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D. Xia, W. Fan, M. Yu, S. Guo, C. Zhu and Y. Gan, J. Mater. Chem. B, 2015, DOI: 10.1039/C5TB01425E.

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1

ORIGINAL RESEARCH

Poly-L-glutamic acid functionalized nanocomplex for improved oral

drug absorption

Quanlei Zhu1,2, Wenyi Song1, Dengning Xia1, Weiwei Fan1,2, Miaorong Yu1,2, Shiyan

Guo1, Chunliu Zhu1, Yong Gan1*

1Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203,

China

2University of Chinese Academy of Sciences, Beijing 100049, China

*Corresponding author:

Yong Gan

Shanghai Institute of Materia Medica, Chinese Academy of Sciences

NO. 501 Haike Road

Shanghai 201203, People’s Republic of China

Tel.: +86-21-20231000-1425

Fax: +86-21-20231000-1425

E-mail: [email protected]

Page 1 of 40 Journal of Materials Chemistry B

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View Article OnlineDOI: 10.1039/C5TB01425E


2

Abstract

Poor permeability to cross the intestinal epithelium limits the oral absorption of many drugs. Here,

poly-L-glutamic acid (PGA)-based functional ternary nanocomplex (TC) was reported for enhancing

intestinal absorption of poorly permeable drug doxorubicin hydrochloride (Dox·HCl). The particle

size and zeta potential for TC were 189.3 ± 13.7 nm and -29.1 ± 7.4 mV, respectively. TC showed to

be more stable in simulated gastrointestinal changing pH or electrolyte content conditions than the

binary nanocomplex Dox•HCl/PGA. Cellular uptake and apparent permeability coefficient value

(Papp) of TC were determined to be 5.2- and 4.6-fold higher than that of Dox•HCl solutions,

respectively. Mechanism studies showed that active endocytosis caused by specific interactions

between γ-glutamyl terminal groups of PGA and membrane-bound γ-glutamyl transferase

contributed much to the TC-dependent Dox•HCl absorption. Studies in rat model also

demonstrated the highest efficiency for Dox•HCl absorption by TC throughout the intestinal tract,

with 2.6- and 4.2-fold higher in Cmax and AUC0–24h values compared to Dox•HCl solutions. In

conclusion, TC was a promising carrier in improving Dox•HCl intestinal absorption, and the rational

design of carriers with functional polymer PGA could implement the efficient active absorption of

poorly permeable drugs.

Keywords: Doxorubicin hydrochloride; absorption; PGA; nanocomplex; intestinal

delivery

Page 2 of 40Journal of Materials Chemistry B

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1. Introduction

Oral administration is the most convenient route for drug delivery. However, intestinal

epithelium composed of continuous sheets of tightly linked cells has formed a

formidable permeability barrier for drugs oral absorption into systemic circulation.1, 2

Low absorption due to the intrinsic poor permeability to penetrate the lipid bilayer of

the epithelial cell membrane limits the oral bioavailability of many drugs.3, 4

There has been a long-standing interest in using polymeric nanoparticles as drug

carriers for overcoming intestinal absorption obstacle5. Several groups have shown

improved oral bioavailability of encapsulated drugs using nanoparticles formulated

with polymers such as poly(lactic-co-glycolic acid) 6-8 and polyallylamine

hydrochloride.9, 10 It is generally assumed that these polymeric nanoparticle-

dependent improvement in oral bioavailability is because of their capacity to be non-

specific uptake into enterocytes.6, 9, 11 Nevertheless, current progresses in design of

drug delivery system are more and more concentrated on the concept of targeting to

epithelium-bound proteins. Through specific binding to the certain membrane-bound

proteins such as folic transporter/receptor12, 13 and bile acid transporters,14, 15 more

efficient specific endocytosis was achieved for these novel delivery systems compared

to traditional carriers, and thus enhanced drug absorption.

PGA, a naturally-occurring anionic polypeptide of L-glutamic acid, was shown to

have excellent biocompatibility and biodegradability properties. Terminal γ-glutamyl

groups endow the polymer with the potential to attach to intestinal membrane-bound

γ-glutamyl transferases (γ-GT),16, 17 which are highly expressed in intestine participating


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in the peptides digestion and absorption.17, 18 Specific interactions between PGA and

cell membrane are expected to implement the PGA-based particle-cell specific

interactions,16, 19 and thus the efficient epithelial absorption for particle encapsulated

drugs. In fact, previous studies have shown that PGA, as an adjuvant in nanoparticles

has great potential in enhancing cellular uptake of genes.20 However, to the best of our

knowledge, the usage of PGA as specific epithelium targeted polymer for enhancing

intestinal absorption of poorly permeable drugs has not been proposed until now.

The purpose of our study was to investigate the potential of PGA-based

nanoparticles in improving the oral bioavailability of Dox•HCl, a drug with poor oral

absorption due to its low permeability. The natural fluorescent property of Dox•HCl

facilitates its detection and quantification by enabling us to visually trace the

compound in cells and tissues.6, 21, 22 In this work, binary polyion nanocomplex

Dox•HCl/PGA (BC) was firstly developed. Then, another polymer, Poly(β-amino esters)

(PAE), was introduced into BC to form ternary polyion nanocomplex Dox•HCl/PGA/PAE

(TC) for optimizing the surface properties and stability of BC to ensure the efficient

nanoparticle-cell binding capacity in vivo. Caco-2 cell monolayers were used as the

intestinal barrier model to determine the effect of PGA based TC on Dox•HCl

absorption. Enhancement of overall Dox•HCl absorption from TC was also confirmed in

vivo using rat model.


