Treatment of chemotherapy-induced neutropenia in a rat model by using multiple daily doses of oral...

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Treatment of chemotherapy-induced neutropenia in a rat model byusing multiple daily doses of oral administration of G-CSF-containingnanoparticles

Fang-Yi Su a,b,1, Er-Yuan Chuang a,b,1, Po-Yen Lin a,b,1, Yi-Chun Chou a,b, Chiung-Tong Chen c,Fwu-Long Mi d, Shiaw-Pyng Weye, Tzu-Chen Yen f, Kun-Ju Lin e,f,**, Hsing-Wen Sung a,b,*

aDepartment of Chemical Engineering, National Tsing Hua University, Hsinchu, Taiwan, ROCb Institute of Biomedical Engineering, National Tsing Hua University, Hsinchu, Taiwan, ROCc Institute of Biotechnology and Pharmaceutical Research, National Health Research Institutes, Zhunan, Miaoli, Taiwan, ROCdDepartment of Biochemistry, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan, ROCeDepartment of Medical Imaging and Radiological Sciences, Chang Gung University, Taoyuan, Taiwan, ROCfDepartment of Nuclear Medicine and Molecular Imaging Center, Chang Gung Memorial Hospital, Taoyuan, Taiwan, ROC

a r t i c l e i n f o

Article history:Received 24 December 2013Accepted 8 January 2014Available online 27 January 2014

Keywords:Chemotherapy-induced neutropeniaTranslational medicineOral protein deliveryBiodistributionGlucose utilization

a b s t r a c t

Chemotherapy-induced neutropenia often increases the likelihood of life-threatening infections. In thisstudy, a nanoparticle (NP) system composed of chitosan and poly(g-glutamic acid) conjugated withdiethylene triamine pentaacetic acid (gPGA-DTPA) was prepared for oral delivery of granulocyte colony-stimulating factor (G-CSF), a hematopoietic growth factor. The therapeutic potential of this NP system fordaily administration of G-CSF to treat neutropenia associated with chemotherapy was evaluated in a ratmodel. In vitro results indicate that the procedures of NP loading and release preserved the structuralintegrity and bioactivity of the G-CSF molecules adequately. Those results further demonstrated theenzymatic inhibition activity of gPGA-DTPA towards G-CSF against intestinal proteases. Additionally, thein vivo biodistribution study clearly identified accumulations of G-CSF in the heart, liver, bone marrow,and urinary bladder, an indication of systemic absorption of G-CSF; its relative bioavailability wasapproximately 13.6%. Moreover, significant glucose uptake was observed in bone marrow during G-CSFtreatment, suggesting increased bone marrow metabolism and neutrophil production. Consequently,neutrophil count in the blood increased in a sustained manner; this fact may help a patient’s immunesystem recover from the side effects of chemotherapy.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction

Cytotoxic chemotherapy suppresses the production of neutro-phils, increasing the likelihood of life-threatening infections [1]. Asa protein produced by recombinant DNA technology, granulocytecolony-stimulating factor (G-CSF) has been increasingly used totreat chemotherapy-induced neutropenia [2]. As a hematopoieticgrowth factor, G-CSF predominantly promotes the differentiationand proliferation of neutrophilic granulocyte progenitor cells by

interacting with their cell surface G-CSF receptor (G-CSFR) [3]. Tomaintain its therapeutic effects, G-CSF is normally administered viadaily injections repeated over several days during chemotherapy[4]. However, frequent injection is inconvenient and painful forpatients, possibly causing infections at the injection site [5].

As an attractive alternative to parenteral injection, oral route ofdrug administration both avoids the pain and side effects associ-ated with daily injections, as well as offers a self-administrationcapability with a high degree of patient compliance [5]. However,orally administering protein drugs often creates a limitedbioavailability, owing to their presystemic degradation and inade-quate permeation through the intestinal epithelium [6]. Conse-quently, an adequate delivery vehicle is necessary to protect thetherapeutic proteins from inactivation in the gastrointestinal (GI)tract and improve their oral bioavailability.

Our recent work presented a pH-responsive nanoparticle (NP)vehicle composed of chitosan (CS) and poly(g-glutamic acid) con-jugated with diethylene triamine pentaacetic acid (gPGA-DTPA) for

* Corresponding author. Department of Chemical Engineering, National TsingHua University, Hsinchu 30013, Taiwan, ROC. Tel.: þ886 3 574 2504.** Corresponding author. Department of Medical Imaging and Radiological Sci-ences, Chang Gung University, Taoyuan, Taiwan, ROC.

E-mail addresses: [email protected] (K.-J. Lin), [email protected] (H.-W. Sung).

