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Biomaterials 34 (2013) 9678e9687

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Biomaterials

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Intestinal mucosa permeability following oral insulin delivery usingcore shell corona nanolipoparticles

Xiuying Li a,b, Shiyan Guo a, Chunliu Zhu a, Quanlei Zhu a, Yong Gan a,*, Jukka Rantanen b,Ulrik Lytt Rahbek c, Lars Hovgaard d, Mingshi Yang b,**

a Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 501 Haike Road, Shanghai 201203, ChinabDepartment of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen 2100, DenmarkcADME Department, Novo Nordisk A/S, Maalov 2760, DenmarkdOral Formulation Development, Novo Nordisk A/S, Malov 2760, Denmark

a r t i c l e i n f o

Article history:Received 28 July 2013Accepted 19 August 2013Available online 7 September 2013

Keywords:Oral protein deliveryCore shell coronaInsulinMucus penetrating particlesCellular uptake

* Corresponding author. Tel.: þ86 21 20231000 1424** Corresponding author. Tel.: þ45 35336141.

E-mail addresses: simm2122@vip.sina.com (Y. Ga(M. Yang).

0142-9612/$ e see front matter � 2013 Elsevier Ltd.http://dx.doi.org/10.1016/j.biomaterials.2013.08.048

a b s t r a c t

Chitosan nanoparticles (NC) have excellent capacity for protein entrapment, favorable epithelialpermeability, and are regarded as promising nanocarriers for oral protein delivery. Herein, we designedand evaluated a class of core shell corona nanolipoparticles (CSC) to further improve the absorptionthrough enhanced intestinal mucus penetration. CSC contains chitosan nanoparticles as a core compo-nent and pluronic F127-lipid vesicles as a shell with hydrophilic chain and polyethylene oxide PEO as acorona. These particles were developed by hydration of a dry pluronic F127-lipid film with NC sus-pensions followed by extrusion. Insulin nested inside CSC was well protected from enzymatic degra-dation. Compared with NC, CSC exhibited significantly higher efficiency of mucosal penetration and,consequently, higher cellular internalization of insulin in mucus secreting E12 cells. The cellular level ofinsulin after CSC treatment was 36-fold higher compared to treatment with free insulin, and 10-foldhigher compared to NC. CSC significantly facilitated the permeation of insulin across the ileumepithelia, as demonstrated in an ex vivo study and an in vivo absorption study. CSC pharmacologicalstudies in diabetic rats showed that the hypoglycemic effects of orally administrated CSC were 2.5-foldhigher compared to NC. In conclusion, CSC is a promising oral protein delivery system to enhance thestability, intestinal mucosal permeability, and oral absorption of insulin.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Despite rapid progress made in the development of moderndrug delivery technologies, an efficient oral delivery of therapeuticproteins and peptides remains to be achieved. Oral delivery ofprotein drugs faces several barriers, including pre-systemicdegradation, limited mucosal diffusion, and poor intestinalepithelial membrane permeability [1e3]. A variety of innovativeapproaches have been developed to tackle these challenges,including the use of small molecule permeation enhancers, enzymeinhibitors, and the encapsulation of protein drugs into micro-spheres or nanoparticles [4,5]. The development of nanoparticleswith biodegradable and non-toxic polymers has become one of themajor focal areas in this field due to their ability to protect proteins

; fax: þ86 21 20231000 1425.

n), mingshi.yang@sund.ku.dk

All rights reserved.

from degradation in harsh pH environment, by enzymes in thegastrointestinal (GI) tract [6e9], and their ability to modulatephysicochemical characteristics, drug release, and biologicalbehavior [10,11].

Among those polymeric nanoparticles, chitosan and chitosanderivative based nanocarriers (NCs), appear to be particularlypromising [12]. They can significantly enhance the oral absorptionof peptides [13] and have excellent capacity for protein entrapmentand low toxicity [14]. They enhance the oral absorption of proteinsby several mechanisms, such as bioadhesion with the negativecharged cell membrane, transient widening of tight junctions andincreasing cell permeability by affecting paracellular and intracel-lular pathways without changing junctional morphology [15].However, they cannot effectively transport therapeutic proteinsacross the mucus barrier due to the electrostatic interaction be-tween the anionic mucus gel and cationic nanoparticles [16]. It alsohas been reported that the binding of NCs to the surfaces ofepithelial cells and subsequent absorption-enhancing effects aresignificantly reduced by mucus [16e18]. Although the adhesion of

X. Li et al. / Biomaterials 34 (2013) 9678e9687 9679

NCs to mucus can slow particle transit time through the GI tract,and thus enhance drug absorption, the absorption enhancement islimited by mucus turnover [19].

Recent studies have revealed that nanoparticles coated withhydrophilic polymers exhibit decreased adhesion to mucus com-ponents [19]. Our previous work also shows that Pluronic F127modified lipid vesicles have superior mucus penetration, comparedto unmodified lipid vesicles [20]. To further improve the mucuspenetration of chitosan nanoparticles, we attempted to design acore shell corona nanolipoparticles (CSC), using chitosan nano-particles as a core component and F127-lipid vesicles as a shell withpolyethylene oxide PEO chains as the corona. By enveloping theF127-lipid shell, the positively charged chitosan nanoparticles (NC)would be shielded, ensuring free diffusion of NC in mucus. Addi-tionally, protein nested in the CSC core would be better protectedfrom enzymatic degradation, and therefore the efficacy of drugdelivery could be further enhanced. To the best of our knowledge,the CSC particles, which were designed to feature advantages ofboth NC and Pluronic F127-lipid vesicles, are the newly designednano-size lipoparticles for oral protein delivery with significantlyenhanced mucus penetration.