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2. Experimental

2.1. Materials

Dox•HCl (purity 98.5%) was a generous gift from Shang Hai Pharma (Shanghai, China).

PGA was a gift from Shanghai Jiuqian Chemical Co., Ltd (Shanghai, China). PAE (Mn

~8,600; Mw ~11,300; 1H NMR (DMSO-d 6), 4.03 (m, 4H), 3.34 (q, 2H). 2.90 (m, 2H),

1.57 (t, 4H)) was a gift from Chen Shi biotech Co., Ltd. (Shanghai, China).

Paraformaldehyde was purchased from Sinopharm Chemical Reagent Co., Ltd.

(Shanghai, China). Dulbecco's Modified Eagle’s Medium (DMEM) and 2-(4-

amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) were purchased from

Invitrogen (Burlington, ON, Canada). All other reagents were of analytical grade.

2.2. Preparation of nanocomplex

As illustrated in Figure 1, Dox•HCl/PGA (BC) and Dox•HCl/PGA/PAE (TC) were prepared

through a self-assembly process. Aqueous solution of Dox•HCl (1.3 mg/mL) was

premixed with PGA solution (1 mg/mL) under magnetic stirring for 5 min to form BC.

PAE (0.5 mg/mL) was subsequently blended with the mixture under constant stirring

for 5 min at pH 5.5 ± 0.5 to form TC. Self-assembled TC were collected and washed

thrice with distilled water by centrifugation at 24,000 g on a 10 μL glycerol bed for 30

min.23 The centrifuged TC were then re-dispersed in distilled water and stored at 4 °C

until use.

The loading efficiency (LE) and loading content (LC) of Dox•HCl in TC were

determined by subtracting the amount of free Dox•HCl in supernatants quantified by a


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Synergy H1m microplate reader (BioTek, Winooski, VT, USA) at 480 nm (excitation) and

600 nm (emission) from the amount added，using the following equation:

0

0

% 100%SC C

LEC

−= ×

Eq. (1)

% 100%t s

t

m mLC

m

−= ×

Eq. (2)

where C0 is the initial drug concentration, Cs is the concentration of free drug in the

supernatant, C0-CS is the concentration of encapsulated drug, mt is the total mass of

drug and polymer, ms is the mass of free drug in the supernatant, and mt-mS is the

mass of encapsulated drug.

2.3. Characterization of nanocomplex

The particle size, size distribution [polydispersity index (PDI)] and zeta potential of

nanocomplex were investigated using dynamic light scattering (DLS; Nano ZS, Malvern,

UK). The morphology of nanocomplex was observed using a transmission electron

microscope (TEM) Tecnai G2 Spirit (FEI, Netherlands). Characterization of

nanocomplex by proton nuclear magnetic resonance (1H NMR) spectroscopy, fourier

transform infrared spectroscopy (FT-IR) and X-ray diffraction（XRD）were also

conducted.

2.4. Effect of simulated physiological pH and ionic strength conditions on the

stability of nanocomplex

The stability of polyion nanocomplex is greatly influenced by various factors involving

their compositions and their surrounding environment, in particular, by pH value or

ionic strength because of their shielding effect on the electrostatic interactions.24 This


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is important for oral delivered polyion nanocomplex, because they are exposed to

changing pH conditions, ranging from the acidic medium in the stomach to pH of

approximately 7 in the intestinal lumen, which is filled with various electrolytes. Thus,

the stability of TC in simulated physiological pH and ionic strength conditions was

investigated.

To evaluate the effects of pH conditions along GIT on the stability of the

nanocomplex, BC (1 mg/mL PGA) or TC (1 mg/mL PGA) were mixed with equal volumes

of pure water, and the pH of the mixture was titrated by Malvern MPT-2 autotitrator.

The particle sizes of nanocomplex at various pH values were tested by Malvern

Zetasizer Nano for measuring their stability. Experiments were performed in triplicate.

The stability of the nanocomplex in 0.01 M Phosphate-buffered saline (PBS) was

evaluated by measuring changes in nanocomplex size over time.24 BC (1 mg/mL PGA)

or TC (1 mg/mL PGA) were mixed with 0.01 M PBS at pH 5.5 at 1:20 volume ratio, and

the mixtures were placed in an orbital incubator at 37 °C. The size of nanocomplex was

measured by DLS at determined time points. Experiments were performed in triplicate.

To investigate the effects of physiological pH and ionic strength on the stability of

nanocomplex, 2 mL of BC (1 mg/mL PGA) or TC (1 mg/mL PGA) were loaded into a

dialysis bag with a 10,000 MW cut-off against 40 mL pH 7.4 PBS (0.01 M).

Nanocomplex was incubated in PBS (pH 7.4) and imaged at determined time points.

2.5. Gut cell (Caco-2) studies

Caco-2 gut cells were used as the model to study the permeability of the drugs to cross

the intestinal epithelial barrier. Caco-2 cell line (American Type Culture Collection,

Manassas, VA, USA; passages 30–40) was cultured in DMEM supplemented with 10%


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heat-inactivated fetal bovine serum, 1% penicillin, and 1% streptomycin. Cells were

cultured with the density of 1 × 106 cells in a 75 cm2 culture flask in a humidified

incubator under 5% CO2/95% air atmosphere at 37 °C. Culture medium was changed

every 1-2 days and the cells were harvested at 80% confluence with trypsin-EDTA.

