Biomimetic Viruslike and Charge Reversible ... - ACS Publications

Mar 5, 2018 - R8 peptide and induced efficient cell-penetrating peptide (CPP)-mediated ... insulin-loaded P-R8-Pho NPs enabled to induce a preferable ...
0 downloads 0 Views 2MB Size
Subscriber access provided by UNIVERSITY OF ADELAIDE LIBRARIES

Biological and Medical Applications of Materials and Interfaces

Biomimetic virus-like and charge reversible nanoparticles to sequentially overcome mucus and epithelial barriers for oral insulin delivery Jiawei Wu, Yaxian Zheng, Min Liu, Wei Shan, Zhirong Zhang, and Yuan Huang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b16524 • Publication Date (Web): 05 Mar 2018 Downloaded from http://pubs.acs.org on March 6, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Materials & Interfaces

Biomimetic virus-like and charge reversible nanoparticles to sequentially overcome mucus and epithelial barriers for oral insulin delivery

Jiawei Wu, Yaxian Zheng, Min Liu, Wei Shan, Zhirong Zhang and Yuan Huang*

Key Laboratory of Drug Targeting and Drug Delivery System (Ministry of Education), West China School of Pharmacy, Sichuan University. No. 17, Block 3, South Renmin Road, Chengdu 610041, P.R. China

*Corresponding author: Prof. Yuan Huang, Tel/Fax: +86-028-85501617. E-mail: [email protected]

1

ACS Paragon Plus Environment

ACS Applied Materials & Interfaces 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract Nanoparticles (NPs) for oral delivery of peptide/protein drugs are largely limited due to the co-existence of intestinal mucus and epithelial barriers. Sequentially overcoming these two barriers is intractable to for a single nanovehicle due to the requirements of different or even contradictory surface properties of NPs. To solve this dilemma, a mucus penetrating virus-inspired biomimetic NPs with charge reversal ability (P-R8-Pho NPs) was developed by densely coating PLGA NPs with cationic octa-arginine (R8) peptide and specific anionic phosphoserine (Pho). The small size (81.81 nm) and virus-like neutral charged surface (-2.39 mV) of the biomimetic NPs achieved rapid mucus penetrating, which was almost equal to that of the conventional PEGylated mucus penetrating nanoparticles. The hydrolysis of surface-anchored anionic Pho was achieved by intestinal alkaline phosphatase (IAP), which led to the turnover of zeta potential to positive (+7.37 mV). This timely charge reversal behavior also exposed cationic R8 peptide and induced efficient CPPs-mediated cellular uptake and transepithelial transport on Caco-2/E12 co-cultured cell model. What’s more, P-R8-Pho NPs showed excellent stability in simulated GI conditions and enhanced absorption in intestine in vivo. Finally oral administration of insulin-loaded P-R8-Pho NPs was able to induce a preferable hypoglycemic effect and a 1.9-fold higher oral bioavailability was achieved compared with single CPPs-modified P-R8 NPs on diabetic rats. The combinative application of biomimetic mucus penetrating strategy and enzyme-responsive charge reversal strategy in a single nanovehicle could sequentially overcome mucus and epithelial barriers, thus show great potential for the oral peptide/protein delivery.

Key words: oral nanoparticles, mucus and epithelial barriers, biomimetic virus-like, charge reversal, hypoglycemic effect

2

ACS Paragon Plus Environment

Page 2 of 36

Page 3 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Materials & Interfaces

1. Introduction With the advent of biotechnology, the therapeutic peptide/protein drugs have received a great deal of attention for treating numerous diseases due to their high potency and specificity 1.The administration of peptide/protein drugs via oral route is preferred to other routes due to the convenience and good patient compliance, especially for the treatment of chronic conditions. However, the oral bioavailability of peptide/protein drugs is extremely limited by their inherent poor stability in the gastrointestinal (GI) tract and low permeability across the intestinal epithelium 2-3

.Nanoparticles (NPs) based drug delivery systems including liposomes, micelles

and polymeric nanoparticles have been developed as potential vehicles for oral delivery of peptide/protein drugs 4. Different strategies have been employed, such as ligand modification, to augment the transepithelial transport and absorption of nanoparticles, thus to improve the oral bioavailability of the encapsulated drugs 3, 5. However, oral delivery of NPs may be greatly influenced by mucus layer that continuously secreted and covered above the epithelium. Intestinal mucus is a robust barrier which can immobilize and remove pathogens, bacteria and exotic particles, especially for those with cationic and hydrophobic properties, before they reach epithelial surfaces

6-7

. In order to overcome the mucus barrier, the mucus penetrating

particles (MPPs) with small particle size as well as the hydrophilic and neutral charged properties (e.g. PEGylation), called “mucus-inert” surface, were developed with the ability to diffuse unhindered through mucus

8-9

.Nevertheless, the

“mucus-inert” surface of NPs would reduce the affinity with lipophilic and negatively charged cell membrane thus lead to inefficient cellular uptake

10-11

. Therefore,

efficient oral peptide/protein drug delivery NPs should have the ability to sequentially conquer the mucus and epithelial barriers 12-14. It is noteworthy that NPs designed to overcome the two barriers require different or even contradictory surface properties

15

, that is hydrophilic and neutral charged

surface for the mucus barrier while hydrophobic and cationic surface for the epithelial 3

ACS Paragon Plus Environment

ACS Applied Materials & Interfaces 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 36

barrier, making it intractable in a single nanovehicle. Cell-penetrating peptides (CPPs) such as penetratin and oligoarginine are developed to be useful tools for intracellular delivery of various cargos through the membranes of numerous cells

16

. But the

cationic property of these CPPs will have strong interaction with anionic mucin in the mucus, leading to the immobilization of CPPs-modified NPs (CPPs-NPs) and reduce cellular uptake efficiency. Many viruses are capable of diffusing in mucus as fast as in water due to their unique surface properties which are densely coated with both cationic and anionic groups to possess a densely charged yet neutral surface

6, 17

.

Besides the “muco-inert” property, the surface of above mentioned viruses usually contains evolved specialized proteins which enable their binding and invasion to the host cells by either fusing the viral envelope with a host cell membrane or forming membrane pores

18

. In a similar way, the CPPs (e.g. arginine-rich R8 peptide) was

also able to translocate into cells through membrane pore-opening mechanism as reported

19

. Inspired by this, anionic groups could be introduced into the cationic

CPPs-NPs to imitate the surface properties of viruses (biomimetic NPs) for rapidly diffusing in mucus and then directly translocating into epithelium. However, it arised another dilemma that the virus-like neutral charged surface was conducive to mucus penetrating but unfavourable for the cellular uptake because the cationic property of CPPs was shielded

20

. So if the designed virus-like NPs owned

the ability to appropriately change its surface properties (e.g. surface charge) after penetrating through mucus, it would be promising to conquer the following epithelial barrier 12-13. Intestinal alkaline phosphatase (IAP) is a brush border enzyme expressed in the intestinal epithelium and functioned as catalyst for the hydrolysis of monophosphate esters (as IAP substrates) resulting in the dissociation of the anionic phosphoric acid

21-22

. Based on this, if the IAP substrate was chosen as the specific

anionic group to form the virus-like NPs, it could timely expose the modified-CPPs and induce an increase of surface charge upon reaching the IAP expressing epithelium after penetrating mucus layer. This charge reversal property would be favorable for the cellular internalization 23. By the combination of these two strategies, we herein reported a biomimetic 4

ACS Paragon Plus Environment

Page 5 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Materials & Interfaces

virus-like NPs meanwhile with the charge reversal ability to sequentially overcome mucus and epithelial barriers for oral insulin delivery (Figure 1. Schematic diagram). DSPE-PEG2000 was applied as the bridge to anchor both the cationic octa-arginine (R8) peptide, one kind of the CPPs, and anionic phosphoserine (Pho), one of the substrates of IAP, on the surface of PLGA NPs, thus obtained the virus-like densely charged yet neutral surface NPs (Figure 2A, structure of P-R8-Pho NPs). We hypothesized that at first, this virus-like hydrophilic and neutral charged surface of NPs could facilitate the mucus permeation, subsequently the enzyme-responsive hydrolysis of Pho led to the exposure of the cationic R8 peptide (charge reversal). Then the exposed CPPs could mediate the cellular uptake and transepithelial transport of NPs across the epithelium. The stability and mucus penetrating ability of NPs were investigated as well as the cellular uptake and transepithelial transport efficiencies in both mucus-secret and non-mucus-secret cell models. The intestinal absorption in vivo was investigated as well. Finally the in vivo hypoglycemic effect and pharmacokinetic study of insulin-loaded NPs via oral administration were performed in diabetic rats.