1 The first three authors (F.Y. Su, E.Y. Chuang, and P.Y. Lin) contributed equally tothis work.

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Biomaterials

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0142-9612/$ e see front matter � 2014 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.biomaterials.2014.01.020

Biomaterials 35 (2014) 3641e3649


the oral delivery of insulin [7]. The CS can adhere to the intestinalsurface to impart transient opening of the tight junctions (TJs)between epithelial cells, thereby increasing its paracellularpermeability [8]. As a well-known complexing agent, DTPA canchelate divalent metal ions, subsequently inhibiting intestinalproteases and disrupting epithelial TJs [9,10]. Our experimentalresults demonstrated the effectiveness of CS/gPGA-DTPA NPs aspermeation enhancers and their ability to significantly enhance theintestinal absorption of insulin, ultimately lowering the bloodglucose in diabetic rats.

By extending the results of above research, this work evaluatesan approach in which the CS/gPGA-DTPA NP system is used fordaily administration of G-CSF via an oral route during one cycle ofchemotherapy in a rat model. The effectiveness of gPGA-DTPA inprotecting G-CSF from intestinal enzymatic degradation is evalu-ated. Additionally, the structural integrity of G-CSF moleculesreleased from test NPs is investigated by determining their mo-lecular weight. Meanwhile, their bioactivity is assessed by a NFS-60cell proliferation assay, in which free-form G-CSF is used as acontrol. Previous research has used NFS-60 cells, a murine myelo-blastic cell line with the G-CSFR on their cell surface, as an in vitromodel to evaluate the G-CSF bioactivity [11,12]. The biodistributionof G-CSF, as orally delivered via CS/gPGA-DTPA NPs, is examined bysingle-photon emission computed tomography (SPECT)/computedtomography (CT). Its subsequent stimulation of glucose uptake inbone marrow is observed using positron emission tomography(PET)/CT. Moreover, their pharmacokinetic (PK) profiles of plasmaG-CSF level and pharmacodynamic (PD) effects on producing asustained increase in circulating neutrophilic granulocytes areanalyzed as well.

2. Materials and methods

2.1. Materials

The 85%-deacetylated CS (MW 60 kDa) was obtained from Koyo Chemical(Japan), while gPGA (MW 55 kDa) was purchased from Vedan (Taichung, Taiwan).The G-CSF was acquired from Mycenax Biotech (Miaoli, Taiwan). Other chemicalsand reagents used were of analytical grade and obtained from SigmaeAldrich (St.Louis, MO, USA).

2.2. Inhibition of G-CSF degradation by gPGA-DTPA

The gPGA-DTPA was synthesized based on a method described in our previouspublication [7]. Its ability in protecting G-CSF from enzymatic degradation wasstudied using the proximal portion of an intestinal tract freshly isolated from rats(Wistar, about 250 g). The isolated intestinal segments were longitudinally dissected(1 cm2), followed by mounting onto a modified Franz diffusion cell [13]. The intes-tinal tissues were then incubated with a KrebseRinger buffer solution containingfree-form G-CSF (1.1 mg/mL, 0.5 mL) and gPGA-DTPA (11 mg/mL, 0.5 mL) at 37 �C.Next, the amount of intact G-CSF remaining was quantified by withdrawing a 50 mLof test solution at different time intervals and immediate analysis by high-performance liquid chromatography (HPLC; LC-NetII/ADC, JASCO, Tokyo, Japan).The G-CSF solution without gPGA-DTPA was used as a control.

2.3. Preparation and characterization of G-CSF-containing NPs

During preparation of G-CSF-containing NPs (G-CSF NPs), an aqueous G-CSF(1.1 mg/mL, 0.25 mL, pH 4.0) was premixed with an aqueous CS solution (1.2 mg/mL, 2.5 mL, pH 6.0) in the presence of sodium tripolyphosphate (TPP, 1.25 mg/mL,0.25 mL) by magnetic stirring at 4 �C for 6 h. An aqueous gPGA-DTPA (10 mg/mL,75 mL, pH 7.4) was subsequently added into the mixed solution and blendedthoroughly at room temperature in order to form the CS/gPGA-DTPA NPs con-taining G-CSF; the obtained G-CSF NPs were collected by centrifugation(10,000 rpm at 4 �C for 50 min). The loading efficiency (LE) and content (LC) of G-CSF in the as-prepared NPs were determined by assaying the amount of free G-CSFin the supernatants using HPLC [14]. To reveal their intestinal stability, the particlesize and zeta potential value of G-CSF NPs at distinct pH values, in which the pHenvironments in the GI tract were simulated, were assessed using a Zetasizer(3000HS, Malvern Instruments, Worcestershire, UK); meanwhile, changes in theirmorphology were examined by transmission electron microscopy (TEM, JEOL,Tokyo, Japan).