2.5.1. MTT cytotoxicity assay of PGA/PAE complex in Caco-2 cell

Caco-2 cells were seeded at a density of 104 cells/well in 96-well plates, and incubated

for 3 days. Cultured cells were washed 3 times with Hank's balanced salt solution

(HBSS) and used for MTT assay. HBSS (200 μL) was added to each well of the 96-well

plates and incubated for 30 min. HBSS solutions and PGA/PAE suspensions were

subsequently added to each well at a range of concentrations of PGA. After 2 h

incubation at 37 °C, incubation medium was discarded and 100 μL of MTT solution (0.5

mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide in PBS) was

added to each well. Following 2 h incubation at 37 °C with MTT solution, 50 μL

dimethylsulfoxide was added to dissolve MTT formazan, and the cells were incubated

in an orbital shaker at 37 °C for 20 min. The absorbance was measured at 490 nm using

Synergy H1m microplate reader.

2.5.2. Cellular uptake study

Caco-2 cells were seeded onto 0.4 µm pore transwell inserts (Costar Corning, NY, USA)

by adding 0.5 mL of cell suspension at a density of 1.0 × 105 cells/mL. Cells were

cultured for 21 days with 0.5 mL of culture medium in donor chamber and 1 mL in the

acceptor chamber under culture conditions described above. The integrity of cell

monolayers was evaluated prior to the cellular studies by measuring transepithelial


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electrical resistance (TEER). Cell monolayers were considered intact and suitable for

use when TEER values were 400–600 Ω▪cm2.

To investigate the effect of PGA-based nanocomplex on drug intake capacity,

cellular uptake efficiency from Dox•HCl solution, BC, and TC were compared. Prior to

the uptake study, the donor chamber of transwell inserts was incubated with HBSS for

30 min while the culture medium was removed from the acceptor chamber. The

uptake was subsequently initiated by replacing the contents of the donor chambers

with test solutions, and cells were incubated for 1 h at 37 °C.25 Following incubation,

cell monolayers were washed 3 times with HBSS. Cell monolayers were handled and

intracellular Dox•HCl concentrations were measured using previously reported

methods, with minor modifications.11, 26 The cell monolayer was lysed，and samples

were collected and diluted in ethanol solutions containing 6% hydrochloric acid.

Diluted samples were left overnight and then centrifuged at 12,000 × g for 15 min.

Aliquots (200 μL) of supernatant were analyzed at 480/600 nm (excitation/emission)

using the Synergy H1m microplate reader.

For analysis by laser scanning confocal microscopy (LSCM, FV1000, Olympus, Tokyo,

Japan) , Caco-2 cell monolayers on transwell inserts were immediately washed 3 times

with a large amount of HBSS following the uptake incubation. Polycarbonate

membranes with cleaned cell monolayers were cut and attached to glass slides. Cells

were subsequently fixed with 4% paraformaldehyde solution, stained with DAPI, and

embedded in a phosphate-buffered saline/glycerol (1:9) mixture. Samples were

observed using LSCM. Dox•HCl and DAPI were evaluated at 590/618 nm and 359/461

nm (excitation/emission), respectively.


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2.5.3. Transport study

Cell monolayers were incubated with the HBSS in both donor chamber and acceptor

chamber for 30 min. Transport studies were initiated by replacing the contents of the

donor chambers with test solutions. Acceptor chambers were sampled at

predetermined time points, and equivalent amount of fresh HBSS was supplemented

maintaining a constant volume. Collected samples were transferred into a 96-well

plate, and the Dox•HCl concentration was detected using a microplate reader.

Apparent permeability coefficient (Papp) was calculated using the following equation:

0

1= ×

×app

dQP

dt A C Eq. (3)

where dQ/dt is the flux rate of Dox•HCl from the apical side to the basolateral side, C0

is the initial concentration of Dox•HCl in the apical compartment, and A is the

membrane area (cm2).

2.5.4. Mechanisms Evaluation study

The potential role of PGA-dependent particle uptake was evaluated using free PGA as

the competitive intake inhibitor in uptake studies. Prior to the uptake study, the donor

chamber was incubated with HBSS in the presence of PGA for 30 min, while culture

medium was removed from the acceptor chamber. Uptake was initiated by replacing

the contents of the donor chamber with test solutions in the presence of PGA, and

incubated for 1 h at 37 °C. Following incubation, the cell monolayers were washed 3

times with HBSS and cell samples were handled as described above for the uptake

study to measure Dox•HCl concentration.


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To investigate the possible role of epithelium-bound γ-GT in the uptake of Dox•HCl-

loaded PGA nanocomplex, uptake studies using acivicin as the γ-GT inhibitor was

conducted in Caco-2 cell monolayers.27 The endocytosis mechanism for nanocomplex

loaded Dox•HCl into cells was also investigated in the presence of chlorpromazine,

nystatin, and methyl-beta-cyclodextrin (MβCD). Chlorpromazine was shown to

dissociate clathrin from the surface membrane, inhibiting clathrin-mediated

endocytosis.28 Nystatin is an inhibitor of caveolae-mediated endocytosis. MβCD, a

reagent that extracts cholesterol from the plasma membrane, is an inhibitor of lipid

raft-dependent endocytosis, including cholesterol-associated clathrin and caveolae-

mediated endocytosis.28-30 These inhibitors were used at the following concentrations:

10 μg/mL of chlorpromazine, 13.2 mg/mL of methyl-beta-cyclodextrin (MβCD, 10

mmol), 25 μg/mL of nystatin. All these uptake inhibition studies were conducted

according to the uptake study method described above.