In the present study, insulin was chosen as a model biomole-cule for testing the CSC system. Insulin has currently attractedgreat interest in developing protein delivery approach, due to itswide clinical use, but limited routes of administration. Herein, CSCwas prepared by hydration of a polymer-lipid film with NC sus-pension to form coreeshell structure and they were then char-acterized in terms of particle size, zeta potential, and morphology.Their ability to protect the encapsulated insulin from enzymaticdegradation, mucus penetration property, and cell uptake effi-ciency and permeability were evaluated in HT29-MTX-E12 (E12)cells. Pharmacological and pharmacokinetic studies were con-ducted in streptozotocin (STZ) induced diabetic Sprague-Dawleyrats.

2. Materials and methods

2.1. Materials

Protasan UP CL113 was purchased from Nova Matrix (Drammen, Norway). Eggphosphatidylcholine (EPC) was purchased from Q.P. Corp (Tokyo, Japan). PluronicF127 was donated by BASF (Ludwigshafen, Germany). RPMI1640 medium, AlexaFluor-555-labeled wheat germ agglutinin and 0.25% trypsin-0.53 mmol/L EDTAwere purchased from Invitrogen (Ontario, Canada). Paraformaldehyde was pur-chased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) was purchased fromBeyotime Institute of Biotechnology (Jiangsu, China). All the other reagents were ofanalytical grade. Alexa 488 labeled insulin was synthesized by Novo Nordisk A/S(Copenhagen, Denmark). HT29-MTX-E12 (E12) cells (52nde56th passages) culturedfor 14e18 days were supplied by the ADME Department of Novo Nordisk A/S. Caco-2cell lines were obtained from the American Type culture collection (ATCC, Manassas,VA, USA). Cells from the 40th �43rd passages were used in the present study.

2.2. Preparation and characterization of nanocarriers

2.2.1. Preparation and characterization of chitosan nanoparticlesChitosan nanoparticles (NC) were prepared using a previously reported proce-

dure [21], based on the ionotropic gelation of chitosan with sodium tripolyphos-phate (TPP) where the positively charged amino groups of chitosan interact with thenegative charged TPP. Briefly, TPP (1.0 mg/mL) was added to chitosan solution(1.0 mg/mL) under magnetic stirring at room temperature to produce NC at a finalchitosan/TPPweight ratio of 6:1. The insulin-loaded NCwere obtained bymixing theinsulin solution in 0.01 M NaOH (3.75 mg/mL) with NC solution at a theoreticalcontent of 30% (w/w) of insulin. Self-assembled NC were collected and washedthrice with distilled water by centrifugation at 16,000 g on a 10-mL glycerol bed for30 min. The centrifuged NC were then re-dispersed in distilled water and stored at4 �C until use.

The association efficiency and loading capacity of insulin in NCwere determinedby subtracting the amounts of free insulin in supernatants quantified using highperformance liquid chromatography (HPLC) from the amounts added, using thefollowing equation:

Association efficiency ¼ total amount of insulin added� free insulintotal amount of insulin added

(1)

Loading capacity ¼ total amount of insulin added� free insulinweight of nanoparticles

(2)

2.2.2. Preparation of core shell nanolipoparticlesThe homogeneous F127-lipid filmwas prepared by drying a chloroform solution

containing egg phosphatidylcholine with F127 (4:1, mol/mol) and then hydratingthe filmwith NC suspensions (lipids/NP¼ 6:1, w/w) for 30min at room temperature,followed by 6 rounds of extrusion through a polycarbonate membrane with 200-nmpores (Avestin Inc., Canada). As a control, core shell nanolipoparticles (CS) withouthydrophilic coronawas prepared by encapsulating NC in a pure lipid vesicle withoutF127.

2.2.3. Characterization of nanocarriersThe mean particle sizes and zeta potential values of different nanocarriers were

measured using a Malvern Zetasizer NanoZS (Malvern Instruments, London, U.K.).Each measurement was made in triplicate. A transmission electron microscope(TEM, CM-200, Philips, Netherlands) was used to observe their morphology. Thesamples were stained with an aqueous solution of phosphotungstic acid (1%, w/v)before observation and the images were taken at 160 kV. The stability of thesenanocarriers in stimulated gastric fluid (SGF) and stimulated intestinal fluid (SIF)were tested by measuring the changes in the particle size and polydispersity index(PDI) after a 2-h incubation.

2.3. In vitro enzymatic degradation assay

The stability of nanocarriers against enzymatic degradation was carried outusing a procedure described previously [22]. In brief, trypsin (2500 IU/mg) andchymotrypsin (800 IU/mg) were dissolved in Tris buffer (pH ¼ 8.0) containing 1 mM

CaCl2 at final concentrations of 250 IU/mL and 50 IU/mL, respectively. Each enzymesolution (50 mL) was separately incubated with 950 mL of test formulations (NC andCSC) containing 5 IU/mL of insulin. These formulations were pre-incubated at 37 �Cfor 15 min. 50 mL of each sample was taken at pre-determined intervals and theenzyme activity was terminated by addition of 50 mL ice cold acetonitrile solutioncontaining 0.1% trifluoroacetic acid. Samples were subsequently treated with TritonX-100 to remove the CSC and CS lipid shell to release insulin. The samples were thenanalyzed using HPLC to determine the amount of insulin. Plain insulin solution andinsulin-loaded NC suspensions were used as control and treated samples under thesame experimental conditions.