Figure 1. Schematic diagram of P-R8-Pho NPs to sequentially overcome both two barriers. 5

ACS Paragon Plus Environment

ACS Applied Materials & Interfaces 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 36

2. Materials and methods 2.1. Materials DSPE-PEG2000-Mal, DSPE-PEG2000-NHS and DSPE-PEG2000-COOH were purchased from Ponsure Biotechnology (Shanghai, China). Poly (lactic-co-glycolic acid) (PLGA) with one methoxy end group was purchased from Lactel absorbable polymers. R8 peptide with one cysteine end (Cys-R8) was chemically synthesized by Ontores Biotechnology Co., Ltd (Hangzhou, China). Porcine insulin (26.5 IU/mg) was

purchased

from

Wanbang

Bio-Chemical

(Jiangsu,

China).

3,3'-Dioctadecyloxacarbocyanine Perchlorate (Dio) was purchased from Invitrogen; porcine mucin and O-Phospho-L-Serine (phosphoserine), from Sigma-Aldrich; alkaline phosphatase from calf intestine (5000 U), from Baiaolaibo Technology Co., Ltd (Beijing, China); phosphatase inhibitor cocktail Ⅱ , from MedChemExpress (MCE, USA). All other solvents and reagents were of analytical grade.

2.2. Synthesis of DSPE-PEG2000-R8 and DSPE-PEG2000-Pho DSPE-PEG2000-R8

was

synthesized

by

conjugating

Cys-R8

to

DSPE-PEG2000-Mal via the specific addition reaction between thiol (SH) group and maleimide (Mal) group. Cys-R8 peptide was reacted with DSPE-PEG2000-Mal (molar ratio of SH group: Mal group = 1:1) in dimethyl sulfoxide (DMSO) for 24 h at room temperature under moderately stirring, then stored for nanoparticles (NPs) preparation. DSPE-PEG2000-Pho was synthesized through covalently attached amino groups of O-phospho-L-serine (phosphorserine) to functional hydroxysuccinimide (NHS) in DSPE-PEG2000-NHS.The reaction was performed within phosphate buffered saline (PBS, pH 7.0) under moderately stirring for 48 h at room temperature. Resulting product was purified by dialysis against distilled water then lyophilized and stored at -20 °C. 6

ACS Paragon Plus Environment

Page 7 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Materials & Interfaces

2.3. Preparation and characterization of nanoparticles (NPs) Nanoparticles (NPs) were manufactured using the self-assembly nanoprecipitation method

24

. In brief, to prepare PLGA-PEG NPs (P NPs), 2mg PLGA, 0.8 mg

DSPE-PEG2000 and 0.4 mg soybean phospholipid were dissolved in 0.2 ml dimethyl sulphoxide (DMSO), then the resulting organic mixture was added dropwisely into 4.0 ml deionized water under magnetic stirring (900 rpm) at room temperature. To prepare PLGA-PEG-R8 NPs (P-R8 NPs), 0.8 mg DSPE-PEG2000 was replaced by 0.2 mg DSPE-PEG2000-R8 and 0.6 mg DSPE-PEG2000 under the same condition. As for PLGA-PEG-R8-Pho NPs (P-R8-Pho NPs), 0.2 mg DSPE-PEG2000-R8 and 0.2, 0.3, 0.4, 0.5 or 0.6 mg DSPE-PEG2000-Pho (the ratio increased from 1:1 to 1:3) were used to screen for the desired nearly neutral charged NPs. The prepared NPs were dialyzed in deionized water to remove the residual DMSO. For the preparation of fluorescence-labled NPs, the hydrophobic fluorescent dye Dio (0.15% w/w) was blended with PLGA prior to addition to deionized water. The particle size and zeta potential of NPs were measured using Malvern Zeta-size NanoZS90. Morphology of NPs were imaged by transmission electron microscopy (TEM, Tecnai G2 F20, FEI, USA).

2.4. Stability study To test the stability of NPs in vitro, freshly preparaed NPs were dispersed in simulated gastric fluids with pepsin (SGF, pH2.0), simulated intestinal fluids with trypsin (SIF, pH6.8) or PBS (pH 7.4), respectively. These suspensions were incubated at 37 °C in a shaker and at predetermined time points the particle size was measured as described above.

7

ACS Paragon Plus Environment

ACS Applied Materials & Interfaces 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

2.5. Mucus permeation studies 2.5.1. Mucin interaction study In order to measure particle-mucin aggregates, fluorescent Dio-labled NPs were dispersed in mucin solution at different concentrations (0.5%, 1.0%, 2.0%, m/v), then vortexed at 100 rpm and further incubated at 37 °C for 30 min under gently shaking. The samples were then centrifuged at 1500 rpm for 3 min to separate mucin-NPs aggregates and unbound NPs remained in supernate. Subsequently the supernate was collected and treated with DMSO to destroy the remaining NPs, so did the initial NPs (NPs without incubation with mucin). Finally the fluorescence intensity in supernate and initial NPs were both measured using Varioskan Flash Multimode Reader. NPs aggregated with mucin was calculated by the following equation: Aggregated (%) =

fluorescence in initial NPs − fluorescence in supernatant fluorescence in initial NPs

2.5.2. Mucus permeation study by Ussing Chamber Ussing Chamber system was applied to investigate the ability of NPs to penetrate across mucus. Briefly, 20 µl of fresh porcine intestinal mucus was added uniformly between a pair of polycarbonate membrane filters (2.0 µm), then fixed to the sliders which were installed in Ussing Chambers. All the chambers were filled with 2.0 ml PBS, oxygenated with 95% O2 and 5% CO2, and stirred with gas flow. After equilibration for 30 min, the donor chambers were filled with 2 mL of PBS buffer containing Dio-labled NPs and the acceptor chambers were filled with the same volume of blank PBS. The samples were continuously aerated with gas and maintained at 37 °C. At predetermined time intervals, an aliquot of samples (200 µl) was withdrawn from acceptor chamber and equal volume of blank PBS were supplemented. Finally DMSO was added to destroy the Dio-NPs and fluorescence intensity was measured. The apparent permeability coefficient (Papp) was calculated using the following equation: 8

ACS Paragon Plus Environment

Page 8 of 36

Page 9 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Materials & Interfaces

Papp =

dQ 1 × dt A × C0

dQ/dt is the flux of Dio-labled NPs from donor chamber to acceptor chamber, C0 is the initial fluorescence intensity of NPs and A is the membrane area (cm2).

2.6. Enzyme-responsive zeta potential switch In order to prove the ability of phosphoserine modified NPs (P-R8-Pho NPs) to change its surface charge under the influence of intestinal alkaline phosphatase (IAP), the zeta potential of NPs was determined before and after incubation with free IAP in vitro. First zeta potential of freshly prepared NPs was measured prior to the addition of isolated IAP. Then NPs were mixed with the IAP solution (10 U/ml) and incubated for 1 h at 37 °C in a shaker. Afterwards, the zeta potential measurements were performed again.

2.7. Cell studies 2.7.1. Cell culture In current studies, the human colonic adenocarcinoma cells (Caco-2) and mucus producing HT29-MTX-E12 cells (E12) were used. Cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco, USA) with high glucose, 10% (v/v) fetal bovine serum (FBS; Gibco, USA), 1% (v/v) non-essential amino acid, 1% (v/v) L -glutamine, 1% penicillin and streptomycin (100 IU/mL) (Hyclone, UT, USA). Cells were incubated at 37 °C and 95% relative humidity with 5% CO2. Caco-2 cells, E12 cells or Caco-2/E12 co-culture cells (ratio as 7:3) were seeded into 96-well plates at 1× 104 cells per well for the detection of IAP expression, cellular uptake and cytotoxicity studies. For the transepithelial transport study, Caco-2/E12 co-culture cells (7:3) were seeded onto the Transwell inserts consisting of the polycarbonate membrane at 4 × 104 cells per well. Cell monolayers were fed with fresh medium on both sides of the membrance every two days. Transepithelial electrical resistance 9

ACS Paragon Plus Environment

ACS Applied Materials & Interfaces 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(TEER) was measured with an electrical resistance meter (Millicell ERS-2, Millipore) to monitor the integrity of the cell monolayers.