For statistical analysis, we analyzed the results by comparing the means of groups in

the in vitro experiments using one- and two-way analyses of variance (ANOVA) tests by

GraphPad Prism. P-values less than 0.05 were considered to be statistically significant.

2.6. Animal studies

2.6.1. Absorption by intestinal villi

The overall absorption at the intestinal villi was investigated in male Sprague-Dawley

rats weighing 260–287 g. All animal procedures performed in this study were

evaluated and approved by the Animal Ethics Committee of Shanghai Institute of

Materia Medica, Chinese Academy of Sciences (IACUC certification number: 2013-04-


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GY-10). Before experiments, rats were fasted for 12 h, but allowed free access to water.

The fluorescence intensities of different formulations were calibrated using solutions

with known Dox•HCl concentrations before the administration. Dox•HCl formulations

(5 mg/kg) were intragastrically administered using an oral feeding needle. After 2 h,

rats were sacrificed and intestinal tissues were excised. The intestinal tissue was

immediately washed 3 times with a large amount of HBSS to remove non-penetrated

nanocomplex. Cleaned intestinal tissues were divided into 3 anatomical regions:

duodenum, jejunum, and ileum. Intestinal tissues were sliced into 20 μm-thick sections

using a cryostat (CM3050S, Leica, Nussloch, Germany) and attached to glass slides.

Tissue slices were subsequently fixed with 4% paraformaldehyde solution, stained with

DAPI, and embedded in PBS/glycerol mixture (1:9). Each sample was observed under

an LSCM (FV1000, Olympus). Drug and DAPI levels were evaluated at 590/618 nm and

359/461 nm (excitation/emission), respectively.

2.6.2. In vivo pharmacokinetic study

Total absorption of Dox•HCl from various formulations was evaluated in male Sprague-

Dawley (SD) rats weighing 290~325 g. 18 rats were randomly distributed into 3 groups

with 6 rats in each group. Rats were fasted for 12 h before the experiments, but

allowed free access to water. Different formulations (with dosage corresponding to

Dox•HCl dose of 15 mg/kg) were administered intragastrically using an oral feeding

needle. Blood samples (0.25 mL) were collected at predetermined time points (1–24 h).

Collected samples were handled and Dox•HCl concentrations detected using

previously reported methods, with minor modifications.6, 9, 11, 21, 31, 32 The method was

validated for specificity, precision, recovery, and linearity. Briefly, freshly collected


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blood samples were centrifuged at 4 °C (12,000 × g, 10 min) to obtain the plasma.

Plasma aliquots (100 µL) were mixed with acetonitrile to precipitate the plasma

proteins. Samples were subsequently centrifuged at 4 ºC (12,000 × g, 10 min) to

extract Dox•HCl. Aliquots (50 μL) of supernatant were analyzed using HPLC by

quantifying its fluorescence intensity at 480 nm/590 nm (excitation/emission

wavelengths). Pharmacokinetic variables, including AUC0-24h, Cmax, and t1/2 were

calculated using pharmacological software Drug and Statistics Software (DAS, version

2.1.1, Mathematical Pharmacology Professional Committee of China).

3. Results and discussion

Dox•HCl has very low oral bioavailability (approximately 1%) because of its limited

intestinal absorption. The poor physicochemical properties (hydrophilic cation; pKa

=9.67, log P =0.5) make Dox•HCl absorption across the intestinal epithelium primarily

via the paracellular pathway other than the transcellular pathway.33 However, the

paracellular route offers a limited window for intestinal absorption since it was

estimated to account for only about 0.01% of the total small intestinal surface area.34

Encapsulation of Dox•HCl in polymeric nanoparticles could potentially implement the

enhancement in transcellular absorption by endocytosis pathway. Studies have shown

that incorporation of PGA in polymeric nanoparticle has great potential in enhancing

cellular uptake of genes.20 The aim of the current work was to explore the effects of

PGA-based TC in delivering of Dox•HCl across the epithelial cell monolayer, and then

enhancing Dox•HCl oral absorption.


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3.1. Preparation and characterization of PGA-based nanocomplex

The number- and weight-average molecular weights (Mn, Mw) for PGA were 122,954 ±

1979.6 and 113,788 ± 1775.1, respectively. The polydispersity index (PDI) was 1.081

(Table S 1 in supporting information). The structures of PGA and PAE were illustrated

in Figure 1. BC and TC were successfully prepared using electrostatic attractions

between oppositely charged components (Figure 1). After mixed with cationic Dox•HCl,

PGA was formed into BC with zeta potential of -69.1 ± 5.4 mV and particle size of 189.3

± 13.7 nm （PDI = 0.211 ± 0.099）(Figure 2). When Dox•HCl was 1.3 mg/mL, 1 mg/mL

of PGA was used. Higher concentration than 1 mg/mL for PGA would lead to the

aggregation of the particles. To further improve the physiochemical properties of BC,

PAE was introduced to form TC. As presented in Table 1 and Figure 2, TC with

Dox•HCl/PGA/PAE/ (w/w/w) composition of 13:10:1.25 was slightly negatively charged

(-29.1 ± 7.4 mV) compared to BC. With PAE content increasing, TC tended to aggregate,

and the particle size could not be accurately detected by Malvern Zetasizer (Table 1). A

reduction of particle size for BC was observed with the increase of PAH concentration

(Table 1). The particle size for TC (179.0 ± 19.7 nm, PDI = 0.171 ± 0.063) was smaller

than that of BC (189.3 ± 13.7 nm, PDI = 0.211±0.099) (Figure 1, Table 1). The possible

reason was the strong ionic interactions between oppositely charged PGA and PAH.