2.4. Cell-based assays

2.4.1. Cytotoxicity of nanocarriersCytotoxicity of various nanocarrier suspensions in Caco-2 cells was evaluated

using the MTT assay. Briefly, Caco-2 cells were seeded onto 96-well plates at adensity of 1.0�104 cells per well. After a 48-h culture, nanocarrier suspensions wereadded to the culture media. After a 2-h incubation at 37 �C, the suspensions werereplaced by 100 mL of MTT solution (0.5 mg/mL in HBSS, pH ¼ 7.4) and incubated foran additional 3 h at 37 �C. Subsequently, the MTT mediumwas removed and 150 mLof DMSO was added to dissolve the formazan crystals. The absorbance of theresultant solutions was measured at 490 nm using a microplate reader (Bio-Rad,USA). Each sample was analyzed in five replicates.

2.4.2. Transport study in E12 cell monolayersThe transport study was conducted following a previously reported procedure

[23]. The E12 cell monolayers were washed thrice with pre-warmed 1� Dulbecco’sphosphate buffered saline (PBS, pH 7.4) and equilibrated in the 1� HBSS (with Ca2þ

andMg2þ, 25mM D-glucose) at 37 �C and 95% relative humidity for 30min. Alexa 488insulin stock solution (0.5 mg/mL) and the Alexa 488 insulin loaded nanocarriersuspensions were diluted with PBS. On the day of the experiment, the donor (apical)solutions were prepared by diluting an aliquot of these solutions into HBSS to makea final Alexa 488 insulin concentration of 40 mg/mL. For all the transport experi-ments, a total of 200 mL of each acceptor (basolateral) sample was removed at 0, 15,30, 45, 60, or 90 min after drug administration. For each acceptor sample taken,200 mL of fresh HBSS was added to the mixture to maintain a constant volume. Thesamples were transferred onto a 96-well titer plate and Alexa 488 insulin wasdetected using a microplate fluorometer (Model 680, Bio-Rad, USA) at an excitationfilter of 485 nm and an emission filter of 530 nm. To eliminate the effects of mucuson the transport of free Alexa 488 insulin, the E12 cells were incubated with 10 mM

N-acetyl cysteine (NAC) in HBSS for 30 min to remove the mucus prior to thetransport study. Apparent permeability coefficient (Papp) and enhancement ratio (R)were calculated using the following equations.

Papp ¼ dQdt

� 1A� C0

(3)

Table 1Particle size, polydispersity index (PDI), z-potential, association efficiency (AE), and loading efficiency (LE) of NC, CS, and CSC.

Formulations Mean Diam (nm) PDI z potential (mV) AE LE

NC 210.5 � 45.3 0.311 � 0.075 þ36.6 � 4.5 76.6 � 5.8% 39.0 � 4.2%CS 202.8 � 22.9 0.175 � 0.069 �7.1 � 3.2CSC 195.3 � 32.9 0.151 � 0.048 �4.3 � 5.4

X. Li et al. / Biomaterials 34 (2013) 9678e96879680

R ¼ Papp�test

Papp�control(4)

Where dQ/dt is the flux of insulin from the apical side to the basolateral side, C0 is theinitial concentration of insulin in the apical compartment, and A is the membranearea (cm2). Papp-test is the Papp of different groups, while Papp-control is the Papp of thosetreated with insulin solutions.

2.4.3. Mucus penetration in E12 cells analyzed by confocal microscopyIn order to assess the interactions between Alexa 488 insulin and the mucus, the

E12 cells grown on Transwell� filter inserts for 14e17 days were treated asdescribed above. The apical incubation buffer, containing Alexa 488 insulin loadednanocarriers, was removed after 60 min of incubation with the formulations. TheE12 monolayers were then washed with PBS and the mucus layer was stained withAlexa Fluor 555 labeled wheat germ agglutinin (Alexa Fluor 555-WGA, 10 mg mL�1)for 10 min at 37 �C [24]. The membranes supporting the cell layers were washedwith PBS, and the cell layers supporting membranes of Transwell inserts were cutfrom the plastic support without fixation, mounted onto microscope slides, andcovered with coverslips. The slides were immediately observed under a confocalmicroscope (LSM 5 Pa, Zeiss, USA) using a 63� oil objective lens. Alexa Fluor 488 andAlexa Fluor 555 were excited with a 488 nm argon laser and 543 nm helium-neonlaser, respectively. Image visualization and processing were performed using theLSM 5 Pa software. To observe the interactions between the different formulationswithmucus in a larger view, a 2D image in the middle of the mucus was taken undera confocal microscope at 5� magnification.

2.4.4. Qualification of mucus entrapment and cellular uptake of Alexa 488 insulinTo differentiate Alexa 488 insulin trapped in the mucus from bound insulin and

insulin that had been taken up by cells, the E12 cells were treated with the differentparticle formulations for 1 h. The mucus layer was removed by washing the cellmonolayers twice with 300 mL of 4% (v/v) formalin solution in PBS (0.1 M, pH 7.4),followed by the washing procedure as reported previously [17]. Then the cell layerswere disrupted with lysis buffer (RAPI), and cell-associated fluorescent protein wasdetermined by fluorescence spectroscopy. The amount of cell-associated Alexa 488labeled insulin was reported as a percentage (%) of the initial amount of Alexa 488labeled insulin.

