Enhanced oral bioavailability of a cancer preventive agent (SR13668) by employing polymeric...

RESEARCH ARTICLE

Enhanced Oral Bioavailability of A Cancer Preventive Agent(SR13668) by Employing Polymeric Nanoparticles with HighDrug Loading

HAO SHEN,1 ARYAMITRA A. BANERJEE,2 PAULINA MLYNARSKA,1 MATHEW HAUTMAN,3 SEUNGPYO HONG,4

IZET M. KAPETANOVIC,5 ALEXANDER V. LYUBIMOV,2 YING LIU1,4

1Department of Chemical Engineering, University of Illinois at Chicago, Chicago, Illinois 60607

2Department of Pharmacology, University of Illinois at Chicago, Chicago, Illinois 60612

3Center for Biomedical Testing, Chicago, Illinois 60612

4Department of Biopharmaceutical Sciences, University of Illinois at Chicago, Chicago, Illinois 60612

5National Cancer Institute, Bethesda, Maryland 20892

Received 15 May 2012; revised 18 June 2012; accepted 29 June 2012

Published online in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.23269

ABSTRACT: SR13668 [2,10-Dicarbethoxy-6-methoxy-5,7-dihydro-indolo-(2,3-b)carbazole] hasbeen proven effective in cancer prevention, but the limited bioavailability has hindered its clin-ical translation. In this study, we have developed a continuous, scalable process to form stablepoly(lactic-co-glycolic acid) nanoparticles encapsulating SR13668, based on understanding ofthe competitive kinetics of nanoprecipitation and spray drying. The optimized formulationachieved high drug loading (33.3 wt %) and small particles (150 nm) with narrow size distribu-tion. The prepared nanoparticle suspensions through flash nanoprecipitation were spray driedto achieve long-term stability and to conveniently adjust the nanoparticle concentration beforeuse. In vitro release of SR13668 from the nanosuspensions was measured in a solution withseparated organic and aqueous phases to overcome the limit of SR13668 low water solubility.Higher oral bioavailability of SR13668 by employing polymeric nanoparticles compared withthe Labrasol R© formulation was demonstrated in a mouse model. © 2012 Wiley Periodicals, Inc.and the American Pharmacists Association J Pharm SciKeywords: bioavailability; nanoparticles; stability; scalable; nanoprecipitation; spray drying;cancer chemoprevention

INTRODUCTION

Nearly 40% of the pharmaceutical compounds on themarket and 90% of the newly developed compoundsare hydrophobic, and therefore, difficult to deliver andto maintain sufficient bioavailability.1–3 Advances innanotechnology have shown innovative solutions fortargeted drug delivery and new methods for fabrica-tion of drug-delivery vehicles.4,5 Nanoscale particlesdramatically increase the surface to volume ratio andparticle solubility, which is higher than the bulk solu-

Correspondence to: Ying Liu (Telephone: +312-996-8249; Fax:+312-996-0808; E-mail: [email protected]); Alexander V. Lyubimov(Telephone: +312-996-9185; Fax: +312-996-7755; E-mail:[email protected])Journal of Pharmaceutical Sciences© 2012 Wiley Periodicals, Inc. and the American Pharmacists Association

bility of the compound. Among many nanostructuresand drug-delivery vehicles, polymeric nanoparticlesserve as promising drug carriers because of their bio-compatibility and biodegradability. A few methodshave been developed to prepare polymeric nanoparti-cles encapsulating hydrophobic drugs, such as super-critical processing,6,7 emulsification,8 sonication,9,10

and nanoprecipitation.11–13 However, limited drugloading (DL) (<15%) has been an issue for most poly-meric particles prepared by the above conventionalprocedures, despite the ideal hydrophobic interactionbetween the drug and the hydrophobic block of thecopolymer. Moreover, producing polymeric nanopar-ticles encapsulating hydrophobic drug compoundsin a scalable and reproducible manner is the keyfor nanoparticles to be practically applied as novelmedicine.

