Novel Solid Lipid Nanoparticle with Endosomal Escape Function for

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A Novel Solid Lipid Nanoparticle with Endosomal Escape Function for Oral Delivery of Insulin Yining Xu, Yaxian Zheng, Lei Wu, Xi Zhu, Zhirong Zhang, and Yuan Huang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b00507 • Publication Date (Web): 27 Feb 2018 Downloaded from http://pubs.acs.org on February 27, 2018

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ACS Applied Materials & Interfaces

A Novel Solid Lipid Nanoparticle with Endosomal Escape Function for Oral Delivery of Insulin

Yining Xu1, Yaxian Zheng1, Lei Wu, Xi Zhu, 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, Southern Renmin Road, Chengdu 610041,China 1

The authors contributed equally to this study and shared first authorship. *Correspondence to: Prof. Yuan Huang, West China School of Pharmacy, Sichuan University, Chengdu 610041, Sichuan, P.R. China. Tel/Fax: +86-28-85501617. E-mail: [email protected]

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ABSTRACT: Although nanoparticles (NPs) have been demonstrated as promising tools for improving oral absorption of biotherapeutics, most of them still have very limited oral bioavailability. Lyso-endosomal degradation in epithelial cells is one of important but often-neglected physiological barriers, limiting the transport of cargoes across the intestinal epithelium. We herein reported a solid lipid nanoparticle (SLN) platform with a unique feature of endosomal escape for oral protein drug delivery. The SLNs consisted of a solid-lipid shell, which contained an endosomal escape agent (GLFEAIEGFIENGWEGMIDGWYG, HA2), and an aqueous core that loaded with insulin (INS HA2-O-SLNs). SLNs without and with HA2 peptide in aqueous core (INS SLNs and INS HA2-W-SLNs, respectively) were used as the control groups. Our study showed that INS HA2-O-SLNs effectively facilitated the escape of the loaded insulin from the acidic endosomes, which preserved the biological activity of insulin to a greater extent during the intracellular transport. The spatial location of HA2 peptide was demonstrated to determine the endosomal escape efficiency. As demonstrated in the intracellular trafficking of SLNs, INS HA2-O-SLNs displayed much less distribution in late endosomes and lysosomes. Meanwhile, insulin in INS HA2-O-SLNs exhibited the highest transepithelial permeation efficiency, with 2.19 folds and 1.72 folds higher accumulated amount in basolateral side as compared to INS SLNs and INS HA2-W-SLNs. In addition, insulin from INS HA2-O-SLNs exhibited the highest insulin permeation in various regions of small intestines. INS HA2-O-SLNs generated an excellent hypoglycemic response following oral administration in diabetic rats. Thus, such functional SLNs demonstrated a great potency for oral delivery of peptide/protein drugs. Keywords: Solid lipid nanoparticles, Insulin, Endosomal escape, HA2 peptides, Oral delivery


