Scalable Manufacturing of Enteric Encapsulation Systems for Site

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Scalable Manufacturing of Enteric Encapsulation Systems for Site-Specific Oral Insulin Delivery Lilong Sun, Zhijia Liu, Houkuan Tian, Zhicheng Le, Lixin Liu, Kam W. Leong, Hai-Quan Mao, and Yongming Chen Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b01530 • Publication Date (Web): 11 Dec 2018 Downloaded from http://pubs.acs.org on December 14, 2018

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Biomacromolecules

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Scalable Manufacturing of Enteric Encapsulation Systems for Site-Specific

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Oral Insulin Delivery

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Lilong Sun,†,# Zhijia Liu,*,† Houkuan Tian,† Zhicheng Le,† Lixin Liu,† Kam W. Leong,Ω Hai-

4

Quan Mao,‡,^ and Yongming Chen*,†

5

†School

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Functional Materials of Ministry of Education, GD Research Center for Functional

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Biomaterials Engineering and Technology, Sun Yat-sen University, Guangzhou 510275, China

8

#Department

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Guangzhou, 510006, China

of Materials Science and Engineering, Key Laboratory for Polymeric Composite and

of Biomedical Engineering, School of Engineering, Sun Yat-sen University,

10

ΩDepartment

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United States

12

‡Institute

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Johns Hopkins University, Baltimore, Maryland 21218, United States

14

^Department

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Hopkins University School of Medicine, Baltimore, Maryland 21287, United States

of Biomedical Engineering, Columbia University, New York, New York 10027,

for NanoBioTechnology and Department of Materials Science and Engineering,

of Biomedical Engineering and Translational Tissue Engineering Center, Johns

16 17 18

Corresponding Authors

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*E-mail: [email protected].

20

*E-mail: [email protected].

21 22

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ABSTRACT

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Oral drug delivery is a more favored mode of administration because of its ease of

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administration, high patient compliance, and low healthcare costs. However, no oral protein

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formulations are commercially available currently due to the hostile gastrointestinal (GI)

5

barriers resulting in insignificant oral bioavailability of macromolecular drugs. Herein, we used

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insulin as a model protein drug, insulin-loaded N-(2-hydroxy)-propyl-3-trimethylammonium

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chloride modified chitosan (HTCC)/sodium tripolyphosphate (TPP) nanocomplex (NC) as a

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nanocore was further encapsulated into enteric Eudragit L100-55 material through two-step

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flash nanocomplexation (FNC) process in a reliable and scalable manner, forming our NC-in-

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Eudragit composite particles (NE). By tailoring particle size and surface properties, our

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optimized NE could protect the loaded insulin from acidic degradation in hostile stomach

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environment, and then achieve intestinal site-specific drug release as well as improve oral

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delivery efficiency of insulin. In addition, oral administration of the optimized NE to type 1

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diabetic rats could induce a very significant hypoglycemic effect with a relative oral

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bioavailability of 13.3%. Our results demonstrated that enteric encapsulation of

16

nanotherapeutics using a FNC apparatus could make drug formulations better size

17

controllability, batch-mode reproducibility and homogenous surface coating, and then

18

significantly enhance oral bioavailability of insulin, indicating its great potential for clinical

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translation of oral protein therapeutics.

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KEYWORDS: Chitosan derivative; Eudragit; Flash nanocomplexation; Insulin; Oral

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delivery

23 24

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Biomacromolecules

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INTRODUCTION

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Biologics including therapeutic proteins, peptides, and antibodies have been explored for

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prevention or treatment of chronic or inflammatory diseases, and cancers.1-3 However, delivery

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of biologics currently has been restricted to the parenteral administration, which is largely

5

resulted from their high molecular mass and hydrophilicity creating big obstacle to penetrate

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across biological barriers such as skin, mucosa or cell membranes.1, 4, 5 Unquestionably, oral

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drug delivery is a more favored modality of administration because of its ease of administration,

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high patient acceptance, and low healthcare costs.6,

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medication for type 1 diabetes management, is now administrated by subcutaneous (s.c.)

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injection in clinical application, which usually leads to very poor patient compliance due to the

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pain and needle phobia.8, 9 If obtainable, orally delivered insulin can significantly improve the

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quality of life of diabetics, and possibly show better effects in the treatment of diabetes by

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closely mimicking the physiological path of pancreatic insulin.10-12 Nevertheless, no oral

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protein formulations are commercially available due to the hostile gastrointestinal (GI) barriers

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resulting in insignificant oral bioavailability of protein drugs.1, 8, 13

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For example, insulin, an essential

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In the past decades, these biocompatible and biodegradable nanoparticulate systems have

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emerged as one of the most promising drug vehicles toward oral delivery of protein

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therapeutics.3, 14-19 Specially, they have been confirmed to exhibit many advantageous features

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such as tailor-made particle size and surface properties, controllable drug release as well as

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improved oral delivery efficiency of drugs.3,

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nanocarriers have been widely studied for oral drug delivery because of their potency to

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improve intestinal permeability by transiently and reversibly opening tight junctions located in

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intestinal epithelium.5, 21-23 For instance, Sonaje et al. have prepared pH-sensitive and mucus-

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adhesive nanoparticles self-assembled by chitosan and poly-γ-glutamic acid for enhancing oral

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insulin delivery by paracellular pathway transport.24 Liu et al. have prepared trimethyl chitosan

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nanoparticles coated with a dissociable “mucus-inert” agent to improve oral absorption of

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insulin through their synergistic effect of efficient mucus-permeation and opening of tight

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junction.25 However, extremely acidic condition of stomach could degrade the encapsulated

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proteins and change native physicochemical properties of nanoparticulate delivery systems,

5, 20

It was known that natural chitosan-based

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which would possibly compromise the desired therapeutic outcome after oral dosing of

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nanotherapeutics.26, 27 To address these challenges, different enteric polymer materials (e.g.,

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Eudragit) have been frequently used to protect protein formulations from burst release,

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denaturation or acidic degradation in stomach environment, as well as achieve drug-controlled