JOURNAL OF PHARMACEUTICAL SCIENCES 1

2 SHEN ET AL.

Figure 1. The steps of producing PLGA nanoparticles encapsulating SR13668 using theMIVM, followed by nanoparticle characterization, spray drying, and resuspension. Similar par-ticle size distributions before and after spray dry were achieved by optimize the processes ofFNP and spray dry.

In this work, a candidate cancer preven-tive agent, 2,10-dicarbethoxy-6-methoxy-5,7-dihydro-indolo-(2,3-b)carbazole (SR13668), was encapsulatedin poly(lactic-co-glycolic acid) (PLGA) nanoparticlesat high drug-loading rate and high encapsulation ef-ficiency (EE) by employing flash nanoprecipitation(FNP) (Fig. 1) to enhance its oral bioavailability.SR13668 is developed from a natural anticancer agentindole-3-carbinol (I3C), which can be found in crucif-erous vegetables, such as broccoli, cauliflower, andcabbage, and has promising anticancer activity bothin vitro and in vivo models.14 Compared with the nat-ural oligomeric products of I3C, SR13668 shows im-proved anticancer activities, negative results in thegenotoxicity battery, and low toxicity in subchronicrat and dog studies.15–17 In the application of preven-tive agents, only noninvasive routes of administration(such as oral administration) are acceptable. How-ever, the solubility of SR13668 in water is 10 ng/mL,18

which results in its poor oral bioavailability. Chemi-cal modification of the molecule to increase its solubil-ity (such as PEGylation) is difficult because the endgroups of the molecule are not active for reactions. The

strategy for increasing oral bioavailability of SR13668is to develop a drug-delivery system, which can en-hance SR13668 aqueous solubility and meanwhile re-main its high permeability through biological mem-branes. Two lipid surfactant-based SR13668 formu-lations, Solutol R© and Labrasol R©, have been used toincrease the compound oral bioavailability.17,19 How-ever, one disadvantage of using surfactant-based for-mulations for sustained drug delivery is that theymay not be as stable as polymeric nanoparticles uponlarge dilution in the blood stream. We have gener-ated polymeric nanoparticles encapsulating SR13668with high DL by employing FNP. Polymeric mi-celles and nanoparticles are more stable becauseof their low critical micelle concentration comparedwith the surfactants.20–22 SR13668 oral bioavailabil-ity in mice is an order of magnitude higher by usingour polymeric nanoparticle formulation, compared tothe Labrasol R©. Unlike other traditional processes inwhich DL depends on the thermodynamic equilibriumof the system, FNP enables rapid mixing to createhigh supersaturation and therefore rapid nucleationand growth of the drug. Previously, we have developed

JOURNAL OF PHARMACEUTICAL SCIENCES DOI 10.1002/jps

ENHANCED ORAL BIOAVAILABILITY OF A CANCER PREVENTIVE AGENT (SR13668) 3

and characterized a multi-inlet vortex mixer(MIVM)23,24 to facilitate the process of FNP. Particlegrowth kinetics at various drug-to-polymer ratios hasbeen studied to optimize the size and DL of the parti-cles. In addition, to increase the long-term stability ofnanoparticles by avoiding Ostwald ripening and par-ticle collision and to adjust the concentration of theparticles conveniently, nanoparticle suspensions werespray dried to be powder format (Fig. 1). By integrat-ing the MIVM with the spray drier, a continuous andscalable process has been achieved to generate andpurify a large amount of nanoparticles. Methods to re-disperse the nanoparticles are studied and reported.

EXPERIMENTAL

Materials

Poly(lactic-co-glycolic acid) [acid terminated, molec-ular weight (MW) 7000–17,000], tetrahydrofuran(THF), dimethyl sulfoxide (DMSO), ethanol, ace-tonitrile [high-performance liquid chromatography(HPLC) grade], ammonium formate, leucine, sucrose,and trehalose were purchased from Sigma–Aldrich(St Louis, MO, USA). Methyl tert-butyl ether (MTBE)(HPLC grade) was purchased from Fisher Scientific(Pittsburgh, PA, USA). SR13668 was provided byNational Cancer Institute (NCI) (Germantown, MD,USA). Labrasol R© was purchased from Gattefosse USA(Paramus, NJ, USA). Unless otherwise stated, allchemicals were purchased at standard grades andused as received.