PGA was self-assembled into nanocomplex by adding of Dox▪HCl which acted as the

linked point between molecular chains of PGA. After adding positively charged

polymer PAE, the complexes might be further condensed or compacted due to strong

ionic interactions between PAE and PGA, leading to a reduced particle size and a better

stability. LE and LC for Dox•HCl in TC were calculated as 75.5 ± 10.3% and 33.6 ± 4.5%,


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respectively. TEM observation showed that sphere-like morphology were obtained for

both BC and TC (Figure 2).

1H NMR spectroscopy was used to characterize the nanocomplex. Figure 3 showed

the 1H NMR spectra of Dox▪HCl, PGA, PAH, Dox▪HCl+PAH (mixture for pure Dox▪HCl

and PAH), BC and TC in D2O. After simple mixing of Dox•HCL with PAH, there was no

significant change in chemical shifts of 1H NMR spectra for Dox▪HCl and PAH compared

to the corresponding chemical shifts for pure Dox▪HCl and PAH. These results

suggested that no strong interactions between Dox▪HCl and PAH were detected.

However, the peaks a, b, c and d of Dox▪HCl in BC and TC disappeared, while the

characteristic peaks of PGA in BC and PAH in TC could be clearly observed. It could be

speculated that Dox▪HCl was strongly interacted with PGA mainly via ionic interactions,

leading to the entangling of PGA molecules and finally the formation of nanocomplex.

Dox▪HCl was completely confined into inner space of BC and TC, leading to a significant

reduction in characteristic peak intensity and even the disappearance of peaks. 35

In TC, compared to BC and pure PAH, the characteristic peaks e and g of PGA were

shifted from 4.11 ppm and 2.01 ppm to 4.20 ppm and 2.10 ppm, respectively.

Meanwhile, the characteristic peaks h, k and l of PAE were shifted from 4.23 ppm, 3.62

ppm, and 3.24 ppm to 4.20 ppm, 3.66 ppm, and 3.22 ppm, respectively. This results

indicated that the group of carboxyl (-COO-), amide(-CO-NH-) in PGA and the group of

ester (-OCO-), tertiary amine and hydroxyl (-OH) in PAH were participated in the

interactions of TC components. Therefore, it could be speculated that the noncovalent

interactions between PGA and PAH included the electrostatic interactions and

hydrogen-bonding.


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Additional data such as XRD and FT-IR also confirmed the existence for interactions

between Dox▪HCl, PGA and PAH. The X-ray diffractograms of drug, polymers and

nanocomplex were shown in Figure S1A (supporting information). Distinctive

crystalline peaks were exhibited in the diffractogram of Dox•HCl, whereas PGA and

PAE showed a typical amorphous pattern. Absence of characteristic peaks for Dox•HCl

and PAE was observed after formed into TC, implying the interactions between these

compositions and PGA. FT-IR analysis (Figure S1B, supporting information) showed

that the formation of TC by Dox•HCl, PGA and PAE was accompanied by changes in

their IR spectra as compared with the individual components. For example, the signals

for broad strong characteristic peak at 3430.74 cm-1 corresponding to -OH stretching

for the pure PGA and the strong characteristic peak at 1729.83 cm-1 corresponding to

C=O stretching for pure Dox•HCl were both shifted in TC, confirming the interactions

involving these characteristic groups for drug and polymers.

3.2. Stability of nanocomplex in simulated physiological pH and ionic strength

conditions of GIT

An important factor for enhancing oral absorption of polyion nanocomplex loaded

drugs is to ensure the integrity of particles and their good dispersion in the harsh gut

conditions, such as the extremes of pH and ionic strength.24 Aggregation of

nanocomplex can lead to the loss of some special properties of nanocomplex, such as

the ability to adhere to the mucosal wall. In our study, the prepared TC were shown to

be relatively more stable than BC in simulated physiological pH and ionic strength

conditions in GIT (Figure 4).