2.5. Animal study

2.5.1. Animal careMale Sprague-Dawley rats, 6e8 weeks old, were provided by the Animal

Experimental Center of Shanghai Institute of Materia Medica, Shanghai, China. Theanimals had free access to rat chow and tap water. All of the animal experimentswere carried out according to the Institutional Animal Care and Use Committee(IACUC) guidelines of Shanghai Institute of Materia Medica.

2.5.2. Absorption studies in the ligated intestinal loops2.5.2.1. Permeation studies ex vivo. The transport ofAlexa 488 insulin loadedCSC andNC across the epithelial mucosa of rat ileum was monitored ex vivo, as described byYin et al. [25]. Briefly, after rats were sacrificed, 5-cm sections of the ileum wereexcised and incubated in 10mLKreb’-Ringer buffer at 37 �Cwith gentle agitation. CSCandNC (0.5mL)were loaded into the loops using a syringe. At various times (0.5,1,1.5and 2 h), 200 mL of the incubation bufferwas collected for determination of Alexa 488labeled insulin using fluorescence spectroscopy. The same volumeof fresh bufferwasadded at each time point. All of the experiments were performed in triplicates.

2.5.2.2. Absorption assay in vivo. The in vivo uptake of CSC and NC were evaluatedusing the ligated intestinal loopsmodel following themethod described by Yun et al.[9]. Sprague-Dwaley ratswere fasted overnight before experiments, but allowed freeaccess towater. The rats were anesthetized with pentobarbital sodium (0.04 mg/kg).After the abdomenwas exposed, 5-cm loops of ileumwere made by ligation at bothends and the tissue was washed with physiological saline. CSC and NC (0.5 mL,100 mg/mL of Alexa 488 insulin) were injected into the loop. To study the effect ofmucus on the absorption of free Alexa 488 insulin, ligated ileum loops were pre-incubated with saline containing 10% N-acetyl cysteine (NAC) for 10 min beforethe absorption study to remove mucus, and then 0.5 mL CSC and NC suspensionswere injected into the loop. Rats were sacrificed after 0.5 h or 2 h and the sections ofeach loop were removed and extensively washed using PBS. Subsequently, theremoved loops were fixed by 4% paraformaldehyde for 2 h and immersed in 30%sucrose at 4 �C overnight. Samples were embedded and frozen at �20 �C (OCT,

Sakura Fine technical Co., Ltd.). Frozen ileum sections (20 mm) were cut using acryostat (Leica CM 1950) and then stained with DAPI. The tissue-sections were thenvisualized using a confocal microscope.

2.5.3. Pharmacological and pharmacokinetic studiesDiabetes was induced in Male Sprague-Dawley rats weighting 180e200 g by an

injection of streptozotocin (65 mg/kg) dissolved in citrate buffer as previouslydescribed [26]. The blood glucose level was determined using a glucose meter (OnCall� EZ II, Acon Biotech.Co.Ltd., Hangzhou, China). Rats were considered to bediabetic when their fasting blood glucose level was higher than 250mg/dL oneweekafter streptozotocin treatment. Sprague-Dwaley rats were fasted overnight beforeexperiments, but allowed free access to water. Various formulations of insulin wereadministered to the diabetic rats by gavage of insulin solution (50 IU/kg), insulinloaded NC (50 IU/kg), or insulin loaded CSC (50 IU/kg) or by subcutaneous injection(SC) of insulin solution (5 IU/kg). Blood samples were collected from the tail veins ofrats prior to drug administration and at various times (1, 2, 4, 6, 8 and 10 h) afterdosing. The blood glucose levels were analyzed and the area under concentration-time curve (AUC) over 12 h was calculated for each group. For the analysis ofserum insulin level, blood samples were centrifuged (3000 rpm, 5 min) and sub-sequently quantified using an appropriate insulin ELISA kit (R&D system, Inc, MN,USA). The total decrease (D%) in serum glucose levels were calculated using amodified method as follows:

D% ¼ AUCðInsulin solutionÞ � AUCðtestÞAUCðInsulin solutionÞ � 100 (5)

The relative bioavailability (F%) of CSC after oral administration was calculatedusing the following formula.

F% ¼ AUCðoralÞ � DoseðscÞAUCðscÞ � DoseðoralÞ � 100 (6)

2.6. Statistical analysis

Comparisons between the two groups were performed using Student’s t-test(SPSS, Chicago, IL) and P < 0.05 was considered statistically significant.

3. Results

3.1. Characterization of nanocarriers

Asshown inTable 1, thenakedNCwere around210� 45nminsizeand had a positive zeta potential of approximately þ36.6 �4.5 mV. The mean association efficiency of insulin to NC was76.6 � 5.8%, and the mean loading efficiency was 39.0 � 4.2%. Theincorporation of NC into vesicles did not have a remarkable effect onthe size, but led to a significant changes in zeta potential (P < 0.05),whichwere reduced toe 7.1�3.2 ande 4.3� 5.4mV for CS and CSC,respectively. The conversion of the zeta potential suggested that theNCwere encapsulated in the vesicles. As shown in Fig.1, the core shellcorona structures were observed for CSC under TEM, indicating thatchitosan nanoparticles were encapsulated into the vesicles under thecurrent experimental conditions. A schematic presentation of thecore shell corona structure is shown in Fig. 1A. CSC formed by thismethod remained stable in SGF and SIF, and the size and PDI did notchange significantly. CS was stable in SGF but the size greatlyincreasedaftera2-h incubation in SIF.Whereas,NCwassoluble inSGFandnoparticle size change could beobserved inSIF (Data not shown).