Nanoparticle Preparation and Size Characterization

Poly(lactic-co-glycolic acid) nanoparticles encapsulat-ing SR13668 were generated by using the MIVM(Fig. 1). Among the four inlet streams, stream 1 waswith organic solution (0.2–0.8 wt % PLGA and 0.2 wt% SR13668 dissolved in THF). The other three inletstreams were Millipore (Billerica, MA, USA) wateras an antisolvent to precipitate the drug compound(SR13668) and the copolymer (PLGA). The volumet-ric flow rate of streams 1 and 2 was 6 mL/min and itwas 54 mL/min for streams 3 and 4.

Traditional nanoprecipitation (TNP) was also usedto generate the PLGA–SR13668 nanoparticles forcomparison. 0.5 mL organic solution (0.4 wt % PLGAand 0.2 wt % SR13668 dissolved in THF) was added to9.5 mL Millipore water with the stirring of a magneticstir bar, followed by rotating on a lab rotator (ThermoScientific, Labquake, Waltham, MA, USA) for 10 min.

Nanoparticle size distributions were measured bydynamic light scattering (DLS) (Zetasizer Nano ZS90;Malvern, Worcestershire, UK). The particle sizes werereported as the intensity-weighted radius.

Measurements of DL

Drug loading and EE were defined as,

DL(%) =Amount of SR13668 encapsulated in nanoparticles smaller than 450 nm

Total weight of nanoparticles

×100%

and

EE(%) =Amount of SR13668 encapsulated in nanoparticles smaller than 450 nm

Feeding amount of SR13668

×100%,

respectively.Drug loading of SR13668 in PLGA–SR13668

nanoparticles was quantified by ultraviolet–visible(UV–vis) spectroscopic measurements at the ab-sorbance wavelength of 310 nm, after the sequen-tial processes of dialysis, filtration through a 0.45mm polyethersulfone filter, freeze drying for 3 days,and being redissolved in DMSO at solid concentra-tion of 2 mg/mL. Therefore, SR13668 in the format oflarge crystal or encapsulated in particles larger than450 nm was excluded when DL was measured.

More details are presented as follows. First, af-ter generated, the nanoparticles were collected intoa dialysis bag (molecular weight cut-off 6000)0 anddialyzed against Millipore water for 24 h. Water waschanged every 30 min for the first hour, every 45 minfor the following 3 h, and four times for the rest of thetime. The dialyzed nanoparticle suspensions were dis-pensed in 20 mL amber vials, and then frozen in anultra-low temperature freezer (MDF-U52VAT; Sanyo,Moriguchi, Japan) for 2 h at −80◦C. The frozen sam-ples were freeze dried for 3 days in a freeze dryer(FreeZone 4.5 Liter Console Freeze Dry Systems; Lab-conco, Kansas City, MO, USA) at a vacuum pressureand −45◦C. The powder was collected and redissolvedin DMSO at solid concentration of 2 mg/mL. Finally,the amount of drug was quantified by UV–vis spectro-scopic measurements at the absorbance wavelength of310 nm. The extinction coefficient of SR13668 at 310nm is 8.8 × 104 M−1 cm−1.

Measurements of Particle Growth Kinetics

The growth kinetics of SR13668, PLGA, and SR13668together with PLGA was measured in situ by usinga DLS (NanoDLS; Brookhaven, Holtsville, NY, USA)at the batch mode. The scanning time was 10 s. Be-fore the measurements, nanosuspensions of SR13668were diluted 10 times, whereas PLGA and PLGA en-capsulating SR13668 were diluted 100 times usingMillipore water to maintain the appropriate countingintensity. Water viscosity and refractive index wereused for the measurements, which are 0.890 cP and1.331, respectively.