2.4. In vitro release study

In vitro release studies were performed by incubating G-CSF NPs, enclosed indialysis bags (cellulose membrane, MWCO 50,000, Sigma), in phosphate bufferedsaline (PBS) with various pH values at 37 �C under mild agitation (100 rpm) in awater bath. At predetermined time intervals, samples were withdrawn from theincubation medium and the amount of G-CSF that was released from test NPs wasquantified by HPLC. The amount of released G-CSF was expressed as a percentage ofthe total G-CSF associated with test NPs as calculated from their LE [14].

2.5. In vitro G-CSF transport study

The ability of CS/gPGA-DTPA NPs to enhance the transport of G-CSF via openingthe epithelial TJs was investigated in intro in Caco-2 cell (human colon adenocar-cinoma) monolayers [14]. The grown cell monolayers were incubated with test NPscontaining G-CSF at distinct pH environments (pH 6.6, 7.0, or 7.4 in the donorcompartment and pH 7.4 in the receiver compartment) to replicate the physiologicalconditions in different segments of the small intestine [15]. Samples (50 mL) werecollected from the receiver compartment at distinct time intervals, and their G-CSFconcentrations were measured by using ELISA (Human G-CSF Instant ELISA; eBio-science, San Diego, CA, USA).

2.6. Structural integrity and bioactivity of the G-CSF released from test NPs

The structural integrity of G-CSF molecules that had been released from the testNPs in a neutral pH environment due to their pH-sensitivity (the released G-CSF)was exposed by measuring their molecular weight, using the matrix-assisted laserdesorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS; Micro-mass, Manchester, UK). TheMALDI-TOFMS allows for characterization of the proteinstructures at various levels, including their covalent structure, conformation, dy-namics and interaction with physiological partners [16]. Free-form G-CSF moleculeswere used as a control group.

This study also attempted to determine their ability to bind to the cell surface G-CSFR, by co-culturing test NPs containing the fluorescein isothiocyanate-labeled G-CSF (FITC-G-CSF) with NFS-60 cells at pH 7.4. Twenty four hours later, cells werewashed three times with PBS, fixed in 4% paraformaldehyde, incubated with a Cy3-conjugated antibody (rabbit anti-G-CSFR; Abcam, Cambridge, MA, USA) overnight at4 �C, and then examined via a fluorescence microscope (Axio Observer Z1, Carl Zeiss,Göttingen, Germany).

Once binding to G-CSFR, bioactivity of the released G-CSF in stimulating theproliferation of NSF-60 cells was also evaluated [17]. Briefly, NFS-60 cells weresuspended in the RPMI 1640 medium containing 10% fetal bovine serum (FBS) andthen aliquoted to 96-well tissue culture plates (50 mL/well, 2 � 105 cells/mL). Todeactivate its containing proteolytic enzymes, the RPMI 1640 medium was heatedfor 1 h at 59 �C right before use followed by cooling to 37 �C for cell culture appli-cation [18]. Free-form G-CSF and G-CSF NPs (0.01, 0.1, or 1.0 ng/mL G-CSF; 50 mL/well) were then individually added to each well of the culture plate and incubated at37 �C in an incubator for 48 h. Next, cell proliferation assay was performed by usingthe CellTiter 96� AQueous One Solution Cell Proliferation Assay kit (Promega, Madi-son,WI, USA) [19]. The absorbance of each test well, which is directly proportional tothe number of living cells in culture, was read at 490 nm using a multi-modemicroplate reader (Molecular Devices, Sunnyvale, CA, USA). The NFS-60 cellswithout any treatment and those treated with empty NPs were used as the controls.

2.7. Animal studies

Animal studies were performed according to the “Guide for the Care and Use ofLaboratory Animals”, as prepared by the Institute of Laboratory Animal Resources,National Research Council and published by the National Academy Press in 1996,and approved by the Institutional Animal Care and Use Committee of National TsingHua University (protocol number 10046).

2.7.1. Biodistribution studyDynamic biodistributions of the G-CSF orally delivered via CS/gPGA-DTPA NPs in

rats were assessed by using a high-resolution dual-modality NanoSPECT/CT system(Bioscan, Washington DC, USA), allowing their non-invasive visualization andcharacterization of biological processes in a temporal manner [20]. In this study, G-CSF was radiolabeled with Iodine-123 (123I) by the iodogen precoated tubes (PierceIodination Tubes, Thermo Fisher Scientific, Rockford, IL, USA) as per the manufac-turer’s instructions. Radiolabeled NPs were subsequently prepared using the as-prepared 123I-G-CSF.