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release in high absorptive region of intestine.26, 28, 29 It is worth noting that enteric coating of

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nanoparticles or microspheres are usually processed with spray-drying technique,

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nanoprecipitation or emulsion-solvent evaporation method in the field of pharmaceuticals

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industry.30-33 These coating techniques were complicated to modulate particle size and

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uniformity, coating layer or surface properties, and they generally involved in utilization of

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organic solvent, high temperature or pressure conditions, in which the aggregation or

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denaturation of therapeutic proteins would be occurred during manufacturing process as

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reported previously. 9, 20, 34, 35

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Recently, we have introduced a process termed flash nanocomplexation (FNC) to produce

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polyelectrolyte nanocomplexes for nanomedicine application in a reliable and scalable manner,

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and the particle formation was triggered by a turbulent mixing of aqueous solutions of two or

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more oppositely-charged polyelectrolytes in a miniature chamber.12, 36, 37 In comparison with

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conventional bulk mixing technique, the nanocomplexes generated by a FNC process have

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presented many unique features including better process control, uniformed particle size, higher

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drug loading level and encapsulation efficiency, and higher bioactivity retention of proteins.

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These have been confirmed to play a crucial role in clinical translation of nanotherapeutics.12,

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37, 38

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aqueous conditions via charge-charge interactions through the FNC process.12, 37, 38 Therefore,

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we considered that enteric encapsulation of nanotherapeutics could also be performed through

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a FNC process depending on the charge neutralization between nanocomplex and enteric

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polymer material, which would possibly exhibit more advantages in the production of well-

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controlled nanotherapeutics with homogenous enteric coating, compared to the conventional

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techniques as described above.

Moreover, the particles could be easily decorated with polysaccharide coating in mildly

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In this study, N-(2-hydroxy)-propyl-3-trimethylammonium chloride (HTCC) modified

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chitosan was synthesized to improve its water-solubility and cellular permeability at neutral 4 ACS Paragon Plus Environment

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Biomacromolecules

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conditions.12,

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HTCC/sodium tripolyphosphate (TPP) nanocomplexes (NC) to enhance permeability of insulin

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across intestinal epithelium. Then the optimized NC was encapsulated within enteric Eudragit

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L100-55 (Eudragit) material to produce NC-in-Eudragit composite particles (NE), which was

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further tailored to effectively protect the loaded insulin from acidic degradation, and achieve

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site-specific drug release. The schematic diagram of particles produced by two-step FNC

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process was shown in Scheme 1. The produced particles were characterized in terms of their

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physicochemical properties, drug release behaviors and trans-epithelial transports. Then, we

9

evaluated in vivo hypoglycemic efficacy and pharmacokinetics after oral administrated tested

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particles to type 1 diabetic rats. Finally, we investigated in vivo biodistribution and biosafety of

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the optimized NE for making a further understanding of oral insulin formulation.

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Insulin as a model protein drug was loaded into positively-charged

12 13

Scheme 1. NC-in-Eudragit composite particles (NE) were produced through two-step FNC

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process. Step I: Insulin-loaded HTCC/TPP nanocomplex was generated by a rapid and efficient

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mixing of insulin/TPP solution and HTCC solution under a miniature chamber; Step II: The

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NC was further encapsulated within enteric Eudragit L100-55 material (NE) through a FNC

17

apparatus again.

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2.

2

2.1.

MATERIALS AND METHODS Materials

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Chitosan (50 kDa, deacetylation degree 95%) and sodium tripolyphosphate (TPP) were

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obtained from Sigma-Aldrich. Glycidyltrimethylammonium chloride was purchased from

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Ladder Industrial Development Co., Ltd. Eudragit L100-55 (Eudragit) material was provided

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from Shanghai Chinewey Pharmaceutical Co., Ltd. Rhodamine 123 (R123) and rhodamine B

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isothiocyanate (RITC) were acquired from Aladdin Biochemical Polytron Technologies Inc.

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Sulfo-cyanine 7 NHS ester (Cy7) was purchased from Little-PA Sciences Co., Ltd. Insulin

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(porcine, 27.4 IU/mg) was purchased from Wanbang Biochemical Co., Ltd. Porcine mucin was

10

obtained from Sigma-Aldrich. 3-(4, 5-dimethyl-thiazol-2-yl)-2, 5-diphenyl tetrazolium

11

bromide (MTT) and Alexa Fluor® 647 conjugate of wheat germ agglutinin (AF-647) were

12

purchased from Abcam Plc. Occludin antibody was obtained from Gene Tex Inc. Anti-rabbit

13

IgG Fab2 Alexa Fluor® 488 (AF-488) Molecular Probe was provided from Cell Signaling

14

Technology Inc. Bicinchoninic acid (BCA) protein assay kit was bought from Thermo Fisher

15

Scientific Inc. Porcine insulin ELISA Kit was obtained from Mercodia Inc. Enzyme assay kits

16

including γ-GT (γ-glutamyl transpeptidase), ALP (alkaline phosphatase), ALT (alanine

17

aminotransferase), and AST (aspartate transaminase) were obtained from Nanjing Jiancheng

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Biotechology Co., Ltd. All chemical reagents used in the study were of analytic grade.

19

2.2.