DOI 10.1002/jps JOURNAL OF PHARMACEUTICAL SCIENCES

4 SHEN ET AL.

Spray Drying of the Nanoparticle Suspensions

Spray drying of the nanoparticle suspensions was car-ried out by integrating the MIVM with a spray dryer(SD-05 Spray Dryer; LabPlant, North Yorkshire,UK). To prevent the nanoparticles from aggregat-ing, various amounts of excipients such as sucrose,trehalose, and leucine were added to the nanoparti-cle suspensions during the spray-drying process. 60%(v/v) ethanol was added to lower the inlet temperatureof the spray dryer. The inlet temperature was set be-tween 70◦C and 150◦C. The feed rate of the solutionswas 10 mL/min. Spray-dried powders were collectedin a glass container at the outlet of the spray dryer.

Nanoparticles were resuspended into sterileMillipore water at SR13668 concentration 0.75 mg/mL at vigorous stirring for 10 min before dosinganimals.

In Vitro Release of SR13668 from PLGA–SR13668Nanoparticle Suspensions

The spray-dried PLGA–SR13668 nanoparticles wereresuspended in 0.1 M phosphate buffered saline(PBS)1 at pH 2, 5, and 7.4 at the concentration of1 mg/mL, followed by the addition of MTBE at 4:1 ra-tio. The solutions were prepared following the sinkcondition that was previously described25 to ensurethat the measurements of SR13668 release kineticsis not limited by its solubility in PBS buffer. Releaseof SR13668 from the particles was measured at 20◦C.Five hundred microliter of samples was taken fromthe extractive solution (MTBE phase) at designedtime points (0, 0.5, 1, 2, 4, 6, 24, 48, and 72 h) and500:L fresh MTBE were added back to the nanosus-pensions every time to keep the constant volume.

SR13668 was measured using a liquidchromatography-mass spectrometry (LC-MS) [6430Triple Quad LC/MS equipped with an ultra HPLC(Agilent 1290 Infinity Binary Pump and Autosam-pler); Agilent, Santa Clara, CA, USA]2. SR13668was monitored at m/z 429.0 → 414.0 with a collisionenergy setting of 20. Samples were prepared forinjection by transferring a 300:L aliquot from eachtime point into the appropriate well of a 2 mL 96-wellpolypropylene plate. The aliquoted samples weredried down under a heated stream of nitrogen (setat 35◦C) using a Zanntek analytical evaporator(ZipVap 96; Glas-Col, Terra Haute, IN, USA). Afterdrying completely the samples were reconstitutedwith 100:L of acetonitrile and mixed well using aMicroplate Genie (USA Scientific, Ocala, FL, USA).Samples were then placed into the appropriate au-tosampler tray position for injection of 20:L sampleand subsequent analysis.

Standard curve samples were analyzed on each dayof analysis and injected throughout the injection se-quence. Standards were prepared by adding the ap-

propriate volume from a 200:g/mL stock solution ofSR13668 (in DMSO) to MTBE. Standard concentra-tions used were 37.5, 75, 150, 300, 600, and 1200 ng/mL. Calibrators were processed for analysis followingthe procedure described above.

The chromatographic column was a Thermo Sci-entific Aquasil C18, 5: , 100 × 2.1 mm2. The columntemperature was maintained at 25◦C, and a flow rateof 0.50 mL/min was used. The mobile phase (MP) con-sisted of 50 mM ammonium formate pH 6.5 (MP A)and 100% acetonitrile (MP B). The MP gradient wasas follows: after injection, initial conditions with MPB at 50% were held for 0.10 min, decreased to 5%and held constant for 2.5 min, returning to 50% at2.51 min for an additional 1.5 min to allow for re-equilibration. The total run time was about 5 min.The limit of quantitation was 37.5 ng/mL.