Following oral administration of test NPs containing the radiolabeled 123I-G-CSFin rats (Wistar,w250 g), dynamic SPECT imageswere acquired. Details of radiotracerdistribution were then examined by collecting additional CT images for anatomicalreference. Rats treated with free-form 123I-G-CSF via oral ingestion or subcutaneous(SC) injection were used as the controls (n ¼ 3 for each group). Animal imaging wasalso performed under the controlled temperature (37 �C) and anesthesia (1.5% iso-flurane in 100% oxygen). Details of the protocol used in the image scanning can befound in our earlier study [21].

F.-Y. Su et al. / Biomaterials 35 (2014) 3641e36493642


2.7.2. PK profilesRats (Wistar, ca. 250 g) were fasted overnight, and fasting continued during the

experiment, although the rats were allowed water ad libitum. The following formu-lations were administered individually to the overnight-fasted rats: oral free-formG-CSF (900 mg G-CSF per kg of rat; 900 mg G-CSF/kg) solution, oral G-CSF NP sus-pension (900 mg G-CSF/kg), and SC injection of G-CSF solution (100 mg G-CSF/kg; n¼ 6for each studied group). The oral formulations were administrated to rats by using ananimal feeding needle. Blood samples were withdrawn from the tail veins of rats.

Plasma concentrations of the absorbed G-CSF were determined by centrifugingblood samples (12,000 rpm, 4 �C, for 10 min) with subsequent quantification usingELISA (Human G-CSF Instant ELISA). The relative bioavailability (BAR) of G-CSF wascalculated using the following formula [22].

BAR ¼ AUCðoralÞ � DoseðSCÞAUCðSCÞ � DoseðoralÞ

� 100%

where AUC represents the total area under the plasma G-CSF concentration vs. timecurve.

2.7.3. PD recovery of chemotherapy-induced neutropeniaThis study also examined how the therapeutic effects of G-CSF NPs stimulate the

proliferation of neutrophils on an animal model of neutropenia induced by an anti-cancer drug, 5-Fluorouracil (5-FU) [23]. Rats (Wistar, approximately 250 g) wereorally treated with 5-FU (100 mg/kg) on day 1 and then randomized into treatmentgroups as follows: oral free-form G-CSF solution (900 mg G-CSF/kg/day), oral G-CSFNP suspension (900 mg G-CSF/kg/day), and SC injection of G-CSF solution (100 mgG-CSF/kg/day) on days 0 through 9 [24]. Rats without 5-FU/G-CSF treatments wereused as a control group (n¼ 6 for each studied group). Blood samples were collecteddaily from tail veins of test rats at 8 h after each G-CSF treatment [25]; the absoluteneutrophil count (ANC) was determined using a hematology analyzer (Hemavet 850FS; CDC Technologies, Oxford, CT, USA).

Upon completion of the sequential G-CSF treatments (day 9), test rats weresacrificed and their vertebral columns were removed for neutrophil immunostain-ing to visualize the neutrophil proliferation in bone marrow. The vertebral columnswere washed with PBS, fixed in paraformaldehyde, and subsequently decalcified viaincubation in a decalcifier solution (Decalcifier II; Leica Biosystems, Richmond, VA,USA). Following decalcification, samples were embedded in paraffin and sectioned(5 mm in thickness). Immunohistochemical staining was then performed by incu-bating sections with a primary antibody (rabbit anti-neutrophil; Millipore, Bedford,MA, USA) at a 1:50 dilution overnight at 37 �C. After rinsing 3 times with PBS, tissuesamples were incubated with an Alexa-Fluor-conjugated secondary antibody(Jackson Immunoresearch Laboratories, West Grove, PA, USA) with a 1:50 dilution.Moreover, samples were counterstained with Hoechst to visualize the nuclei andexamine them by an inverted confocal laser scanning microscope (CLSM, ZeissLSM780, Carl Zeiss, Göttingen, Germany). Finally, the obtained CLSM images wereanalyzed by an image software program (Image J software, National Institute ofHealth, Bethesda, MD, USA).