2.7.2. Detection of IAP expression in Caco-2 and E12 cells The chosen cell models possessing the ability of expressing the intestinal alkaline phosphatase (IAP) was the prerequisite for our study. So the IAP activity in both Caco-2 and E12 cells was evaluated quantitatively using the commercial Alkaline Phosphatase Assay Kit (Beyotime, China). The results were normalized to the total intracellular protein content determined by the bicinchoninic acid (BCA) Protein Assay Kit (Beyotime, China) and the IAP activity was expressed as nanomoles of produced p-nitrophenol per min per mg of protein (nmol/min/mg protein).

2.7.3. Cytotoxicity study The cytotoxicity of NPs on Caco-2 and E12 cells was evaluated using MTT assay. Cells seeded on 96-well plates were incubated with NPs (in PBS) at different PLGA concentrations for 3 h. After that the NPs were removed and replaced with fresh medium for another 24 h, then submitted to the MTT assay. Cells incubated with blank PBS were used as negative control, and cell viability was calculated as the percentage of viable cells by comparing with negative control.

2.7.4. Cellular uptake study To evaluate cellular uptake of NPs, Caco-2 or E12 cells were incubated with fluorescent Dio-labled NPs for 3h. After that cells were lysed by cell lysis buffer, the cell-associated fluorescence intensity was measured as mentioned above, and total cellular protein was determined by BCA assay kit. The amounts of uptake were expressed as the quantity of fluorescence intensity of NPs associated with 1 mg of cellular protein. For mucus-producing E12 cells, to measure the amount of NPs that penetrated the cell mucus layer and entered into cells, mucus and attached NPs were 10

ACS Paragon Plus Environment

Page 10 of 36

Page 11 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Materials & Interfaces

removed by washing cell monolayers thrice with 4% (v/v) formalin solution in PBS before addition of cell lysis buffer 25. To prove the greatest cellular internalization of P-R8-Pho NPs was due to its enzymatic-responsive charge reversal ability induced by intestinal alkaline phosphatase (IAP), an additional NPs, the PLGA-PEG-R8-COOH NPs (P-R8-COOH NPs), was introduced for the cellular uptake investigation on Caco-2 cells. After treatment of different NPs for 3 h, cellular uptake was also measured as the method mentioned above.

2.7.5. Influence of phosphatase inhibitor on cellular uptake To further demonstrate the superior cellular uptake of P-R8-Pho NPs was related to the intestinal alkaline phosphatase (IAP) expressed on the cell membrance, the cellular uptake study was performed with the phosphatase inhibitor, namely phosphatase inhibitor cocktail Ⅱ(MCE, USA). Caco-2 cells were pre-incubated with the inhibitor for 1 h, then Dio-NPs were added for another 3 h in the presence of inhibitor. Quantification of cellular uptake of NPs was performed as mentioned above. Besides, the amount of cellular uptake without addition of inhibitor was applied as a control. The relative uptake was expressed as the percentage of cellular uptake in presence of inhibitor compared with the control group.

2.7.6. Cellular uptake study on Caco-2/E12 co-culture cells To better mimic the intestinal epithelial cells for assessing the cellular uptake and the mucous influence on cellular internalization of NPs, the Caco-2/E12 (7:3) co-culture cell model was applied. Caco-2 and E12 cells were seeded into 96-well plates at the ratio of 7:3 for a total number of 1× 104 cells per well. To differentiate the NPs trapped in mucus from those taken up by cells, the mucus and trapped NPs were removed by washing the cell monolayers thrice with 4% (v/v) formalin solution in PBS after the NPs treatment. Subsequently cells were washed two times with ice-cold PBS, which was used to determine the NPs only internalized 11

ACS Paragon Plus Environment

ACS Applied Materials & Interfaces 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

in cells. The cells only washed by PBS and without treatment of formalin were used to determine NPs trapped in mucus and internalized in cells. The cell-associated fluorescence intensity and protein concentrations were measured as described above. Results were also expressed as the quantity of fluorescence intensity associated with 1 mg of cellular protein.

2.7.7 Transepithelial transport study Transepithelial transport study of NPs was investigated on the Caco-2/E12 (7:3) co-culture monolayers seeded on Transwell inserts fitted with polycarbonate membranes. Prior to the study, medium in the apical and basolateral chambers were replaced with pre-warmed Hank’s balanced salt solution (HBSS). After equilibration at 37 °C for 30 min the apical solution was replaced with 200 µl Dio-labled NPs in HBSS. After incubation for 8 h, 100µl of samples were withdrawn from the basolateral chambers. The amount of transported Dio-NPs was measured and TEER value of each insert was detected during experiment to explore the integrity of cell monolayer.

2.8. In situ absorption study The absorption of Dio-labled NPs was qualitatively analyzed using an in situ absorption study in mice. Sprague-Dawley (SD) rats (200-220 g, seven weeks old and raised in clean laboratory animal room with constant temperature at 25 °C) were fasted overnight but free to water. SD rats were anesthetized by pentobarbital sodium, then a midline laparotomy was conducted to expose the intestine and 2 cm sectioned intestinal loops were ligated at both ends. Subsequently, 200 µl Dio-labled NPs (n=3) was directly administered into the sections. After 3 h of administration, rats were sacrificed and sections of loops were withdrawn and mildly washed with PBS. Then the withdrawn loops were fixed with 4% paraformaldehyde for 4 h and dehydrated in 30% sucrose overnight. Samples were embedded in optimal cutting temperature compound and then sanctioned into 10 µm slices. Cell nucleus and mucus were 12

ACS Paragon Plus Environment

Page 12 of 36

Page 13 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Materials & Interfaces

stained with 4’,6-diamidino-2-phenylindole (DAPI) and rhodamine-conjugated ulex europaeus agglutinin I lectin (Rho-UEA-I), respectively. Finally the slices were visualized using confocal laser scanning microscope (CLSM).

2.9. Preparation and characterization of insulin-loaded NPs 1.2 mg of porcine insulin, 2 mg of PLGA, 0.4 mg of soybean phospholipid and a desired

amount

of

different

DSPE-PEG2000,

DSPE-PEG2000-R8

or

DSPE-PEG2000-Pho (described in “Section 2.3.”) were mixed in 0.2 ml DMSO, then via the self-assembly nanoprecipitation method the insulin-load NPs (INS NPs) were obtained. INS NPs were then collected through centrifugation at 12000 rpm for 60 min. The insulin in the supernatant and inside of NPs were both determined using a reverse-phase high performance liquid chromatography (RP-HPLC) method (Agilent 1200 series, CA, U.S.A.). Separation was achieved on a Diamosil C18 column (150 × 4.6 mm2, 5 µm) with mobile phase of acetonitrile − water (28:72, contained 0.2 M Na2SO4 and the pH was adjusted to 2.3 with phosphoric acid). The detection wavelength was set at 214 nm. The drug encapsulation efficiency (EE %) was calculated according to the following equation:

EE ሺ%ሻ =

Insulin in NPs ×100 Insulin in NPs + Insulin in supernatant

2.10. Drug release and protection from enzymatic degradation For evaluating drug release behavior, INS NPs were added into dialysis units (100 kDa) and first immersed in SGF (pH2.0) under magnetic stirring at 37 °C for 2 h. Then medium was replaced to SIF (pH6.8) and incubated for another 6 h. 200 µl of the samples were withdrawn at certain time points and remaining insulin was measured by RP-HPLC. To assess the ability of NPs to protect the encapsulated insulin from enzymatic degradation, insulin-loaded P-R8-Pho NPs (P-R8-Pho-INS NPs) were added into the SIF (pH6.8, with trypsin) and co-incubated at 37 °C in a shaker. 100 µl aliquots of the 13

ACS Paragon Plus Environment

ACS Applied Materials & Interfaces 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 36

samples were withdrawn at determined time and immediately added 100 µl of 0.1M ice-cold trifluoroacetic acid (TFA) to terminate the enzymatic interaction and destroyed NPs as well. Whereafter, the remaining insulin was measured using RP-HPLC. As a control, free insulin was incubated with SIF and analyzed under the same conditions.

2.11. Bioactivity of encapsulated insulin To evaluate the bioactivity of insulin after being encapsulated, INS NPs were allowed to release insulin at 37 °C. Then the released insulin was collected and analyzed using RP-HPLC to determine its concentration. SD rats (200-220 g, seven weeks old and raised in clean laboratory animal room with constant temperature at 25 °C) were fasted with free access to water overnight. Then they were divided in to 2 groups (n=5) and subcutaneously administered with the released insulin (2 IU/kg) or original free insulin (2 IU/kg). At certain time points, the blood glucose level was determined with JPS-6 blood glucose monitoring system.