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The influence of constantly changing pH on the size of the nanocomplex was

presented in Figure 4A. As pH conditions changed from acidic medium of the stomach

to approximately pH 7 in the intestinal tract, TC were found to be more stable than BC,

especially at the range of pH values above 6.5, where the size of BC could not be

accurately detected due to aggregation. The tested pH value was ranged from 3.0 to

7.0 including the simulated intestinal fluids pH value 6.8 and the simulated gastric

fluids pH value 3.0.36 We didn’t detect the stability of nanocomplex in conditions of pH

< 3.0, because the pKa value for PGA is about 2.27 ̴ 2.9. When pH <3.0, most carboxyl

groups on PGA were in the form of –COOH but not –COO- 37, 38 leading to the reduction

of electrostatic interactions between PAE/Dox•HCl and PGA and the subsequent

disintegration of nanocomplex. The effect of ionic strength on particle stability was

shown in Figure 4B. BC tended to be more sensitive to ionic strength. Following

incubation against 0.01 M PBS, the size of BC increased to more than 800 nm within 1

h and then underwent aggregation or disintegration, as sizes of BC particle could not

be measured accurately by DLS after 2 h incubation. Conversely, the size of TC

incubated with PBS maintained at about 200 nm with minor changes. Figure 4C

presented the representative images for nanocomplex in pH 7.4 PBS. Following 2 and 4

h incubation in dialysis bag against PBS, BC was found to be aggregated. Conversely, TC

was found to be well dispersed in the media. Taken together, TC was more stable in

simulated physiological conditions of GIT, and would be more prone to exert its

capacity to adhere to the mucosal wall, then initiate the further specific particle-cell

interactions.


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3.3. Cellular studies

3.3.1. Evaluation of cell activity by MTT

MTT assay was employed to evaluate the cytotoxicity of PGA-based nanocomplex. As

shown in Figure 5A, the concentrations of each constituent group (PGA concentration

ranging from 70-700 μg/mL) used in this study did not significantly reduce cell viability

(>92% of control).

3.3.2. Cellular uptake and Transport study in Caco-2 cell monolayer

The γ-glutamyl terminal groups of PGA on the surface of TC were expected to allow the

polymer to act as a ligand to specifically attach particles to the intestinal membrane-

bound proteins γ-GT, thereby facilitate the specific active endocytosis 16 for poor

permeable drugs. To verify this hypothesis, confluent Caco-2 cell monolayer on a cell

culture insert filter (Transwell) was used as an in vitro epithelial model of the human

small intestine to predict the absorption of orally administered drugs. When cultured

under specific conditions, Caco-2 cells express tight junctions, microvilli, and a number

of enzymes including γ-GT that are morphologically and functionally resemble of

enterocytes.39-41 In our studies, prepared TC exhibited the most drastic improvement

in cellular uptake and Papp for Dox•HCl compared to BC and Dox•HCl solutions (P <

0.01). TC can be a promising intestinal delivery system for Dox•HCl absorption.

As shown in Figure 5B, BC increased the cellular uptake of Dox•HCl in Caco-2 cell

monolayers. After uptake for 1 h, intracellular concentration of Dox•HCl by BC was 1.7-

fold higher than the concentration measured for Dox•HCl solution. Among the test


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groups, TC was found to be most effective in enhancing cellular uptake, resulting in

5.2-fold higher than Dox•HCl solution. Results observed by LSCM also confirmed the

highest Dox•HCl uptake efficiency by TC (Figure 5C). Compared to Dox•HCl solution

and BC, higher intracellular Dox•HCl fluorescence in cell monolayers were observed

following exposure to Dox•HCl-loaded TC.

The Papp values for Dox•HCl, BC, and TC were calculated as 6.9 × 10-7, 1.9 × 10-6, and

3.2 × 10-6 cm/s, respectively. Compared to the Dox•HCl solution, Papp for Dox•HCl-

loaded BC and TC was 2.8- and 4.6-fold higher, respectively.

3.3.3. Mechanism underlying enhanced drug absorption

To elucidate the mechanisms underlying the enhancement of drug cellular absorption,

competition study (or inhibition study) in Dox•HCl cellular uptake was conducted in

the presence of various inhibitors.

i) PGA-dependent TC endocytosis

To confirm the important role of PGA in PGA-incorporated TC-dependent Dox•HCl

absorption, free PGA was used as the inhibitor in evaluation of Dox•HCl cellular uptake

from BC and TC. As shown in Figure 6A, the uptake efficiency was decreased when cell

monolayer was pre-incubated with PGA. When 200 μg/mL PGA concentration was

used, cellular uptake of Dox•HCl from BC and TC was decreased by approximately 42.1

and 61.4%, respectively. The significant decreases in cellular uptake for Dox•HCl from

BC and TC (P< 0.05) after incubating with 200 μg/mL of free PGA indicated the

important role of PGA-mediated specific endocytosis in epithelial absorption of these

nanocomplex.


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ii) γ-GT-mediated specific endocytosis of PGA-based TC nanocomplex

γ-GT is highly expressed in Caco-2 cells and intestine, playing a key role in

catalyzing the cleavage of γ-glutamyl donor substrates. Thus, specific interactions were

proposed between intestinal epithelium bound-γ-GT and PGA-based nanocomplex

which had γ-glutamyl terminal groups on particle surface16, 40, 42, 43. To elucidate the

possible mechanisms underlying the endocytosis of PGA-based TC in epithelial cells, γ-

GT was inhibited by specific irreversible inhibitor acivicin in cellular uptake studies27.

The intracellular Dox•HCl in Caco-2 cell monolayer from BC and TC was reduced by

37.6 and 51.7% respectively, compared to their control group (Figure 6B). These

results confirmed that γ-GT on Caco-2 cell contributed a lot to the specific binding of

PGA-based nanocomplex to the cell membrane, thereby mediating the specific

endocytosis for the absorption of nanocomplex-loaded Dox•HCl. LSCM observation

results for cell monolayers after uptake for 1 h further confirmed the important role of

γ-GT in delivering of Dox•HCl into the gut cells (Figure 6C). Intracellular Dox•HCl

fluorescence was decreased for both BC and TC in the presence of acivicin compared

to their control groups without treated with acivicin. The results showed that γ-

glutamyl terminal group of PGA on TC surface was a ligand for γ-GT, inducing a specific

interaction between nanocomplex and epithelial membranes, and then the

subsequent efficient absorption for Dox•HCl.