2.7.4. Enhancement of glucose uptakeAs is well known, G-CSF therapy increases glucose uptake in bone marrow [26].

This study examined how G-CSF treatments enhance glucose uptake in bonemarrow by using PET/CT. Following sequential G-CSF treatments in the PD study,rats were injected intravenously with 18F-2-fluoro-2-deoxy-D-glucose (18F-FDG,2 mCi/kg), a radiopharmaceutical analog of glucose for clinical use [27]. Immediatelyafter administration of 18F-FDG, dynamic PET images consisting of 18 time frameswere obtained through a PET scanner (Siemens Medical Solutions, Knoxville, TN,USA). The obtained images were reconstructed using the 2D OSEM method (i.e. 4iterations and 16 subsets) without attenuation corrections. By using a color scale, theradioactivity concentration in each pixel was displayed in terms of the standardizeduptake value (SUV), where SUV was determined as the ratio of the regional radio-activity concentration divided by the injected amount of radioactivity normalized tobody weight [28]. Following PET scanning, whole-body CT images were acquiredusing NanoSPECT/CT. Dynamic 3D images of 123I-G-CSF SPECT/CT and 18F-FDG PETobtained from the same animals were co-registered and fused to their reference CTimages by using an imageworkstation (PMOD Technologies, Zurich, Switzerland), toillustrate the G-CSF-stimulated glucose uptake.

2.8. Statistical analysis

The groups were compared using the one-tailed Student’s t-test (SPSS, Chicago,Ill). All data were presented as a mean value with its standard deviation indicated(mean � SD). Differences were considered statistically significant when the P valueswere less than 0.05.

3. Results and discussion

Although 5-FU is a common chemotherapy drug, previousstudies have described an increased risk of neutropenia after its

administration [29,30]. Exogenous administration of haemopoieticgrowth factors G-CSF increases the number of immune cells foundin bone marrow and peripheral blood, significantly shortening theneutropenia period [31]. Due to its renal elimination and specificdegradation mediated by G-CSFR or neutrophil elastase, the in vivohalf-life of G-CSF is relatively short (approximately 4e8 h) [32].Therefore, multiple injections of G-CSF are required during onecycle of chemotherapy treatment. In this study, DTPA was cova-lently conjugated on gPGA to form NPs with CS for oral G-CSF de-livery. This study demonstrate that this NP system can inhibit theproteolytic degradation and promote the intestinal absorptionability of G-CSF, thus enhancing the oral bioavailability of G-CSF andincreasing the circulation of neutrophilic granulocytes in a sus-tainable manner via multiple-dose treatments.

3.1. Characteristics of test NPs

The as-prepared G-CSF NPs in aqueous suspension had a particlesize of 287.0 � 84.2 nm and a zeta potential of 37.3 � 4.7 mV; theirLE and LC of G-CSF were 70.4 � 5.2% and 21.2 � 1.9%, respectively(n ¼ 6 batches). The pH-sensitivity of these G-CSF NPs was exam-ined at different pH values, representing the corresponding envi-ronments of fasted stomach and small intestine [33]. According toTable 1 and Fig. 2a, test G-CSF NPs swelled gradually with anincreasing pH and eventually disintegrated at pH 7.4. Meanwhile,their zeta potential value decreased markedly (P < 0.05), likelyowing to the deprotonation of CS (pKa 6.5) [34]. Above resultssuggest that the as-prepared NPs may protect the loaded G-CSFmolecules from the attack of gastric acid and release them close tothe intestinal epithelium, where the pH environment is nearneutral [35,36].

Fig. 2b presents the release profiles of G-CSF from test NPs at pH2.0, 6.6, 7.0 and 7.4, simulating the pH conditions in the pH envi-ronments of the fasting stomach, duodenum, jejunum, and thesurface of intestinal epithelia, respectively [15]. At pH 2.0, therelease of G-CSF from test NPs was minimal, suggesting that G-CSFNPs remained intact. Conversely, test NPs became unstable anddisintegrated at pH 7.0e7.4, resulting in a fairly fast release ofG-CSF.

3.2. Inhibition of intestinal proteolytic degradation

Orally administering proteins as drug treatment poses a greatchallenge since they are subject to proteolysis in the GI tract. Mostintestinal proteases are divalent cation-dependent enzymes [9].Complexing agents such as DTPA can inhibit the activity of intes-tinal proteases by depriving the essential cations (e.g. Ca2þ andZn2þ) out of their enzyme structures. Related studies evaluated theinhibitory activity of gPGA-DTPA against proteases by using theproximal intestinal tract freshly isolated from rats, which is knownto have the maximum proteolytic activity [37]. According to Fig. 1,proteolytic enzymes in the small intestine degraded free-fromG-CSF rapidly (approximately 98% degradation within 2 h).Conversely, the addition of gPGA-DTPA induced a pronouncedprotective effect towards G-CSF against proteases (50% intact G-CSF

Table 1Mean particle sizes (nm) and zeta potential values (mV) of G-CSF NPs at distinct pHenvironments (n ¼ 6).

pH value Mean particle size Zeta potential

2.0 311.1 � 195.7 38.1 � 0.76.0 287.0 � 84.2 37.3 � 4.76.6 337.4 � 90.2 11.0 � 1.27.0 521.4 � 180.1 6.4 � 0.17.4 NP disintegration 2.1 � 0.1

F.-Y. Su et al. / Biomaterials 35 (2014) 3641e3649 3643


remaining at 2 h, P < 0.05). Above results imply that the enzymaticinhibition activity of gPGA-DTPA can be executed when G-CSFrelease occurs owing to the disintegration of test NPs.