2.12. In vivo hypoglycemic effect and pharmacokinetic study Diabetes was induced in male SD rats (200-220 g, seven weeks old and raised in clean laboratory animal room with constant temperature at 25 °C) via intraperitoneal injection of streptozotocin (STZ, 65 mg/kg) as previously described

26

. The blood

glucose level was determined using JPS-6 blood glucose monitoring system. Rats with fasting glycemia higher than 300 mg/dL after STZ treatment were considered to be diabetic. The diabetic rats were fasted overnight but allowed free to water. Then rats (n=5 for each group) were administered by oral gavage with P-INS NPs, P-R8-INS NPs, P-R8-Pho-INS NPs and free insulin solution (50 IU/kg), or by subcutaneous injection (sc) with free insulin solution (5 IU/kg). Blood glucose level was determined with a glucose meter. Blood samples were collected from the tail veins at different time points, and the plasma insulin level was measured with the porcine insulin ELISA kit (R&D System, USA). Area under the curve (AUC) of 14

ACS Paragon Plus Environment

Page 15 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Materials & Interfaces

plasma insulin concentration versus time was calculated using DAS software (Drug And Statistics ver2.0) and relative oral bioavailability (F%) was quantitated using the following equation:

F (%) =

AUCoral × Dosesc × 100 AUCsc × Doseoral

All animal related studies were performed in accordance with the Guide for the Care and Use of Laboratory Animals and all procedures were approved by Sichuan University Animal Care and Use Committee.

2.13. Statistical analysis All data were presented as mean ± SD and analyzed using SPSS 16.0 by the two-tailed Student’s t test. All experiments were performed in triplicate unless otherwise stated. Significance differences were defined as *p < 0.05 and **p < 0.01.

3. Results and discussion 3.1.

Synthesis

and

characterization

of

DSPE-PEG2000-R8

and

DSPE-PEG2000-Pho Octa-arginine (R8) peptide, one of the classical cell-penetrating peptides (CPPs), whose cationic property played a vital role on facilitating the cellular internalization was chosen as the cationic part of the NPs 27-28. To enable the anchoring of R8 peptide on the surface of the NPs, we conjugated R8 peptide to the terminal of the amphiphilic polymer, DSPE-PEG2000-Mal, to obtain the DSPE-PEG2000-R8. The reaction was monitored through 1H-NMR spectra (Supporting Information Figure S1), the disappearance of the featured proton peaks from the Mal groups after reacting (showed in red circle) confirmed the successful conjugation of R8 peptide to the DSPE-PEG2000-Mal. Phosphoserine (Pho), a substrate of intestinal alkaline phosphatase (IAP) and a product of natural serine through phosphorylation, was selected as the anionic part 15

ACS Paragon Plus Environment

ACS Applied Materials & Interfaces 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 36

which owned the negative charged monophosphate ester group that could be cleaved by the brush border membrane-bound IAP to realize the overturn of surface charge 23, 29

. Likewise, the phosphoserine was modified to the terminal of another amphiphilic

polymer, DSPE-PEG2000-NHS, to get the DSPE-PEG2000-Pho (Supporting Information Figure S2).

3.2. Preparation and characterization of NPs The biomimetic virus-like nanoparticles (NPs) with hydrophilic and equally cationic and anionic coating (Figure 2A) were prepared by R8 peptide (positive charge) and phosphoserine (Pho, negative charge) modified DSPE-PEG2000 via a self-assembly nanoprecipitation method. In order to obtain the neutral charged P-R8-Pho

NPs,

different

ratios

between

DSPE-PEG2000-R8

and

DSPE-PEG2000-Pho were screened. As shown in Figure S3 (Supporting Information), the zeta potential of P-R8-Pho NPs decreased with the increased amount of DSPE-PEG2000-Pho and the size didn’t change significantly. When the ratio reached 1:3 (P-R8-Pho-3 NPs) the nearly neutral charged NPs (-2.39 mv) were obtained. Hence the ratio of 1:3 (DSPE-PEG2000-R8: DSPE-PEG2000-Pho) was used to prepare the P-R8-Pho NPs for the following studies. By mixing the PLGA with various amounts of DSPE-PEG2000 that possessed different terminal charged groups, the negatively charged PLGA-PEG NPs (P NPs), positively charged PLGA-PEG-R8 NPs (P-R8 NPs) and electrically neutral PLGA-PEG-R8-Pho NPs (P-R8-Pho NPs) were obtained separately. Figure 2B showed that P NPs owned a size of 85.89 nm (PDI 0.175) and negative charge of -28.33 mV. In contrast, P-R8 NPs showed a positive charge of +15.57 mV and a similar size of around 76.41 nm (PDI 0.162). What's more, after the co-modification of cationic R8 peptide and anionic Pho group to the surface of the NPs, the virus-like P-R8-Pho NPs were successfully obtained, showing a nearly neutral surface with zeta potential of -2.39 mV and an analogous particle size of 81.81 nm (PDI 0.191). The transmission electron microscopy (TEM) images of the three kinds of NPs were 16

ACS Paragon Plus Environment

Page 17 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Materials & Interfaces

presented in Figure 2C, they all exhibited a similar well-defined spherical shape and a distinct core-shell (PLGA core and PEG chains) structure.

3.3. Stability study of NPs NPs by oral administration have to pass through gastrointestinal (GI) tract before absorption, so remaining stable in the harsh GI environment is the prerequisite for NPs to take effect. Therefore, we first investigated their stability among simulated gastric fluids (SGF, pH2.0) or simulated intestinal fluids (SIF, pH6.8) in the presence of enzymes (pepsin or trypsin), respectively. As shown in Figure 2D, the three kinds of NPs all exhibited excellent particle stability in SGF (incubation for 2 h) or SIF (incubation for 6 h) containing pepsin or trypsin, with no significant variation of size (P>0.05). Additionally, the cell-based studies were performed in PBS (pH 7.4) to maintain the normal morphology and function of cells, thus the stability of NPs in PBS solution was also necessary. Likewise, results (Figure 2D) showed that all the three kinds of NPs exhibited no significant change of particle size up to 6 h incubation (P>0.05), indicating their superior stability in PBS as well. Above results confirmed that all the P NPs, P-R8 NPs and P-R8-Pho NPs can maintain their particle-structure stable in both in vitro (PBS) and in vivo (SGF and SIF) environment.

17

ACS Paragon Plus Environment

ACS Applied Materials & Interfaces 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 2. (A) Schematic diagram of the structure of virus-like P-R8-Pho NPs. (B) Particle size and zeta potential of different PLGA NPs. (C) TEM images of P NPs, P-R8 NPs and P-R8-Pho NPs. Scale bars, 50 nm. (D) Stability of NPs after incubation in simulated gastric fluid (SGF, pH2.0) for 2 h, simulated intestinal fluid (SIF, pH6.8) for 6 h or in phosphate buffer solution (PBS, pH7.4). Results were expressed as a percentage of the size of initial NPs.

18

ACS Paragon Plus Environment

Page 18 of 36

Page 19 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Materials & Interfaces

3.4. Mucus penetrating ability of NPs For the sake of effective absorption into the blood capillary, oral administrated nanoparticles loaded with peptide/protein drugs have to sequentially overcome mucus and epithelial barriers

14, 30

. In order to penetrate the first obstacle, mucus layer

covered over the intestinal epithelium, NPs ought to possess a hydrophilic and neutral charged surface, at the same time become small enough to minimize the hydrophobic entrapment and electrostatic adhesive interactions with mucus

31-32

. The designed

virus-like small-sized (81.81 nm) P-R8-Pho NPs, with hydrophilic PEG-chain in the peripheral and simultaneously densely coated with equal cation and anion on the surface, was expected to own the superior ability to efficiently penetrate the mucus. To confirm this, two experiments were carried out to assess the mucus penetrating ability of the NPs. First, we investigated the interaction of NPs with mucin which was the major constituent of mucus layer and could trap NPs through hydrophobic and electrostatic interactions

33

. Despite the incubation with mucin at different concentrations, the

P-R8 NPs all showed the highest NPs-mucin aggregation rate (26.58%, 36.88% and 45.16% separately) among these three testing samples (Figure 3A), which might be caused by the electrostatic interaction between positively charged P-R8 NPs and the negatively charged mucin. In comparison, the P-R8-Pho NPs dramatically decreased the NPs-mucin aggregation rate to 15.42%, 24.56% and 30.88% (P<0.05), and with the maximal reduction of 68.4% at a mucin concentration of 2% (w/v) compared with P-R8 NPs. Though the conventional PEGylated mucus penetrating P-NPs had low aggregation rate among the three testing mucin concentrations, there was no significant difference compared with P-R8-Pho NPs (P > 0.05). Above results indicated that the additional decoration of anionic phosphoserine (Pho) upon P-R8 NPs to form the net neutral charged P-R8-Pho NPs could obviously reduce the aggregation of NPs with mucin, which may help the NPs to evade the capture of mucus layer. Then we evaluated the mucus penetrating ability of the NPs using an Ussing 19