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iii) Endocytic mechanism for TC nanocomplex

To further elucidate the mechanisms underlying endocytosis of nanocomplex into

cells following the initiation of TC-cell specific interactions, the effects of various

endocytosis inhibitors in uptake experiments were evaluated. Our results showed that

the uptake of Dox•HCl from BC and TC was significantly decreased at 4 °C (P < 0.01),

and Dox•HCl uptake was only 30% of the uptake observed at 37 °C. It was suggested

that energy-dependent active pathway was involved in Dox•HCl uptake from BC and

TC, as active pinocytic/endocytic uptake is inactivated at 4 °C. Compared to the control

group, no significant difference in Dox•HCl uptake was observed in the presence of

nystatin for BC and TC. After treated with chlorpromazine, however, cellular uptake of

Dox•HCl from BC and TC was decreased to 64.7 (P < 0.05) and 40.7% (P < 0.05),

respectively (Figure 6D). This implied the involvement of clathrin-dependent endocytic

pathway for BC and TC. When MβCD was used as an inhibitor, the cellular uptake of

Dox•HCl from TC was decreased to 23.9%, compared to 65.0% from BC. MβCD can

extract cholesterol from the plasma membrane, thus is an effective inhibitor for the

lipid raft-involved epithelial endocytosis including clathrin- and caveolae-

dependent/independent pathways.44, 45 When MβCD was applied, a significant

reduction of cellular uptake for BC and TC was observed, indicating that cholesterol-

dependent endocytosis pathways were highly involved in the cellular uptake of both

nanoparticles. Nevertheless, the extent of inhibition was different between the BC and

TC. The cellular uptake of TC was more sensitive to the MβCD compared to BC. The


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potential reason was that multiple pathways other than lipid raft dependent pathways

might be involved in the endocytosis of BC, while the endocytosis of TC was mainly

mediated by lipid raft.

3.4. Enhanced in vivo TC-dependent drug absorption in rat models

The aim of this study was to prepare an efficient functional delivery system that could

facilitate the absorption of poorly permeable drug Dox•HCl. To evaluate in vivo

absorption by TC, we assessed the absorption of Dox•HCl in rats.

3.4.1. Absorption across intestinal villi

The absorption of Dox•HCl in different sections of the intestine (duodenum, jejunum,

and ileum) was investigated in male SD rats. As shown in Figure 7, only slight Dox•HCl

fluorescence was observed in duodenum and jejunum of the animals after treated

with Dox•HCl solution. Dox•HCl-loaded BC increased the drug absorption compared to

the Dox•HCl solution, as evidenced by enhanced Dox•HCl fluorescence in the

duodenum and jejunum. Dox•HCl-loaded TC were most effective in enhancing the

intestinal absorption of Dox•HCl. Higher Dox•HCl fluorescence was observed in all

three intestinal sections by Dox•HCl-loaded TC, compared to the other treatment

groups (Figure 7). The results of intestinal villous absorption study were consistent

with the findings of our in vitro analyses, with TC exhibiting the best intestinal Dox•HCl

delivery compared to other groups in all 3 intestinal sections.

3.4.2. Pharmacokinetic study

To investigate the overall absorption of Dox•HCl from the nanocomplex, in vivo drug

bioavailability study was conducted in rats. The concentration-time curve was


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presented in Figure 8. Compared to the Dox•HCl solution, absorption for Dox•HCl in

TC was highly improved, that the relative bioavailability (FR) for TC was increased to

417% compared to Dox•HCl solutions (Table 2). The values of Cmax, t1/2, and AUC0-24h

for Dox•HCl solution, BC and TC were shown in Table 2. In vivo pharmacokinetic data

confirmed that TC exhibited the best Dox•HCl absorption.

TC implemented the specific interaction with intestinal epithelium-bound γ-glutamyl

transferase through γ-glutamyl terminal groups of PGA, which implement efficient

transcellular absorption for Dox•HCl (Figure 9). The studies suggest that PGA-based

functional TC is a potential carrier for Dox•HCl oral delivery.

4. Conclusion

In this work, self-assembled TC (Dox•HCl/PGA/PAE) were prepared for oral delivery of

Dox•HCl. Results confirmed that PGA functionalized TC facilitated particle-dependent

specific interactions with enterocytes and increased the transport of Dox•HCl.

Therefore, efficient intestinal absorption for Dox•HCl was achieved by TC, with 2.6-

and 4.2-folds higher in Cmax and AUC0–24h values compared to Dox•HCl solutions,

respectively. TC can be a promising carrier for oral Dox•HCl delivery. These results can

guide rational design of nanocarriers for oral absorption of poorly permeable drugs,

including the selection of functional polymers and control of physicochemical

properties.

Acknowledgements


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We are grateful for the financial support from the National Natural Science

Foundations of China (grant No. 81373356) and the "Science and technology

innovation action plan for basic research" of Shanghai 2014 (grant No. 14JC1493200).

This work was also partly supported by the National Natural Science Foundation of

China (grant No. 81202468).