3.3. In vitro transport of G-CSF

The ability of CS/gPGA-DTPA NPs to facilitate the intestinaltransport of G-CSF was evaluated in Caco-2 cell monolayers, which

have been widely used as an in vitro model to evaluate the ab-sorption enhancement of macromolecules [38]. The G-CSF has amolecular weight of about 19 kDa and a size of approximately2.0 � 2.5 � 4.5 nm [39]. Meanwhile, as is well known, the width ofTJs opened by absorption enhancers is less than 20 nm [40].

According to Fig. 2c, the amount of G-CSF transported throughCaco-2 cell monolayers was negligible in the presence of free-fromG-CSF (Control). Conversely, when incubated with test NPs, sig-nificant amounts of G-CSF transported through cell monolayerswere observed regardless of their pH environments exposed(P < 0.05). Above results suggest that CS/gPGA-DTPA may facilitatethe intestinal absorption of G-CSF in the pH conditions from theproximal duodenum to the distal ileum along the intestinal tract(pH 6.0e7.4).

3.4. Structural integrity and bioactivity of the G-CSF released fromtest NPs

Comparative structural analysis was made of free-form G-CSFand the G-CSF released from test NPs (the released G-CSF) by usingMALDI-TOF MS, which has been extensively adopted to determinethe exact molecular mass, structural integrity and identity of theprotein [16]. According to Fig. 3a, the molecular weights of the twotest samples were comparable, indicating that the structuralintegrity of the G-CSF molecules that had been through the pro-cedures of NP loading and release still remained intact.

While obtained from leukemia cells infected with the Cas-Br-Mmurine leukemia virus, NFS-60 cells could differentiate into neu-trophils and macrophages in the presence of certain growth factors[17]. The G-CSF can bind to NFS-60 cells via a specific cell surface

Fig. 1. Results of G-CSF degradation in the absence and presence of gPGA-DTPA, usingthe proximal intestinal segments freshly isolated from rats. *Statistical significance at alevel of P < 0.05.

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receptor (G-CSFR) and stimulate their proliferation [3]. This studymore closely examined by evaluating its ability to bind to the G-CSFR on NFS-60 cells. Therefore, G-CSF was conjugated with FITCbefore loading into test NPs. The NPs containing FITC-G-CSF andfree-form FITC-G-CSF were then individually incubated with NFS-60 cells. Similar to that treated with free-form FITC-G-CSF, co-localization of the released FITC-G-CSF (green color (in webversion)) and G-CSFR (red color) was clearly observed (Fig. 3b).Above results demonstrate that following its release from test NPs,the receptor-binding ability of G-CSF was still preserved.

By binding to its receptor, G-CSF activates the downstreamsignaling cascades to stimulate the proliferation of neutrophilicgranulocytes and their progenitor cells [3]. This study also evalu-ated the bioactivity of the released G-CSF by determining its abilityto stimulate the proliferation of NFS-60 cells, which was measuredby the amount of 490 nm absorbance [19]. As compared to thecontrol group without any treatment, the NPs containing no G-CSF(empty NPs) did not affect the proliferation of NFS-60 cells(P > 0.05, Fig. 3c). Conversely, when incubated with free-from G-CSF or the NPs containing G-CSF at neutral pH, the number of NFS-60 cells was significantly increased in a dose-dependent manner(P < 0.05). Additionally, their extents of cell increase were com-parable (P > 0.05), suggesting that the proliferative activity of theG-CSF released from test NPs is well conserved.

3.5. Biodistribution of G-CSF

The SPECT and PET have been used for non-invasive examina-tion of biological processes in living subjects [41]. Fig. 4 presentsthe SPECT/CT images of the radioactivity distribution of 123I-G-CSFin rats administered via different formulations at various time in-tervals in the lateral tomographic view. According to this figure,following oral administration of free-form 123I-G-CSF, the bio-distribution of radioactivity was present only along the GI tract; noapparent systemic absorption of 123I-G-CSF was observed.Conversely, similar to the group receiving SC free-form 123I-G-CSF,high levels of radioactivity accumulated in the heart, liver, bonemarrow (as indicated by the white arrows), and urinary bladderwere found in rats orally treated with 123I-G-CSF NPs, indicative oftheir systemic absorption of 123I-G-CSF.