ACS Paragon Plus Environment

ACS Applied Materials & Interfaces 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Chamber system

12-13

. Porcine intestinal mucus was chosen as in vitro mucus model

because of the similarity with human intestinal mucus in structure and molecular weight 34. As shown in Figure 3B, P-R8 NPs owned the lowest apparent permeability coefficient (Papp) value of 4.97 × 10-6 cm/s, while 14.88 × 10-6 cm/s and 12.57 × 10-6 cm/s were found in P NPs and P-R8-Pho NPs. By comparison, the Papp value of P-R8-Pho NPs was 2.53-fold higher than P-R8 NPs (P<0.05), and just rarely lower than the P NPs (P>0.05). It indicated that the existence of anionic Pho group on the surface of P-R8-Pho NPs could act as the shield for preventing the positively charged R8 from binding to the highly negatively charged mucus gels. Furthermore, the utilization of DSPE-PEG2000 as the anchor to surround the P-R8-Pho NPs with equal densities of cation and anion also endowed it a hydrophilic PEGylation shell. These two tactics ensured the “mucus-inert” surface (virus-like) of P-R8-Pho NPs, which possessed superior ability to penetrate through the mucus layer.

Figure 3. (A) NPs-mucin aggregation in the presence of different mucin concentrations. The percentage of aggregated NPs was expressed as the fluorescence intensity of aggregated NPs relative to initial NPs. *p < 0.05 versus P-R8 NPs. (B) The apparent permeability coefficient (Papp) values of NPs permeation across the mucus tested by Ussing Chamber system. *p < 0.05 versus P-R8 NPs.

20

ACS Paragon Plus Environment

Page 20 of 36

Page 21 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Materials & Interfaces

3.5. Enzyme-responsive zeta potential switch Having proven that the P-R8-Pho NPs could diffuse relatively better across the mucus layer, its ability to overcome the second absorption obstacle, the epithelial cell layer overlaid above blood capillary, remained to be investigated. Nanocarriers with positive surface charge are considered for having a higher cellular uptake efficiency compared with neutral or negative ones

35

. P-R8-Pho NPs with the phosphoserine

(Pho), a substrate of intestinal alkaline phosphatase (IAP), as the phosphorylating agent were considered to be able to change their external charge from weak negative to positive through the dephosphorylation induced by IAP expressed in intestinal epithelium. To testify this, we first evaluated the zeta potential switch ability of NPs using isolated intestinal alkaline phosphatase (IAP). The variations of zeta potential of different NPs after incubation with IAP were shown in Table 1. A conversion of zeta potential from -1.87 mV to +7.37 mV was found in P-R8-Pho NPs (P<0.05), while there was no obvious change for the other three NPs. Similar results were reported in other researches with different types of phosphorylating agents

36

. It suggested that

P-R8-Pho NPs were indeed able to change their neutral surface charge by the IAP-responsive hydrolysis of Pho to become positively charged upon reaching the enzyme-expressing epithelial cells. Moreover, a new PLGA-PEG2000-R8-COOH (P-R8-COOH) NPs that possessed a similar surface property with P-R8-Pho NPs (Pho was replaced by carboxyl as the anionic gruop) was introduced for this and following in vitro experiments to further verify that the charge reversal behavior was induced by Pho group in P-R8-Pho NPs.

Table 1. Zeta potential change of NPs prior and after incubation with isolated IAP. Zeta potential (mV)

Initial

After incubation

P NP

-18.2 ± 0.60

-22.93 ± 1.56

P-R8 NPs

+16.0 ± 0.72

+17.53 ± 0.95

21

ACS Paragon Plus Environment

ACS Applied Materials & Interfaces 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 36

P-R8-COOH NPs

+1.54 ± 0.38

+2.26 ± 0.28

P-R8-Pho NPs

-1.87 ± 2.35

+7.37 ± 2.25

3.6. Cellular uptake and transepithelial transport of NPs Another challenging obstacle for the absorption of oral administrated NPs was the epithelium layer, so we evaluated the ability of our NPs to entry and pass through the epithelium. The human colonic adenocarcinoma (Caco-2) and HT29-MTX-E12 cells (E12) which separately mimicked the absorptive enterocytes and mucus-secreting goblet cells were selected as the in vitro cell models 5. Because the charge reversal ability of NPs depended on the intestinal alkaline phosphatase (IAP), it was considered as a prerequisite for the chosen cell models to own the ability of expressing the IAP. Hence, we first quantitatively analyzed the IAP activity in both Caco-2 and E12 cells using the Alkaline Phosphatase Assay Kit. As shown in Figure 4A, the amount of IAP expressed in Caco-2 cells was around 12.0 nmol/min/mg, while only 0.64 nmol/min/mg in E12 cells. The difference in these two types of cell lines might relate to their own degree of cell differentiation, and similar studies were also demonstrated by other reports 37-38. The cell viability of NPs tested on Caco-2 and E12 cells using MTT assay manifested that there was no significant cytotoxicity for all the formulations within the PLGA concentration range from 25 µg/ml to 300 µg/ml (Supporting Information Figure S4). Then we investigated the cellular uptake of NPs on Caco-2 and E12 cells respectively. As demonstrated in Figure 4B, uptake of P-R8-Pho NPs on Caco-2 cells was about 3-fold higher than the control P NPs (P<0.05) and almost the same level as P-R8 NPs. It indicated that firstly, the modification of cationic R8 peptide over the PLGA NPs (P-R8 NPs) could obviously increase their cellular internalization. Secondly, the addition of anionic Pho group on P-R8-Pho NPs to shield the positive 22

ACS Paragon Plus Environment

Page 23 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Materials & Interfaces

charge of R8 aiming to efficiently penetrate mucus would not influence the cellular uptake efficiency. We speculated that the IAP-responsive cleavage of the phosphate ester led to the exposure of cationic R8 to assist the cellular uptake of NPs. For the mucus-secreting E12 cells shown in Figure 4C, NPs entrapped in cell mucus layer were removed by the formalin solution

13

and the result represented only the NPs

internalized in cells. P-R8-Pho NPs still exhibited the highest cellular uptake which was 1.65-fold (P<0.05) and 3.4-fold (P<0.01) higher than that of P-R8 and P NPs, and P-R8 NPs was only 2-fold higher than that of the control P NPs. These results suggested that P-R8-Pho NPs were also able to be internalized more into cells when mucus layer existed. We assumed that P-R8 NPs were capable of obviously enhancing the cellular uptake (Figure 4B) but exhibited a reductive uptake in the mucus-secreting cells (Figure 4C). It might due to the positively charged P-R8 NPs would be trapped by the mucus layer due to the strong electrostatic interactions which made it unable to reach cell layer, as reported previously

25

. P-R8-Pho NPs could

maintain the highest cellular uptake in both Caco-2 and E12 cells owing to the co-existence of superior mucus penetrating and cellular uptake ability based on its appropriate charge reversal behavior as demonstrated above (Table 1). On the contrary, P NPs only had a good ability to penetrate the mucus barrier (Figure 3) but were hard to be internalized because of the hydrophilic and negatively charged surface, while P-R8 NPs only showed satisfactory cellular uptake but found to be troublesome to pass through the mucus layer due to the electrostatic interaction. To further confirm the great cellular internalization of P-R8-Pho NPs was due to its enzymatic-responsive charge reversal ability induced by intestinal alkaline phosphatase (IAP), an additional PLGA NPs, the PLGA-PEG-R8-COOH NPs (P-R8-COOH NPs), was introduced. It owned the same surface property with P-R8-Pho NPs that was both hydrophilic and neutral charged, except the anionic Pho group was replaced by another anionic group carboxyl (COOH). So it meant that P-R8-COOH NPs didn't possess the charge reversal ability as the P-R8-Pho NPs did. P-R8-COOH NPs showed a comparable size of 82.07 nm (PDI 0.190) and zeta potential of -2.38 mV, whose TEM image also exhibited a spherical shape and distinct 23

ACS Paragon Plus Environment

ACS Applied Materials & Interfaces 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

core-shell structure (Supporting Information Figure S5). First we compared the cellular uptake of these two NPs on Caco-2 cells, the cellular uptake of P-R8-Pho NPs was 2.9-fold (P<0.01) higher than that of the control P NPs while P-R8-COOH NPs only 1.54-fold (P<0.05) higher than that of P NPs (Figure 4D). Besides, the cellular uptake of P-R8-Pho NPs was found 1.87-fold (P<0.05) higher than that of P-R8-COOH NPs (Figure 4D). The relatively lower uptake of P-R8-COOH NPs was caused by its neutrally charged surface without a good affinity to negatively charged cell membrane. In contrast, P-R8-Pho NPs with the charge reversal ability were able to switch to a positively charged surface when interacted with the membrane-bound IAP which gave it a higher affinity to cell membrane and resulted in higher cellular internalization. IAP was a kind of phosphatase expressed in the small-intestinal epithelium 21, whose enzyme activity could be inhibited by many phosphatase inhibitors. Therefore, a phosphatase inhibitor mixture called phosphatase inhibitor cocktail Ⅱ was then applied on cellular uptake study to verify the involvement of IAP. Among all of the three kinds of NPs only P-R8-Pho NPs showed a substantially reduced relative uptake to 71.4% (P<0.05) in the presence of phosphatase inhibitor cocktail Ⅱ (Figure 4E). Above results further demonstrated that P-R8-Pho NPs could interact with the IAP which induced a shift of surface charge to become positive, thus led to a superior cell internalization of NPs.