Preparation of Insulin-Loaded HTCC/TPP Nanocomplexes (NC)

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Firstly, HTCC was produced, and purified using the same method as described previously,12

21

and the obtained 1H-NMR spectra (Figure S1 in Supporting Information) was highly consistent

22

with the data reported in the literatures,12, 23, 34 confirming that the successful quaternization of

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chitosan. The quaternary ammonium degree of HTCC was measured to be about 42.5% by

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conductometric titration method.12, 23 Then, the lyophilized HTCC powder was dissolved in

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deionized water at a concentration of 1.5 mg/mL and the pH was adjusted to 5.5−6.5. 4 mg/mL

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of insulin stock solution was prepared by dissolving the insulin in acidic water (HCl, pH 2.8)

27

and then adjusting the solution to pH = 8. TPP was dissolved in deionized water at a

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concentration of 0.2 mg/mL. The insulin/TPP solution (pH 8) was prepared by equal volume

29

mixing of insulin and TPP stock solution with a final concentration of insulin and TPP of 2

30

mg/mL and 0.1 mg/mL, respectively. 6 ACS Paragon Plus Environment

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Biomacromolecules

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Furthermore, NC was produced through a FNC process using multi-inlet vortex mixer

2

(MIVM) apparatus as reported elsewhere.12 Briefly, insulin/TPP mixed solution was through

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Inlet 1−2, HTCC solution (1.5 mg/mL) was introduced through Inlet 3−4. The NC could be

4

immediately formed by polyelectrolyte complex coacervation through a rapid mixing of

5

negatively-charged insulin/TPP solution and positively-charged HTCC solution in a MIVM

6

device. The volumetric flow rate of four inlets (5−50 mL/min) was held consistent and

7

controlled by programmable digital syringe pumps. Besides, the optimized NC was added to

8

cryoprotectant aqueous solution (1% mannitol, and 1% xylitol), and then snap-frozen by

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treatment of liquid nitrogen. After lyophilization at −30 °C and 0.36 mbar by using a lyophilizer

10

(Martin Christ Inc.), the freeze-dried NC powder was stored at 4 °C.

11

2.3.

Enteric Encapsulation of NC within Eudragit Materials

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Eudragit was dissolved in deionized water (pH 11), and then adjusted the pH to 6.5−5.8 with

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a final Eudragit concentration of 0−0.6 mg/mL. The optimized NC (pH 7.4) produced as

14

mentioned above was introduced through Inlet 1 and 2; Eudragit solution with various pH

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conditions (pH 6.5−5.8) was introduced through Inlet 3 and 4, respectively. By changing the

16

pH conditions and flow rate (5−50 mL/min), the NC-in-Eudragit composite particles (NE) were

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rapidly produced via charge neutralization between positively-charged NC and negatively-

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charged Eudragit through a FNC apparatus again.

19

2.4.

Particle Characterization

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The particle diameter (number-average), polydispersity index (PDI) and surface charge (ζ-

21

potential) were measured using laser light scattering instrument (Zetasizer Nano ZS 90,

22

Malvern) at 25 °C. The morphology of tested particles was observed on a transmission electron

23

microscope (JEM-1400 Plus, JEOL). Free insulin in tested particle solutions were separated by

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ultrafiltration (100 kDa, Millipore) under a speed of 3000 g at 4 °C for 15 min, and then insulin

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level in the filtrates were analyzed by a UV−vis spectrometer at 280 nm (Evolution 201,

26

ThermoFisher). The encapsulation efficiency (EE, %) and loading capacity (LC, %) were

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expressed by the equations 1 and 2 as follows.

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EE (%) = 1 ―

(

)

Amount of free insulin × 100% Total amount of insulin 7 ACS Paragon Plus Environment

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1

LC (%) =

Weight of encapsulated insulin × 100% Total weight of particles

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(2)

2

To confirm successful enteric encapsulation of NC into Eudragit material, their interaction

3

between insulin and Eudragit within NE composite particles were studied by the fluorescence

4

resonance energy transfer (FRET) analysis. R123-labeled Eudragit (R123-Eudragit) and RITC-

5

labeled insulin (RITC-insulin) were used as FRET pairs, fluorescence-labeled particles were

6

produced by the same procedures as described above. Fluorescent spectra of tested particles

7

were collected by measurement of emission spectra from 500 to 700 nm at excitation

8

wavelength of 488 nm (fluorescence spectrometer, RF-5301PC, Shimadzu).

9

2.5.

In Vitro Drug Release Study

10

Drug release profiles from tested particles were examined in various pH environments at 37

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C. Briefly, 1 mL tested particles were dialyzed (50 kDa, cutoff molecular weight) against 20

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mL PBS media with the shaking of 100 rpm during sequential changes in buffer pH (2.5, 6.8,

13

and 7.4) were carried out in consecutive time intervals including 0−2, 2−8 and 8−24 h,

14

respectively. At predetermined time intervals, 1 mL of released media were taken out and equal

15

volume of fresh media were added again. The insulin level in released media was measured by

16

BCA protein assay kit.12

17

2.6.

Cell Culture

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Caco-2 and HT29-MTX-E12 (E12) cells were cultivated with a complete medium consisted

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of Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco) supplemented with high glucose, 1%

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non-essential amino acids, 1% L-glutamine, 1% penicillin and streptomycin and 10% fetal

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bovine serum. The cells were maintained in an incubator instrument at 37 °C with 5% CO2 and

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95% of relative humidity.

23

2.7.

In Vitro Cytotoxicity

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The cell biocompatibility of tested formulations was evaluated with Caco-2 and E12 cells by

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a standard MTT assay. Caco-2 or E12 cells (1 × 104 cells/well) were separately cultivated into

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96-well cell plates and incubated for 24 h, and then the cells were treated with fresh medium

27

containing free insulin and tested particles with a insulin concentration of 10−250 μg/mL. After

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24 h incubation, 20 μL of 5 mg/mL MTT solution was added, and then incubated for another 4

29

h. The cell media were removed and replaced with 100 μL DMSO, and the absorbance was 8 ACS Paragon Plus Environment

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Biomacromolecules

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determined at 570 nm using a microplate reader (Synergy 2, BioTek).

2

2.8.

Trans-Epithelial Transport Study

3

The permeability of insulin across Caco-2 cell monolayer was examined in vitro. Caco-2

4

cells with a density of 1 × 104 cells/well were seeded in 12-Transwell fitted with polycarbonate

5

membranes, and then cultivated for 17−21 days with medium replacement every two days until

6

the TEER values were above 700 Ω·cm2 as described previously.12 Prior to the experiment, the

7

media in the apical and basolateral side were taken out and replaced with pre-warmed Hank’s

8

balanced salt solution (HBSS), respectively, and then maintained at 37 °C for equilibration of

9

30 min. For permeability study, the fresh HBSS containing RITC-labeled tested formulations

10

(200 μL, 250 μg/mL RITC-insulin) was added to the apical chamber with or without covering

11

the mucin gel (1%). At different time points, 100 μL media were removed from basolateral

12

chamber, and then the equal volume of fresh buffer was added again. Fluorescence intensity of

13

transported RITC-insulin was analyzed on a fluorescence microplate reader (Synergy 2,

14

BioTek), and the apparent permeability coefficient (Papp, cm/s) value was calculated as shown

15

in the equation 3.