3.6. PK profiles

After administration of different G-CSF formulations, the plasmaG-CSF level vs. time profiles and their related PK parameters weredetermined. According to Fig. 5a, the rats subcutaneously treatedwith free-form G-CSF resulted in a highest plasma level at 2 h posttreatment. No detectable plasma G-CSF level was observed for therats receiving free-form G-CSF via oral route, suggesting that a

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Fig. 3. Comparative analyses of free-form G-CSF and the G-CSF released from test NPs. Results of their: (a) molecular weight measured by MALDI-TOF MS; (b) binding ability to theG-CSFR on NFS-60 cells examined by immunofluorescence staining; and (c) bioactivity in stimulating the proliferation of NFS-60 cells determined by a cell proliferation assay. N.S.:statistically insignificant. *Statistical significance at a level of P < 0.05.



suitable delivery vehicle is needed for oral G-CSF ingestion.Conversely, orally administering G-CSF NPs revealed a maximum G-CSF level at 8h after treatment; in this case, theAUCwas19.6�0.3ngh/mL, and its corresponding BAR was 13.6% (Table 2). Analytical re-sults demonstrate that CS/gPGA-DTPA NPs can effectively deliver G-CSF into the systemic circulation by an oral route. The observed 6-hdelay in their maximum plasma G-CSF level was owing to thetransit time of CS/gPGA-DTPA NPs spent through the GI tract.

Although a higher dose of active protein drugs is required fororal formulation than parenteral administration, the cost can bepartially offset by removing the requirement for sterile injectablemanufacturing [42]. Additionally, with the advancement in re-combinant DNA technology, the production cost of protein drugs iscontinuously decreasing. Consequently, cost effective oral admin-istration of protein drugs is likely to be available in the foreseeablefuture.

3.7. PD recovery of chemotherapy-induced neutropenia

Due to its short half-life, multiple daily injections of G-CSF arerequired to treat neutropenia occurring during chemotherapy [32].

In this study, the PD recovery of neutrophils was performed with adaily treatment of different G-CSF formulations in a neutropenia ratmodel induced by 5-FU on day 1 following first G-CSF treatment[43]. As expected, no apparent variation in ANCwas detected in thecontrol rats without any treatment throughout the study (Fig. 5b).Conversely, the other three test groups showed a significantamount of neutropenia following their 5-FU treatment. A gradualrecovery of ANC was observed for the group orally receiving free-form G-CSF. Notably, the rats treated with free-form G-CSF via SCinjection or G-CSF NPs through oral ingestion significantlyincreased plasma ANC immediately after their first G-CSF treat-ment. Clinically, G-CSF can be used before chemotherapy to activatethe bone marrow to produce more stem cells from which all otherblood cells are made [44]. Following chemotherapy-induced neu-tropenia, ANC increased in both studied groups in a sustainablemanner, possibly due to their neutrophil productions in bonemarrow stimulated by the absorbed G-CSF (Fig. 5a).

This study also more closely examined the bioactivity of theabsorbed G-CSF in stimulating the neutrophil production by per-forming immunohistochemical analyses on the transverse sectionof the vertebral column of test rats, following the sequential

Free

-form

G

-CSF

(SC

)

Bm

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B

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G-C

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NPs

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l)

H: heart; E: esophagus; L: liver; St: stomach;Bm: bone marrow; B: bladder

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-CSF

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l)

0.5 h 1 h 3 h 4 h

0.2

0.1

0kcps

St

H

L StBm

B

CT

E

E

Fig. 4. Soft-tissue contrast CT images (gray color) superimposed with SPECT images (green color) showing the distribution of 123I-G-CSF in rats administered via different for-mulations at various time points in the lateral tomographic view. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of thisarticle.)



treatments of G-CSF. As is well known, the red bone marrow in thevertebral column is major source for neutrophil production [45]. Incontrast with the group orally treated with free-form G-CSF, asignificant amount of neutrophil production was observed in thegroups receiving SC free-form G-CSF or oral G-CSF NPs (P < 0.05,Fig. 5c). Above results demonstrate that the absorbed G-CSF orallydelivered by CS/gPGA-DTPA NPs can retain its bioactivity, thusaccelerating the recovery of test rats from chemotherapy-inducedneutropenia.

3.8. Enhancement of glucose uptake

Increased glucose uptake of bone marrow is often observedduring and after G-CSF therapy [46]. Tissue accumulation of 18F-FDG in rats was assessed visually using PET/CT, and their intensitywas analyzed semi-quantitatively by determining their SUV in bonemarrow, reflecting their glucose utilization [47]. According to Fig. 6aand b, minimal accumulation of 18F-FDG was found in the rats thatreceived free-form G-CSF orally. This observation is likely owing totheir lack of systemic absorption of G-CSF (Fig. 5a), thereby limitingtheir 18F-FDG utilization. In contrast, glucose uptakes were clearlyidentified in bone marrow (as indicated by the white arrows inFig. 6a) for the other two test groups (i.e. SC free-form G-CSF or oralG-CSF NPs) that had significant absorption of G-CSF systemically.