24

ACS Paragon Plus Environment

Page 24 of 36

Page 25 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Materials & Interfaces

Figure 4. (A) Quantitative assay the intestinal alkaline phosphatase(IAP)activity expressed in Caco-2 and E12 cells using the Alkaline Phosphatase Assay Kit. The result was expressed as nanomoles of produced p-nitrophenol per min per mg of protein. (B) Cellular uptake of NPs into Caco-2 cells after incubation for 3 h. The uptake was expressed as the quantity of fluorescence intensity of NPs associated with 1 mg of cellular protein. *p < 0.05 versus P NPs. (C) Cellular uptake of NPs by E12 cells after treatment for 3 h in the presence of cell mucus layer. Result was expressed as the amount of NPs that penetrated the cell mucus layer and entered the cells. *p < 0.05 versus P-R8-Pho NPs, **p < 0.01 versus P-R8-Pho NPs. (D) Cellular uptake of P, P-R8-Pho and P-R8-COOH NPs on Caco-2 cells. *p < 0.05 and **p < 0.01 versus P NPs, #p < 0.05 compared P-R8-Pho NPs with P-R8-COOH NPs. (E) Caco-2 cell relative uptake of NPs in the presence or absence of phosphatase inhibitor cocktail. Relative uptake was expressed as the percentage of internalized NPs in the presence of inhibitor (NPs + I) compared with corresponding no inhibitor treatment groups (NPs). *p < 0.05.

Intestinal

epithelial cells are

composed

of

absorptive

enterocytes

and

mucus-secreting goblet cells below mucus layer. Compared with single cell model 25

ACS Paragon Plus Environment

ACS Applied Materials & Interfaces 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(Caco-2 or E12), the Caco-2/E12 co-cultured cell model could be closer to real epithelium by imitating the absorptive enterocytes and mucus-secreting goblet cells simultaneously

5, 39

. Hence we applied the Caco-2/E12 (7:3) co-culture cell model to

further evaluate the cellular uptake of NPs and the influence of mucus. Figure 5A demonstrated that for the “NPs” group (red column) representing NPs only internalized in cells by post-removing mucus through formalin, P-R8-Pho NPs had the highest cellular uptake among all four kinds of NPs, which was 2-fold, 1.69-fold and 1.22-fold higher than that of P, P-R8 NPs (P<0.01) and P-R8-COOH NPs (P<0.05), respectively. The “NPs + mucus” group (black column) representing NPs trapped in mucus and internalized in cells was obtained without the post-removing mucus process. By comparison only P-R8 NPs showed an obvious entrapment about 18.6% in mucus (#P<0.05) due to the positive charged property. Besides, the existence of cell mucus layer had no influence on the uptake of other three NPs (P>0.05). Above results were in accordance with former single cell model experiments (Figure 4), all suggesting that P-R8-Pho NPs can efficiently penetrate the mucus barrier and at the same time own favorable cellular uptake capacity. Eventually the in vitro transepithelial transport of NPs was assessed on the Caco-2/E12 co-culture cells as well using Transwell permeable supports. After incubation with cell monolayer for 8 h, the transported NPs in basolateral chamber were measured. As shown in Figure 5B, P-R8-Pho exhibited superior transepithelial transport ability which was 1.7-fold (P<0.05) and 2.4-fold (P<0.01) higher than P-R8 NPs and P NPs. This result was consistent with the cellular uptake study on Caco-2/E12 co-culture cells (Figure 5A). It confirmed that the improved mucus permeation and enhanced cellular uptake could also facilitate the transport of P-R8-Pho NPs through cell monolayer. Besides, the transepithelial electrical resistance (TEER) value had no significant change during the transport process (Figure 5C), suggesting that treatment with the NPs would not destroy the integrity of cell monolayer.

26

ACS Paragon Plus Environment

Page 26 of 36

Page 27 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Materials & Interfaces

Figure 5. (A) Cellular uptake study of NPs using Caco-2/E12 co-culture cells. Cellular uptake for “NPs + mucus” group (black column) represented NPs trapped in mucus and internalized in cells. Cellular uptake for “NPs” group (red column) represented NPs only internalized in cells. *p < 0.05 versus P-R8-Pho NPs, **p < 0.01 versus P-R8-Pho NPs, #p < 0.05. (B) Transepithelial transport study of NPs through Caco-2/E12 co-culture monolayer. Result was expressed as the percentage of transported NPs compared with initial NPs. *p < 0.05 versus P-R8-Pho NPs, **p < 0.01 versus P-R8-Pho NPs. (C) The transepithelial electrical resistance (TEER) value variation after incubation with NPs (as the percentage of initial TEER value).

3.7. In situ absorption of NPs To investigate the absorption behavior of NPs in vivo, the in situ absorption of Dio-labled NPs into intestinal villi was performed on SD rats. After treatment with NP samples the obtained isolated intestinal villi slices were visualized by confocal laser scanning microscope (CLSM). As demonstrated in Figure 6, P-R8-Pho NPs (bottom) showed the highest green fluorescence signal in the interior of intestinal villi (white 27

ACS Paragon Plus Environment

ACS Applied Materials & Interfaces 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

arrows), indicating the ability of the NPs to pass across the mucus layer and epithelium into the blood circulation for oral delivery. In contrast, P-R8 NPs (middle) exhibited a much lower green fluorescence signal in the interior of villi and P NPs (top) showed very few green fluorescence signals. The enhanced absorption of intestine by P-R8-Pho NPs was confirmed and thus showed the potential for the oral drug delivery in vivo.

Figure 6. Representative fluorescence images of intestinal villi after administration of Dio-NPs in Sprague-Dawley (SD) rats. White arrows indicate NPs absorption into the interior of intestinal villi (green fluorescence).

3.8. Characterization of insulin-loaded NPs P-R8-Pho NPs showed excellent mucus penetrating and cellular uptake capabilities in vitro. So for the drug delivery application of this kind of NPs, insulin was selected as the therapeutic protein and insulin-loaded NPs (INS NPs) for diabetic treatment in vivo were evaluated. Firstly, the INS NPs were prepared by co-precipitating insulin and polymers within nanoprecipitation process. All INS NPs possessed insulin 28

ACS Paragon Plus Environment

Page 28 of 36

Page 29 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Materials & Interfaces

encapsulation efficiency (EE %) to approximately 40% (Figure 7A). The in vitro insulin release behavior from NPs was performed in SGF (pH2.0) for first 2 h and then in SIF (pH6.8) for another 6 h to imitate the GI environment. As shown in Figure 7B all three kinds of NPs exhibited comparable sustained release profiles over time with insulin released about 34-42% for the first 2 h in SGF and finally 54-62% for another 6 h in SIF. The stability of insulin in trypsin was demonstrated in Figure 7C, free insulin solution was almost all degraded by trypsin within 1 h, while the insulin loaded in P-R8-Pho NPs (P-R8-Pho-INS NPs) remained 33% (P<0.05) after 1 h incubation with trypsin, due to the protection of the NPs. Besides, other strategies still could be applied to further protect the bioactivity of insulin such as enteric coating or adding enzyme inhibitors 40.