16

17

Papp =

Where

𝑑𝑄 𝑑𝑡

dQ 1 × dt A × C0

(3)

indicates the flux of tested particles prepared with RITC-insulin from the apical

18

to the basolateral chamber, and C0 represents the initial fluorescent intensity in apical chamber,

19

and A is the membrane area (cm2).

20

2.9.

Monitoring of Trans-Epithelial Electrical Resistance (TEER)

21

Caco-2 cell monolayer were acquired as above-described procedures. 12-Transwell was

22

washed thrice with pre-warmed fresh HBSS and then equilibrated for 30 min. 200 μL medium

23

containing tested samples at an insulin of 250 μg/mL were pipetted into the apical chambers.

24

After 2 h of incubation, the cultured media were withdrawn and then replaced with pre-warmed

25

fresh HBSS. At different time intervals, TEER values were measured using a Millicell®-

26

Electrical Resistance System (Millipore, MA). In addition, Caco-2 cell monolayer was fixed

27

with 4% paraformaldehyde solution for 10 min, and subsequently treated with anti-occludin 9 ACS Paragon Plus Environment

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antibody at 25 °C for 30 min, and then treated with anti-rabbit IgG Fab2 AF-488 for another 1

2

h, and then washed thrice with PBS. Tight junction of Caco-2 cell monolayer was visualized

3

using immunofluorescent staining method,12 and observed by a confocal laser scanning

4

microscope (CLSM, SP8, Leica).

5

2.10.

Animal Experiments

6

The male Sprague−Dawley (SD) rats weighing about 220 g were provided by Animal

7

Experimental Centre of Sun Yat-sen University (SYSU, Guangzhou, China). All animal

8

experiments were performed in accordance with the Guide for the Care and Use of Laboratory

9

Animals and were approved by the Institutional Animal Care and Use Committee (IACUC) of

10

Sun Yat-sen University (SYSU). Type 1 diabetic rats were built by intraperitoneal injection of

11

70 mg/kg streptozotocin (STZ) dissolved in 0.1 M pH 4.2 citrate buffer as described in our

12

previous works.12 After feeding for 2 weeks, the diabetic rats were screened by the criteria that

13

fasted blood glucose level was above 16 mM.

14

2.11.

In Vivo Hypoglycemic Efficacy and Pharmacokinetics

15

The diabetic rats were fasted for 12 h with drinking water freely prior to administration. Then

16

the diabetic rats (n = 6) were orally administrated with saline, free insulin and tested

17

formulations at an insulin dose of 80 IU/kg, and also subcutaneously injected with insulin at a

18

dose of 5 IU/kg. At specific time intervals, the blood was sampled from the caudal vein of rats,

19

and the blood glucose level was measured by a glucose meter (Johnson & Johnson), and the

20

insulin level in serum was quantified with a porcine insulin ELISA kit. Both area-above-curve

21

(AAC) of blood glucose level profile and area-under-curve (AUC) of serum insulin level profile

22

were calculated, respectively. Pharmacological availability (PA, %) and relative oral

23

bioavailability (BA, %) were calculated by using the equations 4 and 5.

24

PA (%) =

25

BA (%) =

26 27

2.12.

AACp. o. × Doses.c. AACS.C. × Dosep.o. AUCp. o. × Doses.c. AUCS.C. × Dosep.o.

× 100%

(4)

× 100%

(5)

In Vivo Biodistribution

The biodistribution of optimized NE was evaluated in vivo. Briefly, the male SD rats (n = 3) 10 ACS Paragon Plus Environment

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Biomacromolecules

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were fasted overnight before oral gavage of Cy7-labeled NE or NC (as a control) with an insulin

2

dose of 80 IU/kg. The rats were anaesthetized by isoflurane, and the whole-body imaging of

3

rats was performed through In Vivo Imaging System FXPRO (Woodbridge) at 2, 4, and 6 h

4

after oral administration. Then, the rats were euthanized, and the gastrointestinal tract and

5

organs such as liver and kidney were further harvested for ex vivo imaging analysis.

6

2.13.

In Situ Absorption Study

7

The observation of oral absorption of optimized NE or NC (as a control) was qualitatively

8

analyzed in small intestinal segment of the rats. Briefly, the rats were fasted for 12 h with

9

drinking water freely prior to the experiment, then RITC-labeled NE and NC were

10

intragastrically administrated to the rats at a dose of 80 IU/kg (insulin), respectively. At 6 h of

11

post-treatment, the rats were anesthetized, and a midline laparotomy was carried out to collect

12

ileum segments, and then the rats were sacrificed. Subsequently, the ileum tissues were

13

collected and then treated as described in previous work.12 Finally, the tissue sections were

14

observed by a CLSM.

15

2.14.

In Vivo Toxicity Study

16

In vivo toxicity of oral formulations was evaluated in diabetic rats. The optimized NE was

17

orally administrated to the rats (n = 6) at a daily insulin dose of 80 IU/kg. Both normal and

18

model SD rats were used as the control. After 2 weeks post-treatment, the blood samples were

19

collected, and the bioactivity of serum enzyme including γ-GT, ALT, AST and ALP were

20

assayed. Moreover, the rats were sacrificed and major organs was excised surgically, and then

21

tissue sections were obtained as previously reported procedure.12 After treatment with

22

hematoxylin−eosin staining, histological images were collected using Vectra 3.0 Automated

23

Quantitative Pathology Imaging System (PerkinElmer).

24

2.15.

Statistical Analysis

25

Statistical analysis of all data was carried out by one-way analysis of variance in GraphPad

26

Prism software (version 7). All experiments were performed at least in triplicates unless

27

otherwise stated. All data were expressed as mean ± standard deviation (SD). *P < 0.05 was

28

statistically significant difference.