3.2. Protection of insulin against enzymatic degradation

No significant differencewas observed between CS and CSCwithrespect to protection of insulin from enzymatic degradation bytrypsin and a-chymotrypsin. As shown in Fig. 2, CS and CSC

Fig. 1. Design and evaluation of NC and CSC. A: Schematic illustration of the formation of NC and CSC; B: TEM images of NC and CSC. Red line, the boundary between NC andpolymer-lipid shell. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

X. Li et al. / Biomaterials 34 (2013) 9678e9687 9681

protected 40% and 70% of encapsulated insulin, respectively, fromdegradation by trypsin and a-chymotrypsin after 1 h of incubationwith the intestinal enzyme solutions in vitro, whereas plain insulinwas almost completely degraded under the same conditions. Thissuggested that the core shell structures could prevent insulin frombeing exposed to the enzymes by shielding the protein in the coreof the nanolipoparticles. NC alone, in this case, failed to protect theassociated insulin from enzymatic degradation. Its degradationprofiles were similar to those of naked insulin.

3.3. Results from studies with cell-based models

3.3.1. In vitro cytotoxicityAs shown in Fig. 3, all the three nanocarriers were found to have

no significant cytotoxicity (P > 0.05) at the tested concentrations(0.2, 0.5 and 1.0 mg/mL), although there was a slight reduction incell viability treated with 1.0 mg/mL of NC.

3.3.2. Transport of Alexa 488 insulin loaded nanocarriers across E12cells

The apparent permeability coefficient (Papp) and enhancementratio (R) were used to assess the ability of the nanocarriers to

promote transport of insulin across the E12 cell monolayers. Asshown in Table 2, the Papp obtained with NC was quite similar tothose obtained with insulin HBSS solution. However, after NACtreatment, the insulin Papp was increased 2-fold, indicating that themucus formed a barrier for the transport of insulin. The use of CSCwas found to significantly enhance insulin Papp across the E12monolayers. An R-value of 2.42 was obtained with CSC. This wascomparable to the results obtained using plain Alexa 488 insulinHBSS solution in E12 monolayers after mucus depletion by NAC.The unmodified CS also enhanced insulin transport to a less extentas compared to CSC, with an R-value of 1.63.

3.3.3. Mucus penetration of Alexa 488 insulin in E12 cellsIn order to determine the mucus penetration properties of Alexa

488 insulin, the E12 cell monolayers treated with different for-mulations were imaged without fixation to show the diffusion ofAlexa 488 insulin. The red fluorescence derived from Alexa Fluor555 represents themucus that covers the surface of the cell, and thegreen fluorescent spots derived fromAlexa 488 correspond to Alexa488 insulin. Few green fluorescent spots were observed in themonolayer treated with Alexa 488 insulin HBSS solution, butstronger green fluorescence was observed in monolayers treated

NCS

0 20 40 60 80 100 1200

20

40

60

80

100

% u

ndeg

rada

ted

insu

lin

Time (min)

NCS B

0 20 40 60 80 100 1200

20

40

60

80

100%

und

egra

date

d in

sulin

Time (min)

ACS CSC

CS CSC

Fig. 2. Enzymatic degradation profiles of insulin as a function of time A: Trypsin; B: a-chymotrpsin.

X. Li et al. / Biomaterials 34 (2013) 9678e96879682

with NC, CS, and CSC (Fig. 4A). After NC treatment, the mucus layerwas discontinuously distributed and showed strong yellow spots,but the mucus layers treated with CS and CSC were similar to thosetreated with Alexa 488 insulin solution, except for stronger greenfluorescence. The 2D images from the middle position of the mucuson the surface of the E12 cell monolayer (Fig. 4B) showed that NCclosely interacts with mucus, forming aggregates with some of itscomponents (big yellow spots) and some NC self-aggregates (largegreen spots). This phenomenon was seen much less in E12 cellmonolayers treated with Alexa 488 insulin loaded CS and CSC andthe aggregates were much smaller than those seen in the NC group.

3.3.4. Concentrations of Alexa 488 insulin in the mucus layer andcell layers

To differentiate the fraction of Alexa 488 insulin trapped in themucus layer and the fraction associated with, or internalized in thecells, the mucus layer was washed away using the formalin solu-tion. As shown in Fig. 5, the CSC had the strongest interaction withE12 cells, reaching amaximum of about 9.3% of the initial Alexa 488insulin in the mucus layer and cells. Among these particles, 5.4%were inside in or attached to the E12 cells. The CS had an effectsimilar to that of the CSC, with 8.6% of the initial Alexa 488 insulinin the mucus layer or the E12 cells, but only 1.9% was inside orattached to the E12 cells, far less than that with the CSC. The NC also

0

20

40

60

80

100

120

1.0 mg/mL0.5 mg/mL0.2 mg/mL

Cel

l Via

bilit

y (%

)

NC

Control

CS CSC

Fig. 3. Effect of different formulations on the Caco-2 cell viability.

showed stronger association with the entire cell monolayercompared to the insulin solution, as suggested by the observationthat 4.3% of the initial Alexa 488 insulin was detected in the E12monolayers prior to removal of mucus, but only 0.5% of the initialAlexa 488 insulin was also detected inside the cell. These resultssuggested that the enhanced association efficiency of insulin withcells was not as significant as the association with the entire cellmonolayer with intact mucus.