In Vivo Release and Systemic Levels of SR13668 in Mice

The protocol for the mice study was approved bythe UIC Animal Care Committee before initiation.Three groups of CD-1 mice (Charles River Breed-ing Laboratories, Portage, MI, USA) were used ata weight range of 25–35 g (n = 3/group). Animalswere identified by a facility unique (eartag) numberand were housed in an Association for Assessmentand Accreditation of Laboratory Animal Care Inter-national International-accredited facility in microiso-lator polycarbonate cages with Anderson bed-o’cobs R©

bedding (Heinold, Kankakee, IL, USA) in a temper-ature (64–79oF) and humidity (30%–70%) controlledroom with a 14 h light/10 h dark cycle. Teklad Certi-fied Rodent Chow No. 7012C (Harlan Inc., Madison,WI, USA), pellet form and autoclaved tap water inwater bottles were provided ad libitum from arrivaluntil termination. All animals were quarantined forat least 1 week before the initiation of dosing.

The PLGA particle suspensions encapsulatingSR13668 (nano and microsize particles) were pre-pared by resuspending dried powder in sterilewater and vortexing for 10 min at high speed.PEG400–Labrasol R©-based formulation was preparedby mixing PEG400 and Labrasol R© at 1:1 volume ra-tio followed by vigorous stirring for about 30 min.The appropriate amount of the SR13668 was weighedand added to 75% of required total volume of abovePEG400–Labrasol R©, followed by 120 min of stirringand then 5 min of sonication. Then, the remain-ing 25% volume of PEG400–Labrasol R© was addedto the mixture and stirred for 15 min. The finalPEG400–Labrasol R© formulation was stored at 2–8◦Cuntil dosing of the animals.

The mice were dosed via oral gavage at 5.5 mg/kgdose of SR13668 based on most recent body weight.Blood samples were subsequently withdrawn at threetime points per animal via orbital sinus. Three groups



of mice were dosed with PLGA–SR13668 nanopar-ticles, PEG400–Labrasol R© formulation of SR13668,and microsize PLGA–SR13668 particles, respectively.SR13668 concentrations from both plasma and wholeblood samples were determined using LC–MS methoddescribed above.

RESULTS AND DISCUSSION

Particle Fabrication and Resuspension

We have developed a strategy to use polymericnanoparticles to encapsulate the hydrophobic chemo-preventive agent, SR13668, by employing FNP inthe MIVM. FNP has demonstrated many advantagesover TNP and emulsification in preparation of vari-ous polymeric nanoparticles encapsulating hydropho-bic compounds, in terms of scalability, simplicity,and reproducibility.23,26 In this process, SR13668 andPLGA were dissolved in THF and precipitated by anantisolvent, during which copolymers aggregate andorganic-drug compounds nucleate and grow. We havepreviously demonstrated the particle size distributionas a function of mixing time.26,27 More specifically,fast mixing is essential to achieve high DL of 33.3% asreported in next section. In addition, the scalable fea-ture of FNP enables generation of hundreds of gramsof nanoparticles in our laboratory for large animaltests, which could be difficult to prepare by other lab-scale processes.

To maintain the long-term stability of the nanopar-ticles and to conveniently generate suspensions atvarious nanoparticle concentrations, spray drying, asthe post process after the particles were made, hasbeen applied and optimized. Excipients such as su-crose, trehalose, and leucine were used as “spacers”to prevent permanent aggregation.

Drug Loading

Traditional processes to prepare polymeric nanopar-ticles encapsulating hydrophobic compounds, such asemulsion and TNP, generally have problems of lowDL, broad size distribution, and difficulty of main-taining the stability of the nanoparticle suspensions.On the basis of Flory–Huggins theory and Chi mis-match, at thermodynamic equilibrium, DL driven byentropy change of mixing of the drug with the hy-drophobic core of the micelle is disfavored.28 FNP isa process of kinetic control instead of thermodynamicequilibrium, which overcomes the DL limit.