To further illustrate the G-CSF-enhanced 18F-FDG uptake in bonemarrow for the group orally receiving G-CSF NPs, the Supplemen-tary Information provides a time-lapse 3D video; the group orallytreatedwith free-form G-CSF was used as a control. As illustrated inthe video, oral absorption of G-CSF (green color) NPs to rats shortlybefore PET imaging evaluation with 18F-FDG (rainbow color)markedly increased 18F-FDG accumulation in bone marrow overtime. The increase in 18F-FDG uptake of bone marrow could beexplained by increased bone marrow metabolism and cellularityduring G-CSF NP treatment [48].

0

1

2

3

4

0 2 4 6 8 10 12 14 16 18 20 22 24Plas

ma

G-C

SF L

evel

(ng/

mL)

Time (h)

a Free-form G-CSF (SC, 100 μg/kg)

(n = 6)

Days after First G-CSF Treatment

Abs

olut

e N

eutr

ophi

lC

ount

(x10

3 ce

lls/μ

l)

5-FU Administration

G-CSF Treatments

b

Free-form G-CSF (Oral, 900 μg/kg) G-CSF NPs (Oral, 900 μg/kg)

DIC NeutrophilsNuclei

Free

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G

-CSF

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)G

-CSF

NPs

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l)

c

0123456789

10

(1) 0 1 2 3 4 5 6 7 8 9

Control

Neutrophil Count per Bone Marrow Area (103 cells/mm2)0 10 20 30 40 50 60 70 80

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-form

G

-CSF

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l)

500 μm

Fig. 5. Results observed in chemotherapy-induced neutropenia rats treated with multiple daily doses of different G-CSF formulations: (a) plasma G-CSF level vs. time profiles (PKprofiles); (b) absolute neutrophil count in blood vs. time profiles (PD profiles); and (c) CLSM images of immunohistochemical staining of the transverse section of the vertebralcolumn at the end of the sequential G-CSF treatments.

Table 2Pharmacokinetic parameters of G-CSF in test rats after subcutaneous (SC) injectionof free-form G-CSF, oral ingestion of free-form G-CSF, or oral ingestion of G-CSF NPs.Cmax: maximum plasma concentration; Tmax: time at which Cmax is attained; AUC(0e24 h): area under the plasma concentrationetime curve; BAR: relative bioavailability(n ¼ 6).

Dose(mg/kg)

AUC(0e24 h)

(ng h/mL)Cmax

(ng/mL)Tmax

(h)BAR

(%)

Free-form G-CSF(SC injection)

100 15.9 � 0.3 2.9 � 0.2 2 100

Free-form G-CSF(oral ingestion)

900 1.2 � 0.1 0.2 � 0.1 2 0.8

G-CSF NPs(oral ingestion)

900 19.6 � 0.3 2.6 � 0.2 8 13.6



Supplementary video related to this article can be found athttp://dx.doi.org/10.1016/j.biomaterials.2014.01.020.

4. Conclusions

This study demonstrates that oral ingestion of multiple dailydoses of G-CSF NPs can markedly stimulate the production ofneutrophilic granulocytes in bone marrow and increase theneutrophil count in circulation. This approach is a highly promisingtherapeutic method for treating neutropenia associated withchemotherapy, avoiding its potentially infectious side effects.

Acknowledgments

The authors would like to thank the National Science Council ofthe Republic of China, Taiwan (Contract No. NSC 101-2120-M-007-015-CC1) and Chang Gung Memorial Hospital (Contract No.CMRPG340193) for financially supporting this research.

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0

a

b

010

FDG

SU

V

10 min 30 min 60 min 120 minCT

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G

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(Ora

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ee-fo

rm

G-C

SF (S

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Free-form G-CSF (SC, 100 μg/kg) Free-form G-CSF (Oral, 900 μg/kg) G-CSF NPs (Oral, 900 μg/kg)

St

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H: heart; L: liver; St: stomach; Bm: bone marrow; B: bladder

0

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0 30 60 90 120

Fig. 6. (a) PET/CT images showing the 18F-FDG accumulation in rats following the sequential G-CSF treatments. Images are presented in the lateral tomographic views, in terms ofthe standardized uptake value (SUV); (b) Semi-quantitative radioactivity concentration of 18F-FDG accumulated in bone marrow.



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