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1

3.

RESULTS AND DISCUSSION

2

Insulin-loaded NC was prepared by a rapid and homogenous mixing of positively-charged

3

HTCC (Inlets 1, 2) and insulin/TPP mixed solution (Inlets 3, 4) through a FNC apparatus

4

(Scheme 1, Step I). When the concentration of HTCC (1.5 mg/mL), insulin (2 mg/mL) and TPP

5

(0.1 mg/mL), and final pH of solution (pH = 7.4) were held constant, the flow rate of solution

6

was first optimized in the FNC process. Figure 1A represented that average diameter of NC

7

decreased from about 190 to 90 nm, and PDI of NC decreased from about 0.3 to 0.18 during

8

the flow rate increasing from 5 to 20 mL/min, while insignificant changes of diameter and PDI

9

of NC were observed after further increase of flow rate from 20 to 50 mL/min. Then, the

10

influence of final pH of solution on particle size, drug loading level and encapsulation

11

efficiency were also explored, as exhibited in Figure 1B, average size of NC decreased from

12

about 140 to 90 nm, and their surface charges were higher than 20 mV when adjusting final pH

13

of solution from 6.5 to 7.4. The tested NC also showed a high encapsulation efficiency (> 90%)

14

and loading capacity (> 50%) of insulin at various pH conditions (Figure 1C). Therefore, the

15

prepared parameters of 1.5 mg/mL HTCC, 2 mg/mL insulin, 0.1 mg/mL TPP, 40 mL/min of

16

flow rate, and final pH of solution of 7.4 were used to synthesize the optimized NC because of

17

its smaller particle size (87 nm), higher uniformity (PDI = 0.16), and high encapsulation

18

efficiency (95.3%) and loading capacity (52.9%). To confirm that FNC technique offered more

19

advantages over conventional bulk mixing or dropwise addition process, size distribution

20

curves of prepared NC were made a comparison. Figure 1D suggested that the nanocomplex

21

(NC) generated by a FNC process exhibited a smaller particle diameter and narrower size

22

distribution compared to that produced by bulk mixing or dropwise addition, which was also

23

verified by TEM observation (Figure 1E). It was worth noting that the optimized NC at four

24

different batches almost retained the same physicochemical properties including particle size,

25

PDI, ζ-potential, EE and LC (Table 1), suggesting that the optimized NC showed a high batch-

26

mode repeatability and well-controlled quality after scalable production by the FNC apparatus.

27

Those results were particularly crucial for effective clinical translation of protein

28

nanotherapeutics.

12 ACS Paragon Plus Environment

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Figure 1. Production and characterization of NC. (A) Influence of the flow rate on the diameter

3

and PDI of NC. (B, C) Effect of final pH of solution on (B) diameter and ζ-potential of NC, and

4

(C) encapsulation efficiency (EE) and loading capacity (LC) of insulin. The flow rate was kept

5

at 40 mL/min, and the concentration of HTCC, insulin and TPP was 1.5, 2, and 0.1 mg/mL,

6

respectively. (D) Size distribution curves (particle diameter, FNC: 87 ± 4 nm; bulk mixing: 200

7

± 9 nm; dropwise addition: 176 ± 9 nm) and (E) TEM observations of NC produced by FNC

8

process, bulk mixing or dropwise addition methods, respectively. Scale bars: 200 nm.

9 10

Table 1. Various physicochemical properties of the optimized NC generated by an FNP process

11

at four different batches. Prepared conditions: 1.5 mg/mL HTCC, 2 mg/mL insulin, 0.1 mg/mL

12

TPP, 40 mL/min of flow rate, and final pH of solution was 7.4. batch

diameter (nm)

PDI

ζ-potential

EE

LC

(mV)

(%)

(%)

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1

87 ± 4

0.16 ± 0.01

20.3 ± 0.5

95.3 ± 1.3

52.9 ± 1.2

2

90 ± 3

0.17 ± 0.01

22.5 ± 0.1

95.5 ± 0.4

53.1 ± 0.1

3

85 ± 2

0.19 ± 0.01

19.0 ± 0.8

94.9 ± 0.1

52.7 ± 0.4

4

92 ± 6

0.18 ± 0.02

22.1 ± 0.7

95.7 ± 0.3

53.2 ± 0.2

1 2

For protection of insulin from acidic degradation in stomach condition, enteric encapsulation

3

of the optimized NC within Eudragit material was then performed using a FNC process again

4

depending on their charge neutralization of NC and Eudragit material. As shown in Scheme 1,

5

NC-in-Eudragit composite systems (NE) were prepared by introducing NC solution (Inlet 1, 2)

6

and Eudragit solution (Inlet 3, 4) to the FNC apparatus (Step II). At a prepared condition of 0.5

7

mg/mL Eudragit and final pH of solution of 6.8, the diameter of NE decreased from about 185

8

to 105 nm, and the PDI of NE changed from 0.25 to 0.15, when the flow rate was ranged from

9

5 to 50 mL/min (Figure 2A). We also found that Eudragit concentration significantly affected

10

the formation of NE during the FNC process, as exhibited in Figure 2B, the diameter of NE

11

decreased from 125 to 106 nm, and surface charge of NE were below −20 mV during

12

adjustment of Eudragit concentration from 0.6 to 0.5 mg/mL. Nevertheless, NE would rapidly

13

aggregate when the concentration of Eudragit was below 0.4 mg/mL. Hence, we chose 0.5

14

mg/mL of Eudragit to prepare three size-different composite particles including NE-1 (106 nm),

15

NE-2 (310 nm) and NE-3 (1025 nm) by rationally controlling final pH of solution to 6.8, 6.5

16

and 6.0, respectively (Figure 2C). These NE were demonstrated to exhibit a negative surface

17

charge, high encapsulation efficiency and loading level of insulin (Table 2). Then, we used the

18

FERT analysis to confirm enteric encapsulation of NC into Eudragit, Figure 2D showed that

19

the fluorescence signal of R123-Eudragit decreased at the wavelength of 520 nm, but the signal

20

of RITC-insulin increased at the wavelength of 590 nm, implying that successful encapsulation

21

of NC within Eudragit materials. Besides, the produced NE-1, NE-2 and NE-3 composite

22

particles displayed homogenous spherical structure as observed by TEM images (Figure 2E).