The polymeric nanoparticles encapsulatingSR13668 were prepared using the MIVM at a highReynolds number (Re) over 9000, ensuring that thesolvent replacement started homogeneously andmixing reached self-similarity.27 Definition of Re isconsistent with our previous studies.23 In the processof FNP, to produce stable nanoparticles, the kineticsof polymer aggregation and drug nucleation andgrowth have to be comparable to each other. Acidterminated PLGA (MW 7000–17,000) was chosento encapsulate SR13668 and four drug-to-polymerweight ratios (1:1, 1:2, 1:3, and 1:4) were tested.The optimized DL of 33.3% was achieved at drug-to-polymer 1:2 ratio. Further increasing the amount ofdrug resulted in much bigger particles (Fig. 2).

Stability of Nanoformulations

The growth kinetics of the nanoparticles (pureSR13668, pure PLGA, and SR13668:PLGA at 1:1, 1:2,1:3, and 1:4 ratios) was measured for up to 24 h, asshown in Figure 2. Growth of SR13668 alone in first10 min is in a linear fashion with a slope of about55 nm/min. With the presence of PLGA at 1:1 ra-tio, the polymer was not enough to provide surface

Figure 2. Growth kinetics of SR13668, PLGA, and SR13668 with PLGA (a) in 24 h and(b) in first 10 min (dashed area in Fig. 2a). SR13668 (black): SR13668 alone; PLGA1 1(green): SR13668 to PLGA 1:1 ratio; PLGA1 2 (blue): SR13668 to PLGA 1:2 ratio; PLGA1 3(wine): SR13668 to PLGA 1:3 ratio; PLGA1 4 (magenta): SR13668 to PLGA 1:4 ratio;PLGA1 2 trehalose (orange): PLGA nanoparticles (SR13668 to PLGA 1:2 ratio) in an aque-ous solution with 300 mg/mL trehalose; PLGA (red): PLGA alone.


6 SHEN ET AL.

coverage and to prevent aggregation. The growth ofthe particles behaved similar as the pure SR13668. Athigher PLGA to SR13668 ratios, the growth kineticsof the complex was dominated by polymer aggrega-tion. For the first 3 min, PLGA and PLGA–SR13668particles grew even faster compared with the particlesof pure SR13668 because PLGA has similar aggrega-tion rates but larger radius of gyration. Eventually,the growth of the polymeric particles slowed downdue to the surface charge repulsion. The growth kinet-ics of the polymeric particles in aqueous suspensions(at low SR13668 to PLGA ratios) is in a power-lawfashion. Initial fast growth of the particles is mainlycaused by aggregation because of the frequent par-ticle collision; whereas slower particle growth in thelater time span is predominately contributed by Ost-wald ripening,29 which cannot be prevented with thepresence of solution as the molecular-transport me-dia. To maintain the long-term stability of the parti-cles, the suspensions were then spray dried into theformat of solid powders. With the integrated MIVMand the spray drier, the complete process of producingdried nanoparticles took less than 10 min. Therefore,the growth kinetics of first 10 min is more critical todecide the size of the nanoparticles.

Resuspension of Spray-Dried Nanoparticles

Organic solvents are usually used to dissolvethe hydrophobic compounds and the polymers innanoprecipitation processes, and need to be com-pletely removed before any biomedical applicationis attempted. Moreover, the bulk solubility of thehydrophobic drug increases exponentially with thepresence of the organic solvent. Therefore, thetransport-limited process of Ostwald ripening hap-pens faster and particles keep growing bigger.29 Themost common process to eliminate the organic sol-vents is dialysis, which is time consuming and notscalable. Because a small amount of low-boiling-pointorganic solvent (such as THF) can facilitate solventevaporation during the spray-drying process, dialysisof the nanosuspension is not necessary after we in-tegrated the MIVM with the spray drier, which not

only reduced the preparation time resulting in betterstability but also ensured the scalability of the pro-cess. To prevent permanent aggregation during spraydrying and to be able to resuspend the nanoparticles,sugar molecules were added as the excipients. Addi-tion of sugar molecules also helped to reduce the colli-sion rate of the particles because of the high viscosityof the solution.30 Various types of sugar molecules(i.e., leucine, sucrose, trehalose) at various concen-trations have been tested. The results were reportedin Table 1. Leucine was more effective per mass. At1:5 ratio of nanoparticles to leucine, the nanoparti-cles could be redispersed to 445 nm. However, becausethe solubility of leucine in water is limited at 20 mg/mL, solutions at higher concentrations could not beprepared. By using trehalose and leucine together,the nanoparticle size could be further reduced to be340 nm in diameter, whereas a small peak of about80 nm also appeared. Compared with the original sizeof the particles before spray drying (150 nm), the par-ticles of about 340nm are most likely to be the dimersformed in the resuspension.