3.4. Results from the animal studies

3.4.1. The accumulative transport of insulin ex vivoThe transport of NC and CSC across the intestinal epithelia was

investigated using ex vivo in ligated ileum loops. As shown in Fig. 6,compared to NC, CSC increased the accumulative amount of Alexa488 insulin permeated through the rat ileum. The permeated Alexa488 insulin from CSC at 1 h was 21.75 � 0.86 ng, which was 1.53-fold higher than that from NC (14.18 � 0.59 ng) and the accumu-lative amount of Alexa 488 insulin at 2 h increased to61.74 � 7.39 ng, which was 1.76-fold higher compared to NC. Theseresults revealed that absorption enhancement of CSC across theepithelia was associated with improved mucus penetration andcellular uptake.

3.4.2. Absorption studies in villiThe absorption of NC and CSC was qualitatively observed by a

confocal microscope to be located in the villi of ileum loops. Fig. 7shows the absorption of Alexa 488 insulin from NC and CSC at 0.5and 2.0 h, respectively. The absorption of Alexa 488 insulinincreased with time, and stronger green fluorescence was observedin loops treated with CSC. Alexa 488 insulin loaded CSC permeateddeeply into villi after 2 h, indicating their effective absorptionin vivo. With NAC pre-treatment to remove the mucus, both NC andCSC showed strong green fluorescence intensity. The effect of

Table 2Apparent permeability coefficient (Papp) and enhancement ratio (R) of insulinpermeability across E12 cell monolayers. * P < 0.05, compared with S group.

Formulations Papp*10�7(cm/s) Enhancement ratio

S 4.73 � 0.35 e

S þ NAC 7.71 � 0.72* 2.21NC 4.58 � 0.53 0.97CS NP 7.29 � 0.89* 1.63CSC NP 11.46 � 1.58* 2.42

Fig. 4. Mucus and cell-based evaluation of NC and CSC. A: Confocal microscope images of E12 cell layers treated with different formulations. A: 3D image of mucus penetration. B:2D images of E12 monolayers. Red: mucus covering the cell surface stained with Alexa Fluor 555-WGA. Green: Alexa 488-insulin. (For interpretation of the references to colour inthis figure legend, the reader is referred to the web version of this article.)

X. Li et al. / Biomaterials 34 (2013) 9678e9687 9683

S CS0

2

4

6

8

10

12

Insu

lin b

ound

to a

nd w

ithin

cel

ls a

nd m

ucus

(

% o

f ini

tial)

mucus+cellcell

CSC NC

# ##

**

Fig. 5. Relative amounts of Alexa 488 insulin internalized/attached in E12 cells.*P < 0.05, compared with NC group; #P < 0.05, compared with NC group; ##P < 0.01,compared with the NC group.

X. Li et al. / Biomaterials 34 (2013) 9678e96879684

mucus on the absorption of insulin loaded into NC was much moresignificant than those loaded into CSC. For the NC group, the greenfluorescence was much stronger with NAC treatment, whereas thedifference after NAC treatment was less for the CSC group.

3.4.3. Hypoglycemic effect in vivoThe pharmacological effects of NC and CSC were evaluated in

diabetic rats after oral administration. As shown in Fig. 8A, the plaininsulin solution failed to reduce the blood glucose level; NC slightlyreduced the blood glucose level; and CSC exhibited stronger hy-poglycemic effects than the other two treatments. Compared withthe baseline, the blood glucose level decreased to 77% and thehypoglycemic effect lasted 10 h. The D% of CSC was 2.52-fold highercompared to NC. The serum insulin concentration-time profiles areshown in Fig. 8B. The rats subcutaneously treated with plain insulinsolution resulted in a rapid increase in serum insulin concentration,whereas oral administration of CSC demonstrated a slower rise inserum insulin concentration, reaching a maximum serum concen-tration at 4 h after treatment. Compared to NC and plain insulinsolutions, CSC treatment showed significant higher serum insulin

0.5 1.0 1.5 2.00

25

50

75

Time (h)

NCCSC

Amou

nt o

f ins

ulin

per

mea

ted

(ng)

Fig. 6. Accumulative permeated amount of Alexa 488 insulin across rat ileum from NCand CSC (Mean � SD, n ¼ 3).

concentrations at 1e6 h post administration. As shown in Table 3,the AUC(0e12 h) for CSC was 232.5 � 35.5 mIU*h/mL, with a relativebioavailability of 7.8%; both parameters were significantly greatercompared with NC and plain insulin.

4. Discussion

Pre-systemic enzymatic degradation and poor transmucosalpermeability are the major obstacles to an efficacious oral proteinand peptide delivery [3], which can be overcome by the use ofnanoparticle carriers [7,10]. In the current study, we designed coreshell corona structured nanolipoparticles (CSC), based on a ‘parti-cle-in-particle’ approach. The system was comprised of chitosannanoparticles as a core component and F127-lipid vesicles as ashell, with hydrophilic chain polyethylene oxide (PEO) as a corona.These particles protect fragile biomacromolecules, such as insulin,in the polymer nanoparticle cores, enhancing mucus-penetratingproperties and membrane transportation. CSCs were prepared byhydration of polymer-lipid dry film with NC suspensions. NC weredesigned to be covered by the vesicles via hydrogen bonds betweenthe particle surface and lipid polar heads and electrostatic in-teractions between lipids and the hydrophilic surface [27]. As ex-pected, most NC particles were entrapped in the vesicle as observedby TEM, where the dense black cores of NC were surrounded bygray lipid shells (Fig. 1B). The sizes of CSC were around 200 nm, asshown in TEM. This was also in close agreement with the particlesizes indicated by dynamic light scattering measurements. Uponadsorption of lipid or polymer-lipid layers onto NC, the strongpositive zeta potential of NC changed from þ36 mV to negativecharges (�7.1 and �4.3 mV, respectively), which also showed thatthe cores of NC were encapsulated into the polymer-lipid vesiclesand formed nanolipoparticles. Similar assembly of nanoparticlesand vesicles has been reported by other groups [28,29]. However,most other carriers reported in the literature are macro-sized anddo not feature a hydrophilic polymer chain on the surface, whichare unstable in the GI tract and are not ideal for intestinalabsorption.