23

These results as mentioned above suggested that FNC technique is conducive to generate

24

enteric composite particle with a homogenous coating layer, and modulate particle size and

25

surface properties in a reliable and scalable manner. 14 ACS Paragon Plus Environment

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Biomacromolecules

1 2

Figure 2. Enteric encapsulation of NC within Eudragit material. (A) Effect of flow rate of NC

3

solution and Eudragit solution (0.5 mg/mL) on the diameter and PDI of NE. (B) Influence of

4

Eudragit concentration on particle diameter and ζ-potential of NE, and Flow rate was held

5

constant at 40 mL/min. (C) Effect of various final pH of solution on particle size of NE. The

6

prepared conditions: 0.5 mg/mL Eudragit, 40 mL/min of flow rate. (D) Emission spectra of

7

fluorescence-labeled NE-1 with an excitation wavelength of 488 nm. (E) TEM images of NE-

8

1, NE-2 and NE-3. Scale bars: 500 nm.

9 10

Table 2. Particle diameter, PDI, ζ-potential, drug encapsulation efficiency (EE %) and loading

11

capacity (LC %) of tested composite particles including NE-1, NE-2 and NE-3. particle

diameter

PDI

(nm) NE-1

106 ± 5

0.15 ± 0.02

ζ-potential

EE

LC

(mV)

(%)

(%)

- 24.6 ± 2.7

81.9 ± 1.1

35.6 ± 0.5

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Page 16 of 30

NE-2

310 ± 10

0.16 ± 0.06

- 13.0 ± 1.2

76.1 ± 0.2

33.1 ± 0.1

NE-3

1025 ± 30

0.30 ± 0.06

- 5.5 ± 0.3

70.2 ± 0.3

30.5 ± 0.2

1 2

Next, in vitro drug release behaviors of tested formulations were explored in different pH

3

buffer that simulated pH conditions along GI tract including fasting stomach (pH 1.5−4) and

4

small intestine (pH 6−7.4).12 As shown in Figure 3A, the burst release behavior with 45%

5

accumulative release of insulin was observed for NC within initial 2 h after dialysis against pH

6

2.5 buffer, which was attributed to this possibility that the strong charge repulsion of HTCC as

7

well as loss negative charge of insulin at acidic condition of pH 2.5, resulting in the volume

8

swelling and dissociation of the NC. Contrastively, accumulative release of insulin from NE-1,

9

NE-2 and NE-3 were below 15%, indicating that enteric encapsulation of NC within Eudragit

10

material could significantly retard the release of insulin from NE composite particles because

11

of the formation of crosslinked Eudragit network.28, 39-41 therefore, the enteric encapsulation of

12

NC using Eudragit possibly could reduce acidic degradation of insulin in stomach environment.

13

Subsequently, the release behavior was further explored after consecutive incubation with pH

14

6.8 and 7.4 buffer. Notably, slower insulin release was observed for NE-1, NE-2 and NE-3

15

compared with free insulin and NC, and the NE-3 showed slightly lower release rate of insulin

16

than both NE-1 and NE-2, these demonstrated that Eudragit encapsulation of NC could

17

effectively improve structural stability of composite particles (NE) due to the formation of

18

complexes via charge-charge interaction between positively-charged NC and negatively-

19

charged Eudragit material.

20

Then, Caco-2 cell monolayer was chosen as a model to mimic intestinal epithelium for the

21

study of trans-epithelial transport. As exhibited in Figure 3B, NC, NE-1, NE-2 and NE-3 had

22

higher Papp values than free insulin, and the NC showed the highest permeability of insulin

23

across cell monolayer after incubation without mucin, which was explained that the particles

24

with positive charge surface easily interacted with cell membrane, enhancing the trans-

25

epithelial transport of particles.5, 34, 42-44 However, NE-1 as similar to NC exhibited a higher

26

Papp value compared with NE-2 and NE-3 after cell monolayer treated without or with mucin

27

(Figure 3B), suggesting that enteric encapsulation of NC did not significantly affect the 16 ACS Paragon Plus Environment

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Biomacromolecules

1

permeability of insulin across both mucus layer and intestinal epithelium. Then trans-epithelial

2

electrical resistance (TEER) was monitored after post-treatment as described above, as shown

3

in Figure 3C and D, no obvious change of TEER value was observed for cell monolayer after

4

treated with free insulin as a control, while the tested NC, NE-1, NE-2 and NE-3 resulted in

5

apparent reduction of TEER values after 2 h post-treatment when there was no mucin covering

6

cell monolayer, and both NC and NE-1 showed a better effect in reduction of TEER compared

7

with others. Similarly, the same phenomenon was observed after incubated with cell monolayer

8

in the presence of mucin. Besides, we also observed that TEER values slowly recovered after

9

removal of tested formulations at 2 h of post-treatment. These results suggested that the enteric

10

encapsulation of NC did not significantly influence its ability to open the tight junction, and the

11

composite particles with a smaller size (NE-1) could lead to a lower TEER value during

12

penetrated across Caco-2 cell monolayer. In addition, we chose NE-1 to visualize the opening

13

of tight junction between Caco-2 cells in monolayer model, and immunofluorescence assay of

14

tight junction was performed as reported previously.12 As shown in Figure 3E, the intact and

15

continuous rings of occludin was detected before cell monolayer treated with NE-1, but the

16

fluorescent signal of occludin became very weak and discrete at 2 h of post-treatment, implying

17

that effective opening of tight junction mediated by NE-1 particle. After removal of tested

18

sample for 10 h, fluorescent intensity was gradually recovered, which was consistent with the

19

result of TEER monitoring (Figure 3C and D). Those findings demonstrated that the opening

20

of tight junctions inside cell monolayer was transient and reversible after treated with tested

21

insulin formulations.