Because of the relatively low-glass-transition tem-perature of the PLGA used (∼60◦C), high inlet tem-perature of the spray drier may induce active chainmotion and result the permanent aggregation of theparticles. However, relatively high inlet temperatureis necessary to sufficiently dry the nanoparticles dur-ing the spray drying process. Ethanol is widely used tolower the inlet temperature because of its low boilingpoint. Because neither PLGA nor SR13668 dissolvesin ethanol, addition of ethanol should have minimal(if any) effect on PLGA–SR13668 nanoparticle sta-bility. In our study, 60% ethanol was added to theinlet stream. The inlet temperature was varied from100◦C to 70◦C to optimize the spray-drying process(Table 2). At the inlet temperature of 75◦C, popula-tion of the sub-200 nm particles reached the peak.The PLGA–SR13668 nanoparticles generated underthis condition were used in subsequent in vitro andin vivo release tests. When inlet temperature was fur-ther reduced, the size of the resuspended nanoparti-cles became bigger again, which might be the resultof insufficient drying.

Table 1. The Sizes of the Resuspended Nanoparticles [PLGA (MW 7000–17,000) Encapsulating SR13668at Ratio of 2:1] Depend on the Type and Amount of Excipients

Excipient MoleculeNanoparticle to

Sugar Mass RatioInlet Temperature

(◦C)Particle Size (nm)

After Resuspension

Trehalose 1:150 140 529.1Trehalose 1:200 140 406.3Sucrose 1:200 140 405.2Leucine 1:5 140 445.5Trehalose and leucine 1:200 and 1:5 140 341.1Trehalose and leucine 1:300 and 1:5 140 339.4(85%) and 79(12%)



Table 2. The Sizes of the Resuspended Nanoparticles [PLGA(MW 7000–17,000) Encapsulating SR13668 at Ratio of 2:1]Depend on the Inlet Temperature of the Spray Drier

Inlet Temperature (◦C) Size (nm)

100 526(63%) and 112(37%)90 505(62%) and 112(37%)85 629(41%) and 138(59%)80 600(41%) and 130(59%)75 622(31%) and 139(69%)70 339.4(85%) and 79(12%)

300 times trehalose and five times leucine were added as excipients.60% ethanol was also added to the inlet stream.

In Vitro Release

A phase-separated MTBE–PBS (0.1 M) system wassetup to monitor the in vitro release of SR13668 fromthe PLGA–SR13668 nanoparticles at pH 2, 5, and 7.4and at 20◦C, as shown in Figure 3. SR13668 releasedin aqueous phase were thermodynamically extractedinto the organic phase. Therefore, the measurementswere not limited by the extreme low water solubilityof the compounds. The release profile at all three pHconditions were very similar, which indicates that therelease kinetics was controlled by diffusion instead ofpolymer degradation. This is consistent with a previ-ous study.31 A burst release was observed in the firsthalf an hour, during which about 20% of SR13668 wasreleased from the PLGA–SR13668 nanoparticles. Af-ter that, the PLGA–SR13668 nanoparticles showedsustained release of SR13668. After 72 h, about 85%of the drug was released from the PLGA–SR13668nanoparticles.

Mice Data

Pharmacokinetics and in vivo availability of SR13668were measured in mice whole blood and plasmaafter oral administration of different formulations.

Figure 3. In vitro release of SR13668 from the PLGAnanoparticles in MTBE-aqueous two phase system.

Figure 4. Concentration of SR13668 in mice whole bloodand plasma after oral administration of the formulations.