Fragile biomacromolecules, such as insulin, packaged in thenanolipoparticles with solid cores are expected to be able towithstand the harsh physiologic environment due to the shieldingshell of the polymer-lipid layers, preventing direct contact of thebiomacromolecules with enzymes. Our in vitro forced degradationstudy confirmed this hypothesis. The naked NC failed to protectinsulin from enzymatic degradation; whereas, the enzymaticdegradation of insulin associated with chitosan nanoparticles wassignificantly decreased when they were encapsulated in vesicles.These results suggested that nanolipoparticles with core shellstructures may improve the integrity of fragile biomolecules duringoral delivery.

Whether mucus is also a barrier to the diffusion of protein andpeptide drugs is still debatable. The traditional theory is that thepore size of mucus physically constrains the diffusion of macro-molecules [30,31]. However, in recent years, some emerging evi-dence has supported the opposite theory, that mucus is not adiffusion barrier for protein drugs [32,33]. In our study, only verylittle Alexa 488 insulin was detected in the mucus. As shown in theconfocal microscope images (Fig. 4A), veryweak green fluorescencewas visible, indicating that it was difficult for Alexa 488 insulin topass through the mucin network. The qualitative data (Fig. 5) alsoconfirmed that minimal Alexa 488 insulin was present in themucus. In addition, the Papp value of insulin solution was signifi-cantly increased after the mucus was removed by NAC treatment.These results supported the notion that mucus is a diffusion barrierto insulin and that it is necessary to consider drug delivery strate-gies that would overcome the physical barrier of the mucus layer.

Fig. 7. Distribution of Alexa 488 insulin loaded in NC or CSC in ileum villi at 0.5 and 2 h. Green fluorescence refers to Alexa 488 insulin and blue fluorescence refers to cell nucleus.

X. Li et al. / Biomaterials 34 (2013) 9678e9687 9685

It has been reported that the absorption of insulin can beenhanced by using fusogenic liposomes (FLs) as carriers when theyare directly administered to colonic and rectal loops [34]. However,the enhancement in absorption is significantly decreased whenadministered to the ileum [34]. It has been speculated that themucus layer overlying the ileal epithelia may impair or compromisethe fusion of FL to the intestinal mucosa. It also has been reportedthat trimethyl chitosan (TMC) and chitosan do not enhance theabsorption of insulin in the ileum because of the thick mucus layer[35]. In the current study, we investigated the mucus-penetratingproperties of chitosan nanoparticles, which have strong electro-static interactions with mucus. Strong green fluorescence wasobserved in the mucus network of E12 cells after NC treatment,suggesting that NC could increase the amount of insulin trapped inmucus. However the ability of NC to enhance cellular uptake andcellular transport was less significant than its ability to enhancemucus entrapment. These results suggested that NC could notreadily improve the absorption. This could be because most of theNC is trapped in the mucus due to the strong electrostatic in-teractions between chitosan and mucin [16]. NC were unable toreach the epithelial surface, and so failed to improve the absorptionof encapsulated proteins. Considering the mucus turnover in vivo,such a protein delivery system could be rapidly cleared togetherwith the mucus. Therefore, an ideal pharmacological efficacy willbe difficult to obtain. Unlike NC, CSC not only significantlyenhanced the mucus penetration of insulin but also greatlyenhanced the efficiency of cellular associations. As shown in theLSM images, the mucus of E12 cells treated with CSC was homo-genously distributed. The cell monolayers treated with CSC showedstrong green spots, indicating that more protein molecules wereable to reach the cell surface and promote cellular uptake.

In addition to their ability to enhance mucus penetration, CSCmay also enhance cellular uptake of insulin by surface modificationof the nanolipoparticles with F127 polymers. F127 has been re-ported to stabilize and seal damaged lipid bilayer membranes [36e38]. This may reinforce the association of the nanolipoparticleswith the cell membrane. The increased level of cellular insulin

uptake observed with CSC in E12 cells showed 10-fold higher up-take compared to NC. Although CSC have been found to improveinsulin transport through E12 cells as compared to insulin solutionand naked NC (Table 2), their impacts on the Papp values are notcomparable to their effects on the efficiency of cellular uptake.Similar results have been reported by Anchalee et al. and Thompsonet al.: the chitosan nanocomplexes could improve cellular uptake ofinsulin with E12 cells, but no insulin was detected on the baso-lateral sides of the E12 cell monolayer [23,39].

In the present ex vivo transport study across ligated ileum loops,we found that CSC significantly enhanced the permeability of Alexa488 insulin compared with NC. When reaching the small intestine,NC were mostly immobilized in the mucin network, but CSC couldpenetrate through the mucus and thus more Alexa 488 insulincould reach the epithelium surface and be transported across theintestinal epithelium via the paracellular pathway, transcytosis orreceptor-mediated transcytosis. The absorption enhancement ef-fect of CSC was further confirmed by in vivo absorption. Alexa 488insulin could clearly be observed in the villi after 0.5 h treatment ofligated ileum loops treated with CSC, while minimal Alexa 488insulin could be seen in the NC group. The green fluorescence in theCSC group was significant stronger compared to the NC group at 2 hpost administration of CSC. With NAC pre-treatment to remove themucus, ligated loops treated with NC showed significantly strongergreen fluorescence intensity compared to the non-NAC treated NCgroup, while only slightly stronger fluorescence was observed inthe CSC group compared to non-NAC treated CSC group. These re-sults indicated that the effect of mucus on the absorption of insulinloaded in NC was much more significant than those loaded in CSC,suggesting enhanced mucus penetration of NC by polymer-lipidvesicle encapsulation.