17 ACS Paragon Plus Environment

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Page 18 of 30

1 2

Figure 3. (A) In vitro drug release profiles of free insulin, NC, NE-1, NE-2 or NE-3 after

3

consecutive incubation with pH 2.5, 6.8, and 7.4 buffer. (B) Apparent permeability coefficients

4

(Papp) of RITC-labeled insulin penetrated across cell monolayer after treated with tested

5

samples. *P < 0.05 vs insulin under cell monolayer without mucin, #P < 0.05 vs insulin under

6

cell monolayer with mucin. (C, D) Relative change of TEER values as a function of time after

7

cell monolayer (C) without or (D) with mucin treated with free insulin, NC, NE-1, NE-2 or NE-

8

3, respectively. (E) Tight junctions observed by occludin immunofluorescent staining at

9

different time points after treated with NE-1 in cell monolayer with mucin. Scale bars: 10 μm.

10 11

In vivo hypoglycemic efficacy and pharmacokinetics of tested formulations were evaluated

12

by using type 1 diabetic rats. Figure 4A displayed that s.c. injection with a 5 IU/kg dose of

13

insulin as a positive control could induce a sharp reduction of blood glucose to approximate 20%

14

of basal level within 2 h, and then blood glucose level was gradually recovered after 2 h post18 ACS Paragon Plus Environment

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Biomacromolecules

1

injection. However, oral delivered insulin (80 IU/kg) as similar to the saline failed to generate

2

an obvious hypoglycemic effect. Contrastively, NC, NE-1, NE-2 and NE-3 exhibited the

3

gradual reduction of blood glucose level after oral gavage at the same insulin dose of 80 IU/kg,

4

and NE-1 with reduction of glucose level to about 18.6% of basal level within 7 h was superior

5

to NC, NE-2 and NE-3 groups with 60.7%, 21.1% and 28.0%, respectively. In vivo

6

pharmacokinetics revealed that orally administrated NE-1 showed a sustained rising of insulin

7

level within 4 h compared to a rapid change and clearance of serum insulin after s.c. injection

8

(Figure 4B), and the detailed pharmacokinetic parameters of tested insulin formulations were

9

exhibited in Table 3. The pharmacological availability and relative oral bioavailability of

10

optimized NE-1 was calculated to be 4.49% and 13.3%, respectively. Those results

11

demonstrated that our optimized NE-1 formulation could effectively improve oral delivery

12

efficiency and bioavailability of insulin after oral dosing.

13 14

Figure 4. (A) Glucose changes (% base level) of diabetic rats after oral gavage of saline (as a

15

control), free insulin, NC, NE-1, NE-2, and NE-3 at an insulin dose of 80 IU/kg, as well as s.c.

16

injection of insulin at a dose of 5 IU/kg. (B) Changes of serum insulin level after oral

17

administrated with free insulin (80 IU/kg) and NE-1 (80 IU/kg), as well as s.c. injection of

18

insulin (5 IU/kg).

19 20

Table 3. Various pharmacokinetic parameters of tested formulations after administration in

21

type 1 diabetic rats. formulation

dose (IU/kg)

AUC (mIU ∙ h/L)

PA (%)

BA (%)

Insulin (s.c.)

5

153.49 ± 54.26



100

Insulin (p.o.)

80

8.93 ± 7.81

0.85 ± 0.65

0.36 ± 0.32

19 ACS Paragon Plus Environment

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NE-1 (p.o.)

80

326.53 ± 95.22

Page 20 of 30

4.49 ± 0.70

13.3 ± 3.88

1 2

To better understand the mechanism for the excellent hypoglycemic efficacy of optimized

3

composite particle (NE-1), we observed in vivo biodistribution and in situ oral absorption of

4

NE-1 prepared with Cy7-labeled insulin, and the NC was chosen as a control. As shown in

5

Figure 5A, NE-1 displayed a stronger fluorescence signal in whole-body imaging of rats,

6

indicating that NE-1 could more slowly release the loaded insulin from composite particles

7

compared to the NC, which was consistent with the data as described in Figure 3A. Then, major

8

organs including stomach, small intestine, liver and kidney were collected for ex vivo

9

fluorescence imaging after 6 h post-administration (Figure 5A), compared with the NC, the

10

NE-1 with stronger fluorescence intensity was observed in small intestine, especially in ileum

11

segment, suggesting that the enteric encapsulation system could protect the loaded insulin from

12

denaturation and acidic degradation in stomach environment, then achieve intestinal site-

13

specific drug release after oral dosing. Moreover, NE-1 also exhibited slightly stronger

14

fluorescent intensity in the organs of liver and kidney compared with NC group, which

15

indirectly reflected that the NE-1 had a higher oral delivery efficiency during penetrated across

16

intestinal epithelium. Furthermore, in situ visualization of absorption of NE-1 and NC in ileum

17

segment were explored by CLSM observation. Figure 5B showed that compared with weak

18

signal detected in intestinal microvilli for NC, NE-1 exhibited a stronger fluorescence signal

19

within microvilli at 6 h after oral dosing, which was similar to the result analysis of in vivo

20

biodistribution. Therefore, we concluded that the optimized NE-1 could achieve better control

21

of blood glucose in diabetic rats mainly due to its unique advantages in protection of the protein

22

cargo from acidic degradation in stomach, and control intestinal site-specific drug release, as

23

well as improvement of trans-epithelial transport together.

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Biomacromolecules

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Figure 5. (A) Whole-body imaging of the rats after oral administrated NC and NE-1 (insulin,

3

80 IU/kg) at predetermined time points and ex vivo imaging of major organs including 1,

4

stomach; 2, duodenum; 3, jejunum; 4, ileum; 5, liver; 6, kidney after 6 h post-administration,

5

insulin was labeled with Cy-7. (B) Observation of oral absorption of NC and NE-1 (RITC-

6

labeled insulin) in ileum segment using a CLSM at 6 h of post-administration. Scale bars: 50

7

μm.