3.9. In vivo hypoglycemic effect and pharmacokinetic study For the in vivo application of NPs, we first verified the bioactivity of insulin released from the NPs

14, 24

. The releasing insulin and free insulin were separately

administrated by subcutaneous injection to rats and the blood glucose was monitored over time. The hypoglycemic effect of released insulin (2 IU/kg) was consistent with equivalent dose of free insulin (Figure 7D), indicating that the bioactivity of insulin was maintained during the encapsulation and release process. Then we investigated the hypoglycemic effect and pharmacokinetic of INS NPs via oral administration to the diabetic rats 41. As shown in Figure 7E, free insulin solution (5 IU/kg) by subcutaneous injection showed a rapid decrease of blood glucose level, whereas free insulin solution via oral administration (50 IU/kg) showed no hypoglycemic response, indicating that oral delivery of free insulin was in vain. The control P-INS NPs via oral administration (50 IU/kg) also failed to reduce the blood glucose level which might be due to its inferior cellular uptake and intestinal absorption. And for P-R8-INS NPs (50 IU/kg), it only exhibited a small extent of hypoglycemic effect for a maximal decrease of ~15%. In comparison, oral administration of P-R8-Pho-INS NPs generated the most prominent hypoglycemic 29

ACS Paragon Plus Environment

ACS Applied Materials & Interfaces 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

response and induced a maximal blood glucose reduction of 32%. The blood glucose level after oral administration of P-R8-Pho-INS NPs was significantly lower than that of free insulin group at 1-6 h (*p < 0.05), P-INS NPs at 2-6 h (&p < 0.05) and P-R8-INS NPs at 3-4 h (#p < 0.05). The pharmacokinetic parameters of NPs were listed in Table 2. P-R8-Pho-INS NPs still exhibited the highest relative oral bioavailability (F %) of 5.96%, which was found ~14.9 fold higher than that of oral free insulin group (0.40%), ~2.3 fold higher than that of P-INS NPs (2.61%) and ~1.9 fold higher than that of P-R8-INS NPs (3.13%), respectively.

Figure 7. (A) Encapsulation efficiency of insulin-loaded NPs. (B) In vitro release profiles of insulin from NPs in SGF for 2 h and SIF for 6 h. (C) Trypsin degradation profiles of free insulin and insulin encapsulated in P-R8-Pho NPs. *p < 0.05 versus free insulin group in 0.5 and 1 h. (D) Blood glucose level of normal SD rats after subcutaneous injection of equivalent doses (2 IU/kg) of original insulin or insulin released from NPs. (E) Blood glucose level in fasted diabetic rats via oral administration of the insulin-loaded NPs and free insulin (50 IU/kg) or subcutaneous injection of free insulin (5 IU/kg). *p < 0.05 versus oral free insulin group, &p < 0.05 30

ACS Paragon Plus Environment

Page 30 of 36

Page 31 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Materials & Interfaces

versus P NPs, #p < 0.05 versus P-R8 NPs.

Table 2. Pharmacokinetic parameters of free insulin and insulin-loaded NPs by subcutaneous (sc) or oral administration. AUC: area under the curve of plasma insulin concentration; F(%): relative bioavailability (n=5). Samples

Dose (IU/kg)

AUC (mIU·h/L)

F (%)

P NPs

50

58.52 ± 6.42

2.61 ± 0.28

P-R8 NPs

50

70.01 ± 7.21

3.13 ± 0.32

P-R8-Pho NPs

50

133.51 ± 20.85

5.96 ± 0.93

Free INS (oral)

50

8.94 ± 3.44

0.40 ± 0.15

Free INS (sc)

5

223.81 ± 23.78

100

Overall, the in vivo hypoglycemic effect and pharmacokinetic study showed good correlations with intestinal absorption, in vitro mucus penetrating and cellular studies. All suggested that P-R8-Pho NPs owned the capability of sequentially overcoming mucus and epithelial barriers to achieve a promising hypoglycemic effect.

4. Conclusion In this study, a novel biomimetic virus-like oral delivery system co-operative with charge reversal ability (P-R8-Pho NPs) had been developed aiming to sequentially overcome mucus and epithelial barriers. It showed excellent stability under both SGF and SIF conditions (simulated GI environment) in vitro. The small size (81.81 nm) 31

ACS Paragon Plus Environment

ACS Applied Materials & Interfaces 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 32 of 36

and designed virus-like surface property of P-R8-Pho NPs could facilitate rapid mucus penetrating, which was almost equal to conventional PEGylated mucus penetrating nanoparticles. Besides, the charge reversal ability was confirmed by co-incubation with isolated IAP in vitro. The timely charge reversal behavior to expose cationic R8 peptide induced efficient CPPs-mediated cellular uptake and transepithelial transport on Caco-2/E12 co-cultured cell model. What’s more, enhanced absorption of intestine in vivo was observed as well. Finally oral administration of insulin-loaded P-R8-Pho NPs was able to induce preferable hypoglycemic effect and 1.9-fold higher oral bioavailability compared with single CPPs-modified P-R8 NPs on diabetic rats. These results suggested that the combinative

application

of

biomimetic

mucus

penetrating

strategy

and

enzyme-responsive charge reversal strategy in one single nanovehicle to sequentially overcome mucus and epithelial barriers had great potential for the oral peptide/protein delivery.

Acknowledgement We gratefully acknowledge financial support from the National Science Foundation for Distinguished Young Scholars (81625023) and the Major Research Plan of National Natural Science Foundation of China (81690261).

Supporting information Supporting Information Available: 1H-NMR spectra of DSPE-PEG2000-R8 and DSPE-PEG2000-Pho, Screening of ratio (DSPE-PEG2000-R8: DSPE-PEG2000-Pho) for P-R8-Pho NPs, Caco-2 or E12 cell viability of NPs, TEM image of P-R8-COOH NPs. This material is available free of charge via the Internet at http://pubs.acs.org.

32

ACS Paragon Plus Environment

Page 33 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Materials & Interfaces

References (1) Khafagy, E.-S.; Morishita, M.; Onuki, Y.; Takayama, K. Current Challenges in Non-Invasive Insulin Delivery Systems: A Comparative Review. Adv. Drug Delivery Rev. 2007, 59, 1521-1546. (2) Park, K.; Kwon, I. C.; Park, K. Oral Protein Delivery: Current Status and Future Prospect. React. Funct. Polym. 2011, 71, 280-287. (3) Yun, Y.; Cho, Y. W.; Park, K. Nanoparticles for Oral Delivery: Targeted Nanoparticles with Peptidic Ligands for Oral Protein Delivery. Adv. Drug Delivery Rev. 2013, 65, 822-832. (4) Yu, M.; Wu, J.; Shi, J.; Farokhzad, O. C. Nanotechnology for Protein Delivery: Overview and Perspectives. J. Controlled Release 2016, 240, 24-37. (5) Jin, Y.; Song, Y.; Zhu, X.; Zhou, D.; Chen, C.; Zhang, Z.; Huang, Y. Goblet Cell-Targeting Nanoparticles for Oral Insulin Delivery and the Influence of Mucus on Insulin Transport. Biomaterials 2012, 33, 1573-1582. (6) Cone, R. A. Barrier Properties of Mucus. Adv. Drug Delivery Rev 2009, 61, 75-85. (7) Liu, M.; Zhang, J.; Shan, W.; Huang, Y. Developments of Mucus Penetrating Nanoparticles. Asian J. Pharm. Sci. 2015, 10, 275-282. (8) Ensign, L. M.; Schneider, C.; Suk, J. S.; Cone, R.; Hanes, J. Mucus Penetrating Nanoparticles: Biophysical Tool and Method of Drug and Gene Delivery. Adv. Mater. 2012, 24, 3887-3894. (9) Mert, O.; Lai, S. K.; Ensign, L.; Yang, M.; Wang, Y.-Y.; Wood, J.; Hanes, J. A Poly (Ethylene Glycol)-Based Surfactant for Formulation of Drug-Loaded Mucus Penetrating Particles. J. Controlled Release 2012, 157, 455-460. (10) Verma, A.; Stellacci, F. Effect of Surface Properties on Nanoparticle–Cell Interactions. Small 2010, 6, 12-21. (11) Oh, E.; Delehanty, J. B.; Sapsford, K. E.; Susumu, K.; Goswami, R.; Blanco-Canosa, J. B.; Dawson, P. E.; Granek, J.; Shoff, M.; Zhang, Q. Cellular Uptake and Fate of Pegylated Gold Nanoparticles Is Dependent on Both Cell-Penetration Peptides and Particle Size. Acs Nano 2011, 5, 6434-6448. (12) Shan, W.; Zhu, X.; Liu, M.; Li, L.; Zhong, J.; Sun, W.; Zhang, Z.; Huang, Y. Overcoming the Diffusion Barrier of Mucus and Absorption Barrier of Epithelium by Self-Assembled Nanoparticles for Oral Delivery of Insulin. ACS nano 2015, 9, 2345-2356. (13) Liu, M.; Zhang, J.; Zhu, X.; Shan, W.; Li, L.; Zhong, J.; Zhang, Z.; Huang, Y. Efficient Mucus Permeation and Tight Junction Opening by Dissociable “Mucus-Inert” Agent Coated Trimethyl Chitosan Nanoparticles for Oral Insulin Delivery. J. Controlled Release 2016, 222, 67-77. 33