Oral drug delivery presents more biological barri-ers before a pharmaceutical molecule reaches theblood circulation:32 (1) gastrointestinal (GI) track haswide pH span—from extremely acidic conditions ofthe stomach (pH 2.5–3.5) to neutral or slightly ba-sic conditions of the small intestine (pH 7–7.4); (2)the enzymes along the lumen of the intestine coulddegrade the potential pharmaceutical molecules; (3)the tenacious, viscous, elastic, and adhesive mucuslayer coating the intestinal epithelium is hard topass through by diffusion; and (4) the epithelium it-self is another barrier to prevent molecules to en-ter the blood circulation.32–34 PLGA nanoparticlesare promising carriers for oral drug delivery becausethey have been shown to prevent the degradation ofpharmaceutical molecule in the stomach and lumenof the intestine,32,35 and also promote mucus pene-tration and epithelial cell endocytosis by optimiza-tion of their size and surface properties. The sizeof the nanoparticles plays a key role in penetratingthe mucus layer and epithelial cells. Nanoparticlesbetween 50 and 200 nm show the best cell uptakein general,36 because they exhibit efficient interfa-cial interaction with the cell membrane.33,34 In ad-dition to the size of nanoparticles, the nanoparticlesurface properties, including surface charges, target-ing ligands, and hydrophilicity/hydrophobicity, alsoaffect the GI uptake.37,38 The tests on mice demon-strated a significant increase of SR13668 both inwhole blood and in plasma upon use of the smallerPLGA–SR13668 nanoparticles, as shown in Figure 4.Compared with the Labrasol R© formulation, the peakconcentration of SR13668 was increased more thanseven times in whole blood and about three times inplasma by employing nanoparticles with average di-ameter of 150 nm. In order for nanoparticles to en-hance the systemic availability of the hydrophobic


8 SHEN ET AL.

SR13668, PLGA–SR13668 particle size has to bewell controlled and high DL has to be achieved.As a comparison, micron-size PLGA–SR13668 par-ticles presented no help in increasing the oralbioavailability.

CONCLUSIONS

Polymeric nanoparticles have shown attractive po-tentials for many biomedical and biological applica-tions. It is important to elucidate the fundamentalthermodynamics and kinetics to guide the produc-tion of the particles in a repeatable and scalablemanner. We have developed a continuous process togenerate stable polymeric particles encapsulating thehydrophobic cancer-preventive compound, SR13668,with high DL of 33.3%. The procedure can be usedfor encapsulating other hydrophobic compounds withminimal modifications based on the solubility andprecipitation kinetics of the drug. It was demon-strated that instantaneous mixing ensures the higherDL. With the same composition, TNP could neverreach the same DL as achieved by FNP. PLGA wasused to encapsulate SR13668 because of its similarprecipitation kinetics compared with SR13668 andits property of mucoadhesion. The growth kineticsof the PLGA–SR13668 complex is mainly dominatedby PLGA, which significantly decreases the parti-cle growth rate of pure SR13668. At a SR13668 toPLGA ratio lower than 1:2, the size of the nanoparti-cles could be below 200 nm before completely quench-ing particle growth by spray drying. The optimizedspray drying parameters were found on the basisof the type and amount of excipients and the inlettemperatures—trehalose and leucine at concentra-tions 300 and five times of the nanoparticles and 75◦Cinlet temperatures. The spray dryer was integratedwith the MIVM, which reduced the preparation steps(such as dialysis) and therefore minimized the chancefor particles to grow.

PLGA–SR13668 nanoparticles significantly en-hanced the oral bioavailability of SR13668. Comparedwith the Labrasol R© formulation, the peak concentra-tion of SR13668 was increased more than seven timesin whole blood and about three times in plasma byusing PLGA–SR13668 nanoparticles.

ACKNOWLEDGMENTS

The study was supported by National Cancer Insti-tute, Department of Health and Human Services (con-tract number N01-CN-43306). The authors are grate-ful to Dr. Yoon Yeo at Purdue University for the accessof the spray dryer in her laboratory.

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