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Tables

Table 1 The optimization of ternary nanocomplex (TC) composition

Parameter

Dox•HCl:

PGA:PAE

(w/w/w) =

13:10:0

Dox•HCl:

PGA:PAE

(w/w/w) =

13:10:0.5

Dox•HCl:

PGA:PAE

(w/w/w) =

13:10:1

Dox•HCl:

PGA:PAE

(w/w/w) =

13:10:1.25

Dox•HCl:

PGA:PAE

(w/w/w)=

13:10:1.5

Dox•HCl:

PGA:PAE

(w/w/w)

= 13:10:2

Zeta (mV) -69.1 ± 5.4 -57.2 ± 7.3 -41.7 ± 4.7 -29.1 ± 7.4 / /

Size (nm) 189.3±13.7 187.2±10.4 181.4 ± 7.7 179 ± 19.7 / /

PDI 0.211±0.099 0.200±0.080 0.159±0.100 0.171±0.063 / /

Dox•HCl , doxorubicin hydrochloride; PGA, poly-(l-glutamic acid); PAE, poly-(β-amino ester); PDI, polydispersity index; /, size and zeta potential could not be accurately detected by Malvern Zetasizer due to particle aggregation (n=3).


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Table 2 Pharmacokinetic parameters of doxorubicin hydrochlorides (Dox•HCl) following oral administration of different formulations

Parameter Unit Dox•HCl BC TC

t1/2 h 5.6 8.2 17.2

Cmax μg/L 979.8± 94.2 1317.3± 144.7 2572.9 ± 531.3

AUC0-24h μg/L*h 9017.8± 2047.1 15693.7± 3734.2 37614.3 ± 9491.3

FR % 100 174 417

Dox•HCl, doxorubicin solution; BC, Dox•HCl-loaded binary nanocomplex; TC, Dox•HCl-loaded ternary nanocomplex; Cmax: maximum serum concentration; Tmax: time at which Cmax is attained; FR: relative bioavailability.


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29

Figure legends

Figure 1: Schematic of the structure of poly-(L-glutamic acid) (PGA) and poly-(β-amino

ester) (PAE), and the preparation process for nanocomplex.

Figure 2: Particle size, zeta potential and morphology of binary (BC) and ternary

nanocomplex (TC), evaluated by dynamic light scattering (DLS) and transmission

electron microscopy (TEM).

Figure 3: 1H NMR spectra for Dox▪HCl, PGA, PAH, Dox▪HCl+PAH (mixture for pure

Dox▪HCl and PAH ), BC and TC in D2O.

Figure 4: The influence of the harsh gastrointestinal tract (GIT) conditions on particle

stability. (A) Constantly-changing pH conditions on the stability of nanocomplex; (B)

electrolytes content solutions (0.01 M phosphate-buffered saline (PBS), pH 5.5) on the

stability of nanocomplex; (C) Representative images of the state of the nanocomplex

following incubation in PBS (pH 7.4) for 2 and 4 h.

Figure 5: A) Viability of cells by MTT assay following treatment with a range of

concentrations of PGA/PAE nanocomplex (n=5); B) Cellular uptake of Dox•HCl (n=3) in

21 days-cultured cell monolayers; C) Representative images for Dox•HCl cellular

uptake by LSCM in 21 days-cultured cell monolayers. TC exhibited the most effective

cellular uptake for Dox•HCl (Blue fluorescence showed the cell nuclei morphology by

DAPI staining, and the red fluorescence between cell nuclei was Dox•HCl in cytoplasm).

Figure 6: A) PGA-dependent cellular uptake for nanocomplex loaded Dox•HCl was

determined by using free PGA as the cellular uptake inhibitor; B) Epithelium-bound γ-


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30

GT-mediated cellular uptake was confirmed by using acivicin as γ-GT inhibitor; (C) 3D

reconstruction of Z-stack images by LSCM for intracellular fluorescence of Dox•HCl in

Caco-2 cell monolayer after uptake using acivicin as γ-GT inhibitor; D) Results of

endocytosis mechanisms for Dox•HCl absorption showing that more than one active

endocytosis pathways were participated in the uptake for PGA-based nanocomplex

loaded Dox•HCl.

Figure 7: Absorption at intestinal villi by LSCM after intragastrically administered for 2

h. Doxorubicin hydrochloride (Dox•HCl)-loaded TC exhibited improved intestinal

absorption of the drug in all studied intestinal sections, with more drug fluorescence

observed under LSCM. The white arrows indicated the absorbed drugs with red

fluorescence under LSCM.

Figure 8: Enhanced in vivo absorption. Doxorubicin hydrochloride (Dox•HCl) plasma

concentration AUC0-24h value from TC was 4.2 times higher than that of Dox•HCl

solution.

Figure 9: Schematic representation of proposed mechanism underlying the

endocytosis of poly-(L-glutamic acid) (PGA)-based nanocomplex. γ-Glutamyl, the

terminal group of PGA and a ligand for γ-GT, induced specific particle-cell interactions

that result in active endocytosis.


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Figure 1


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Figure 2


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Figure 3


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Figure 4


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Figure 5


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Figure 6


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Figure 7


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Figure 8


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Figure 9


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Graphic abstract (7.22 cm*4 cm)

PGA-based complex enhanced intestinal absorption due to the improved

active epithelial endocytosis through specific interactions with

epthelium-bound γ-GT.


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Documents

ZQL-journal of material chemistry B校稿前版