The in vivo pharmacological and pharmacokinetic resultsshowed good correlations with the improved absorption andtransport of insulin in the ligated intestinal loop and cell studiesin vitro. As expected, the oral administration of plain insulin solutiondid not lead to significant hypoglycemic effects, because of the rapiddegradation in the gastric fluid and poor permeability through

0 2 4 6 8 10 120

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Fig. 8. A Blood glucose levels in diabetic rats following oral administration of insulinloaded NC, CSC, and insulin solution (S, 50 IU/kg) and subcutaneous injection of insulinsolution (S-SC, 5.0 IU/kg) (Mean � SD, n ¼ 4). *P < 0.05, compared with S group;**P < 0.01, compared with S group; #P < 0.05, compared with the NC group. B: Seruminsulin level in diabetic rats following oral administration of insulin loaded NC, CSCand insulin insulin solution (S, 50 IU/kg) and subcutaneous injection of insulin solution(S-SC, 5.0 IU/kg) as positive control (Mean � SD, n ¼ 4). *P < 0.05, compared with theNC group.

X. Li et al. / Biomaterials 34 (2013) 9678e96879686

epithelia. Insulin loaded NC had limited efficiency to reduce theblood glucose level, partially because of the instability of NC as seenin in vitro studies. Strong adhesion to the mucus, instead of pene-trating through themucus, also contributed to the ineffectiveness ofinsulin loaded NC.Whereas, CSC exhibited improved stability in theGI tract, enhanced mucus penetration, and membrane transport,leading to significantlymorepotent andprolongedpharmacologicalefficacies. The onset of hyperglycemic effect occurred at 2 h post

Table 3Pharmacokinetic parameters of insulin in diabetic rats after subcutaneous injectionof insulin solution (5.0 IU/kg) and oral administration of insulin solution, NC and CSC(50 IU/kg). Cmax: maximum serum concentration; Tmax: time at which Cmax isattained; FR: relative bioavailability.

INSsolution (s.c.)

CSC (i.g.) CS (i.g.) INSsolution (i.g.)

Dose (IU/kg) 5.0 50.0 50.0 50.0AUC (mIU*h/mL) 299.3 � 49.2 232.5 � 35.5 111.4 � 19.3 36.3 � 4.9Cmax (mIU/mL) 128.6 � 22.5 40.0 � 10.1 16.3 � 3.7 6.0 � 0.5Tmax (h) 1 3.3 3 2.7FR % 100% 7.8% 3.7% 1.2%

administration and lasted for 10 h. During this experiment, thefasting food blood glucose did not return to the initial level after10 h, similar as other previous reports, which might be the conse-quence of the combined effects of hunger and hypoglycemic agents[9,21]. The quick absorption of insulin suggested the fast diffusion ofCSC through the mucosal barrier while the long-acting effectrevealed the slow release of insulin from the core shell coronastructure. Furthermore, the animals treated with CSC exhibitedsignificantly higher serum insulin levels with a significantlyincreased relative bioavailability compared with the NC and freeinsulin groups. In the clinical practice, sc. administration of insulincan be associated with severe hypoglycemic shock and patientinconvenience. Our results demonstrated that insulin encapsulatedin CSC exhibited prolongedhypoglycemic activitywithout the initialhypoglycemia seenwith sc. administration, suggesting that CSCmaybe a promising strategy for clinical treatment of diabetes.

5. Conclusions

In the present study, we designed core shell corona nano-lipoparticles to improve the intestinal mucosal permeability of in-sulin. The insulin loaded chitosan nanoparticles formed the core,and polymer-lipid vesicles formed the shell, with hydrophilic chainPEO as the corona. Experiments performed with mucus-secretingHT29-MTX-E12 cells suggested that mucus might constitute adiffusion barrier for insulin molecules and prevent them fromreaching the cell membrane. The naked NC were found to increasethe amount of protein entrapped in themucus, while CSC improvedthe ability of insulin to penetrate the mucus and increased cellularuptake of insulin. Enhanced permeability of insulin was confirmedby a transport study using ex vivo intestinal tissue. Enhanced ab-sorption of insulin in intestinal villi by CSC was also observed inin vivo absorption study. The CSC treatment resulted in an elevatedserum insulin concentration and improved hypoglycemic effect indiabetic rats, suggesting that NC encapsulated in F127-lipid vesiclescould be a promising nanocarrier for oral delivery of insulin, as wellas other peptides and proteins.

Acknowledgments

This workwas supported by the NovoNordisk-Chinese Academyof Science (CAS) Research Foundation (No. NNCAS-2009-10) andthe National Science and Technology Major Project, “Key New DrugCreation and Manufacturing Program” (2012ZX09301001-001). Wethank Erik Wisaeus and Pia Wahlberg of the Danish TechnologicalInstitute, Denmark, for their excellent assistance on TEM, which issupported by The Innovation Consortium NanoMorph (952320/2009) funded by The Danish Council for Technology and Innova-tion. We also thank the ADME department of Novo Nordisk A/S,Denmark, for excellent assistance in HT29-MTX-E12 cell studies.

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