 320 nm hydrogels (no drug) in cell lines of interest: A549(non-small cell lung), SKOV-3 (ovarian), and MDA-MB-468 (HER2-enriched breast).

3.4. Maximum tolerated dose of drug and particles in naïve mice

The maximum tolerated dose (MTD) is also an important factor indetermining the dose of a formulation and can be a predictor of clinicalsuccess [55]. It is also the first step in determining toxicity in a pre-clinical model. As shown in Fig. 6, the in vivo toxicity of cisplatin andPRINT-Platin was determined by monitoring the body weight of micedosed at varying levels of drug (3, 5, 6mg/kg and 5, 10, 15mg/kg for cis-platin and PRINT-Platin, respectively) over the course of six weekly in-jections. Soluble cisplatin was well-tolerated at 3 and 5 mg/kg dosesfor the entirety of the study. Following the fourth dose, however, all ofthe 6 mg/kg mice dropped below the acceptable weight loss. This wasin agreement with the MTD of cisplatin from other studies, where sub-stantial variation was shown based on dose schedule and mouse strain(ranging from 9 mg/kg for a single dose to 3–6 mg/kg for repeateddoses) [16,56,57]. PRINT-Platin was tolerated at higher doses thandrug alone; mice dosed at 5 and 10 mg/kg equivalent drug presentedminimal weight loss over the course of the six-week treatment. Othernanoparticle formulations have been shown to also have a higherMTD, as systemic exposure of drug is reduced in an encapsulated form

[58–60]. The highest dose, 15 mg/kg, was tolerated through fourdoses. Shortly after the fifth dose, however, all mice experienced signif-icant weight loss, indicating significant toxicity. Finally, a blank, non-loaded particle (equivalent concentration of the highest PRINT-Platindose) showed no decline in weight. This bolstered the in vitro results,providing further evidence that the carrier itself has no toxic side effects.

3.5. Tumor accumulation

A study tomeasure the tumor delivery of cisplatin via particle or sol-uble formwas undertaken. The ability to selectively accumulate drug atthe site of cancer can be a predictor of efficacy [61]. To that end, passivedelivery of cisplatin was examined in an orthotopic model of non-smallcell lung cancer and a subcutaneous model of ovarian cancer. In bothnon-small cell lung-derived and ovarian tumormodels, a trend towards

Fig. 5. Cisplatin and PRINT-Platin circulation behavior in plasma measured by inductively-coupled plasma mass spectroscopy. The plasma profile (A) and pharmacokinetic parameters(B) of cisplatin were significantly improved in particle form compared to soluble drug (* = p b 0.05; one-way ANOVA). Particle-bound versus released cisplatin in plasma over time(C) shows that less than 10% of drug is released from particle while in circulation. Accumulation of platinum in kidney (D) shows higher levels of drug for cisplatin compared toPRINT-Platin, suggesting renal protection.

75M.P. Kai et al. / Journal of Controlled Release 204 (2015) 70–77

higher accumulation of PRINT-Platin over cisplatin was observed(Fig. 7). Drug was dosed at the MTD of each formulation: 5 mg/kg forcisplatin and 10 mg/kg for PRINT-Platin. As expected, the data showedthat tumor exposure of cisplatin delivered via PRINT-Platin was at

Fra

ctio

n c

han

ge

in b

od

y w

eig

ht

Fig. 6.Change in bodyweight of nudemice dosedwith cisplatin, saline, PRINT-Platin (dosebased on cisplatin loading), andblank PRINT nanoparticles (sameparticle concentration as15 mg/kg PRINT-Platin).

least twice as high as the soluble form. Of interest was the 2.40-timeshigher AUC for PRINT-Platin in the ovarian model, indicative of an en-hanced accumulation of cisplatin when delivered in particulate form.In both types of tumors, the ability to transportmore drugwith less kid-ney accumulation would result in a higher therapeutic index. This

A5 4 9

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p ic

SK

OV

3s u b -q

0

1

2

3

Re

lati

ve

Tu

mo

rA

UC

C is p la tin

P R IN T -P la tin

Fig. 7. Relative exposure of cisplatin versus PRINT-Platin in two tumor models measuredby inductively-coupled plasma mass spectroscopy (sub-q = subcutaneous).

76 M.P. Kai et al. / Journal of Controlled Release 204 (2015) 70–77

suggests the potential for PRINT-Platin to have better efficacy over cis-platin in these models.

4. Conclusions

Cisplatin is a potent chemotherapeutic for a wide variety of tumormodels, but its dose-limiting toxicity—specifically renal failure—preventsit from beingmore efficacious. Furthermore, its continued use diminishesthe patient's standard-of-living due to neuro- and ototoxicity. The poten-tial benefits of formulating the drug into a nanoparticle include increasedtumor accumulation, prolonged circulation, and renal protection,resulting in a remedy formany of the traditional drawbacks to prescribingcisplatin.

We demonstrated a surface PEGylation density-dependent loadingof drug for PRINT hydrogel nanoparticles. For studies in a biological set-ting, this loading was compared to the circulation pharmacokinetic pa-rameters to find the optimal formulation. The ability to safely deliver alarger amount of drug is promising for antitumor studies. PRINT-Platinhas the potential to mitigate the dose-limiting toxicity associated withcisplatin, leading tomore efficacious treatment and a higher therapeuticindex. Future investigation into the in vivo anti-cancer effects of PRINT-Platin would reveal the potential for therapeutic applications.

Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.jconrel.2015.03.001.

Acknowledgments

The authors would like to thank Charlene Santos,Mark Ross, and theUNC Animal Studies Core for their support and indispensable help withthe murine experiments. They also thank Dr. Stuart Dunn, Dr. KevinChu, Yancey Luft, and Thomas Flannery for their valuable contributionsto this work, and Dr. Ashish Pandya for the synthesis of HP4A. All au-thors have given approval to the final version of the manuscript. Thiswork was supported by Liquidia Technologies and the Carolina Centerfor Cancer Nanotechnology Excellence (U54CA151652).

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