ACS Paragon Plus Environment

ACS Applied Materials & Interfaces 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 34 of 36

(14) Zhu, X.; Wu, J.; Shan, W.; Zhou, Z.; Liu, M.; Huang, Y. Sub‐50 Nm Nanoparticles with Biomimetic Surfaces to Sequentially Overcome the Mucosal Diffusion Barrier and the Epithelial Absorption Barrier. Adv. Funct. Mater. 2016, 26, 2728-2738. (15) Ensign, L. M.; Cone, R.; Hanes, J. Oral Drug Delivery with Polymeric Nanoparticles: The Gastrointestinal Mucus Barriers. Adv. Drug Delivery Rev 2012, 64, 557-570. (16) Khafagy, E.-S.; Morishita, M. Oral Biodrug Delivery Using Cell-Penetrating Peptide. Adv. Drug Delivery Rev 2012, 64, 531-539. (17) Lai, S. K.; Wang, Y.-Y.; Hanes, J. Mucus-Penetrating Nanoparticles for Drug and Gene Delivery to Mucosal Tissues. Adv. Drug Delivery Rev 2009, 61, 158-171. (18) Pöhlmann, S.; Simmons, G., Viral Entry into Host Cells. Adv. Exp. Med. Biol., Springer Science+Business Media 2013, volume 790. (19) Herce, H. D.; Garcia, A. E.; Litt, J.; Kane, R. S.; Martin, P.; Enrique, N.; Rebolledo, A.; Milesi, V. Arginine-Rich Peptides Destabilize the Plasma Membrane, Consistent with a Pore Formation Translocation Mechanism of Cell-Penetrating Peptides. Biophys. J. 2009, 97, 1917-1925. (20) Madani, F.; Lindberg, S.; Langel, Ü.; Futaki, S.; Gräslund, A. Mechanisms of Cellular Uptake of Cell-Penetrating Peptides. J. Biophys. 2011, 2011, 414729. (21) Lallès, J. P. Intestinal Alkaline Phosphatase: Multiple Biological Roles in Maintenance of Intestinal Homeostasis and Modulation by Diet. Nutr. Rev. 2010, 68, 323-332. (22) Jedrzejas,

M.

J.;

Setlow,

P.

Comparison

of

the

Binuclear

Metalloenzymes

Diphosphoglycerate-Independent Phosphoglycerate Mutase and Alkaline Phosphatase: Their Mechanism of Catalysis Via a Phosphoserine Intermediate. Chem. Rev. 2001, 101, 607-618. (23) Perera, G.; Zipser, M.; Bonengel, S.; Salvenmoser, W.; Bernkop-Schnürch, A. Development of Phosphorylated Nanoparticles as Zeta Potential Inverting Systems. Eur. J. Pharm. Biopharm. 2015, 97, 250-256. (24) Shan, W.; Zhu, X.; Tao, W.; Cui, Y.; Liu, M.; Wu, L.; Li, L.; Zheng, Y.; Huang, Y. Enhanced Oral Delivery of Protein Drugs Using Zwitterion-Functionalized Nanoparticles to Overcome Both the Diffusion and Absorption Barriers. ACS Appl. Mater. Interfaces 2016, 8, 25444-25453. (25) Li, X.; Guo, S.; Zhu, C.; Zhu, Q.; Gan, Y.; Rantanen, J.; Rahbek, U. L.; Hovgaard, L.; Yang, M. Intestinal Mucosa Permeability Following Oral Insulin Delivery Using Core Shell Corona Nanolipoparticles. Biomaterials 2013, 34, 9678-9687. (26) Arora, S.; Ojha, S. K.; Vohora, D. Characterisation of Streptozotocin Induced Diabetes Mellitus in Swiss Albino Mice. Global J. Pharmacol. 2009, 3, 81-84. (27) Nakase, I.; Tanaka, G.; Futaki, S. Cell-Penetrating Peptides (Cpps) as a Vector for the Delivery of Sirnas into Cells. Mol. BioSyst. 2013, 9, 855-861. (28) Milletti, F. Cell-Penetrating Peptides: Classes, Origin, and Current Landscape. Drug discovery today 2012, 17, 850-860. (29) Coleman, J. E. Structure and Mechanism of Alkaline Phosphatase. Annu. Rev. Biophys. Biomol. Struct. 1992, 21, 441-483. (30) Liu, M.; Wu, L.; Zhu, X.; Shan, W.; Li, L.; Cui, Y.; Huang, Y. Core–Shell Stability of Nanoparticles Plays an Important Role for Overcoming the Intestinal Mucus and Epithelium Barrier. J. Mater. Chem. B 2016, 4, 5831-5841. (31) Tang, B. C.; Dawson, M.; Lai, S. K.; Wang, Y.-Y.; Suk, J. S.; Yang, M.; Zeitlin, P.; Boyle, M. P.; Fu, J.; Hanes, J. Biodegradable Polymer Nanoparticles That Rapidly Penetrate the Human Mucus Barrier. Proc. Natl. Acad. Sci. 2009, 106, 19268-19273. 34

ACS Paragon Plus Environment

Page 35 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Materials & Interfaces

(32) Maisel, K.; Ensign, L.; Reddy, M.; Cone, R.; Hanes, J. Effect of Surface Chemistry on Nanoparticle Interaction with Gastrointestinal Mucus and Distribution in the Gastrointestinal Tract Following Oral and Rectal Administration in the Mouse. J. Controlled Release 2015, 197, 48-57. (33) McGuckin, M. A.; Lindén, S. K.; Sutton, P.; Florin, T. H. Mucin Dynamics and Enteric Pathogens. Nat. Rev. Microbiol. 2011, 9, 265-278. (34) Groo, A.-C.; Lagarce, F. Mucus Models to Evaluate Nanomedicines for Diffusion. Drug discovery today 2014, 19, 1097-1108. (35) Honary, S.; Zahir, F. Effect of Zeta Potential on the Properties of Nano-Drug Delivery Systems-a Review (Part 1). Trop. J. Pharm. Res. 2013, 12, 255-264. (36) Bonengel, S.; Prüfert, F.; Perera, G.; Schauer, J.; Bernkop-Schnürch, A. Polyethylene Imine-6-Phosphogluconic Acid Nanoparticles–a Novel Zeta Potential Changing System. Int. J. Pharm. 2015, 483, 19-25. (37) Gao, A.; Hang, R.; Huang, X.; Zhao, L.; Zhang, X.; Wang, L.; Tang, B.; Ma, S.; Chu, P. K. The Effects of Titania Nanotubes with Embedded Silver Oxide Nanoparticles on Bacteria and Osteoblasts. Biomaterials 2014, 35, 4223-4235. (38) Xie, Q.; Wang, Z.; Huang, Y.; Bi, X.; Zhou, H.; Lin, M.; Yu, Z.; Wang, Y.; Ni, N.; Sun, J. Characterization of Human Ethmoid Sinus Mucosa Derived Mesenchymal Stem Cells (Hesmscs) and the Application of Hesmscs Cell Sheets in Bone Regeneration. Biomaterials 2015, 66, 67-82. (39) Zhang, P.; Xu, Y.; Zhu, X.; Huang, Y. Goblet Cell Targeting Nanoparticle Containing Drug-Loaded Micelle Cores for Oral Delivery of Insulin. Int. J. Pharm. 2015, 496, 993-1005. (40) Carino, G. P.; Mathiowitz, E. Oral Insulin Delivery. Adv. Drug Delivery Rev. 1999, 35, 249-257. (41) Cui, Y.; Shan, W.; Liu, M.; Wu, L.; Huang, Y. A Strategy for Developing Effective Orally-Delivered Nanoparticles through Modulation of the Surface “Hydrophilicity/Hydrophobicity Balance”. J. Mater. Chem. B 2017, 5, 1302-1314.

35

ACS Paragon Plus Environment

ACS Applied Materials & Interfaces 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Graphical abstract

36

ACS Paragon Plus Environment

Page 36 of 36