8 9

Finally, biosafety of the optimized NE-1 formulation was examined by in vitro and in vivo

10

studies. we first evaluated the viability of Caco-2 and E12 cells after treated with tested

11

formulations at a different concentration of insulin using a standard MTT assay, and the culture

12

media was as a control. As showed in Figure 6A and B, positively-charged NC exhibited slight

13

cytotoxicity against Caco-2 or E12 cells at higher insulin concentration of 250 μg/mL, while

14

the NE-1 had no inhibition of cell proliferation after 24 h incubation, indicating that NE-1 was 21 ACS Paragon Plus Environment

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Page 22 of 30

1

biocompatible. It is known that the width of tight junction in small intestinal epithelium has

2

been verified to be less than 1 nm,3, 21 and only hydrophilic small molecule drugs could transport

3

across epithelial barriers by paracellular pathway at normal GI conditions.3,

4

proteins (e.g., insulin) have been reported to effectively penetrate across intestinal epithelium

5

through opening of tight junction in the presence of permeation enhancers, however, which

6

would possibly alter the physiological functions of GI tract and then result in the potential risk

7

of bacterial toxins entering into systemic circulation.5, 21 Herein, chitosan or its derivatives used

8

as the absorption enhancers have been widely utilized to mediate the oral delivery of biologics

9

by reversible opening of tight junction, which have been confirmed to show a good biosafety

10

in vivo as described in many literatures.5, 21, 42, 45, 46 Besides, Eudragit was approved by FDA to

11

act as enteric coating materials in pharmaceutics industry for a long time.47, 48 Although good

12

biocompatibility of these carrier materials used in this study, in vivo toxicity of NE-1

13

formulation after oral dosing was still studied by blood analysis and histological staining,

14

respectively. Figure 6C showed that no significance changes of tested enzyme activity

15

including γ-GT, ALT, AST and ALP were detected after oral administrated NE-1 at a dose of

16

80 IU/kg insulin for total 2 weeks, indicating that NE-1 had no apparent hepatotoxicity after

17

oral administration. Histological analysis also confirmed that NE-1 formulation induced no

18

damage for major organs as observed in Figure 6D. These results collectively confirmed that

19

the optimized NE-1 formulation had no toxicity in vivo after long-term oral dosing, suggesting

20

its great potential as a vehicle for oral delivery of insulin.

22 ACS Paragon Plus Environment

21

Therapeutic

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Biomacromolecules

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Figure 6. (A, B) In vitro cytotoxicity against (A) Caco-2 cells and (B) E12 cells after treated

3

with insulin, NC, NE-1 for 24 h. (C) Serum enzyme activity of γ-GT, ALT, AST, and ALP in

4

rats and (D) histological analysis of major organs after oral gavage of NE-1 formulation at a

5

daily dose of 80 IU/kg to diabetic rats lasted for 2 weeks, and normal rats were used as a control.

6 7 8

4. CONCLUSION

9

In this study, we have tailored an enteric encapsulation system with the aim to effectively

10

protect the loaded protein drugs from acidic degradation in stomach environment, and achieve

11

intestinal site-specific drug release as well as improve oral absorption and bioavailability of

12

protein therapeutics. We chose insulin as a model protein, and then insulin-loaded HTCC/TPP

13

nanocomplex (NC) as a nanocore was encapsulated into an enteric polymer material, Eudragit

14

L100-55, to produce NC-in-Eudragit composite particles (NE) in a reliable and scalable manner,

15

through a two-step FNC process. In vitro and in vivo studies indicated that the optimized

16

composite particle, NE-1, showed ileum site-specific drug release, improved tans-epithelial 23 ACS Paragon Plus Environment

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1

transport as well as oral delivery efficiency of insulin. In addition, NE-1 formulation exhibited

2

the most significant hypoglycemic effect with a relative oral bioavailability of 13.3%. These

3

finding demonstrated that our enteric encapsulation system as a promising formulation for

4

mediating oral delivery of protein drugs.

5 6 7

ASSOCIATED CONTENT

8

Supporting Information

9

The supporting information is available free of charge on the ACS Publications website at xxx.

10

Synthetic method of HTCC, 1H-NMR characterization of HTCC (PDF)

11 12

AUTHOR INFORMATION

13

Corresponding Authors

14

*E-mail: [email protected].

15

*E-mail: [email protected].

16

Notes

17

The authors declare no competing financial interest.

18 19

ACKNOWLEDGMENTS

20

Finical supports were provided by Natural Science Foundation of China (No. 51820105004,

21

No. 51803243), Guangdong Innovative and Entrepreneurial Research Team Program (No.

22

2013S086), Natural Science Foundation of Guangdong Province (No. 2014A030312018) and

23

Fundamental Research Funds for the Central Universities (No. 17lgpy06).

24 25

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Mitragotri, S.; Burke, P. A.; Langer, R. Overcoming the Challenges in Administering

Zelikin, A. N.; Ehrhardt, C.; Healy, A. M. Materials and Methods for Delivery of 24 ACS Paragon Plus Environment

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Using Drug-Device Combinations. Curr. Opin. Pharmacol. 2017, 36, 8-13.

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Chitosan/Poly(Gamma-Glutamic acid) Nanoparticles for Oral Insulin Delivery. Biomaterials

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10. Shrestha, N.; Araujo, F.; Shahbazi, M.-A.; Makila, E.; Gomes, M. J.; Herranz-Blanco, B.;

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Lindgren, R.; Granroth, S.; Kukk, E.; Salonen, J.; Hirvonen, J.; Sarmento, B.; Santos, H. A.

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Thiolation and Cell-Penetrating Peptide Surface Functionalization of Porous Silicon

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Nanoparticles for Oral Delivery of Insulin. Adv. Funct. Mater. 2016, 26 (20), 3405-3416.

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11. Lopes, M.; Shrestha, N.; Correia, A.; Shahbazi, M.-A.; Sarmento, B.; Hirvonen, J.; Veiga,

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F.; Seica, R.; Ribeiro, A.; Santos, H. A. Dual Chitosan/Albumin-Coated Alginate/Dextran

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Sulfate Nanoparticles for Enhanced Oral Delivery of Insulin. J. Control. Release 2016, 232,

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