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1. Introduction Therapeutic peptides and proteins have been extensively utilized in the treatment of numerous diseases for their specificity and high potency1. However, these biotherapeutics are currently only limited to parenteral administration, which result in unwanted pain, poor patient compliance and several side effects. Although oral administration is much more preferred, bioavailability of these drugs are often limited by several physiological barriers in gastrointestinal tract (GIT)2-3. Since 1980s, nanotechnology has been reported to protect drugs from harsh conditions in GIT4-6. However, it is still a big challenge to improve the drug permeability across the intestinal epithelium7-9. Endocytic pathway is the major route for most nanoparticle (NP) uptake10-11. However, the amount of NPs across the intestinal epithelium was always much less than those engulfed in cells. The dilemma of “easy endocytosis and hard transepithelial transport” is one of the major problem that hinders the therapeutic effect of proteins in circulation. After internalization, these nanoparticles are commonly entrapped in endosomes and eventually degraded in lysosomes, which are the terminal enzymatic degradative compartments12. This lysosomal degradation would drastically disrupt the protein integrity, and thus impeded the transport of drugs into blood circulation2. Therefore, lyso-endosomal entrapment is a formidable but often neglected barrier. An innovative and practical strategy utilizing endosomal escape might be desired for oral protein delivery. To date, extensive efforts for endosomal escape have been made on cancer therapies and gene transfer during the past years13-15. However, few researches have been focused on the nanoplatforms combining the endosomal escaping strategy to improve the oral bioavailability of biotherapeutics16. Hemagglutinin-2 (GLFEAIEGFIENGWEGMIDGWYG, HA2) peptide, derived from influenza virus coat, was introduced as a non-toxic fusogenic agent. The protonation of HA2 peptide could induce a conformational change to α-helixes in response to endosomal acidification and destabilize the membrane. The contents were supposed to avoid lysosomal degradation, and then the transepithelial transport of drugs would be further improved17. Therefore, we hypothesized that a well-designed nanocarrier with HA2 peptide might held out a considerable promise for oral delivery of biomacromolecules. Herein, a novel delivery system was constructed with the function of endosomal escape for orally administered protein drugs aiming to protect these drugs from lysosomal degradation and enhance their oral bioavailability. The water in oil in water (W/O/W) double emulsion was used to co-encapsulate hydrophilic insulin (INS, model protein drug) and hydrophobic HA2 peptide (endosomal escape agent) in solid lipid nanoparticles (SLNs). INS HA2-W-SLNs (HA2 peptide loaded in inner aqueous phase of SLNs) and INS HA2-O-SLNs (HA2 peptide mainly loaded in outer oil phase of SLNs) were respectively prepared and supposed to have different release kinetics of the fusogenic agent. The effect of spatial location of HA2 peptide in SLNs was thoroughly investigated via cellular uptake and



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intracellular trafficking. Furthermore, transepithelial transport efficiency of formulations was also conducted in vitro as well as ex vivo. Finally, in vivo studies were performed in diabetic rats to elucidate the pharmacological and pharmacokinetics of INS SLNs with HA2 peptide after oral administration. 2. Materials and methods 2.1. Materials Porcine

insulin

was

gained

from

Wanbang

Bio-Chemical

Co.,

Ltd.

GLFEAIEGFIENGWEGMIDGWYG (HA2) peptide was gained from Chinese Peptide Co., Ltd. Soybean lecithin was supplied by Taiwei Pharmaceutical Co., Ltd. Glycerol tripalmitate was gained from Alfa Aesar Co., Ltd. Pluronic F68 was gained from Nanjing Weier Chemical Co., Ltd. Stearic acid was gained from Huzhou Zhanwang Pharmaceutical Co., Ltd. Fluorescein isothiocyanate (FITC) and Tetramethyl rhodamin isothiocyanate (TRITC) were gained from Sigma-Aldrich. Rabbit anti-Rab5 and mouse anti-Rab7 antibodies were obtained from Abcam. Lyso-Tracker Red was purchased from Invitrogen. Other chemicals were of analytic grade. 2.2. Preparation and characterization of SLNs Insulin loaded SLNs (INS SLNs) as well as the two kinds of HA2 peptide and insulin dual-loaded SLNs were produced by micelle-double emulsion method18-19. As for the preparation of INS SLNs, 5 mg insulin and 25 mg sodium cholate were dissolved in pH 2 hydrochloric acid (HCl) solution. Then 1 ml dichloromethane containing 15 mg soybean lecithin, 15 mg tripalmitin and 1 mg stearic acid was added. The mixture was emulsified by probe sonication for each ultrasound 5 s, interval 5 s at 80 W within 2 min. 4 ml pluronic F68 solution (2%, w/v) was then added and sonicated in the same way. After eliminating organic solvent by evaporation for 30 min at 37 °C, this emulsion was diluted with pluronic F68 solution. INS HA2-W-SLNs or INS HA2-O-SLNs were prepared by using the aforementioned method, besides that HA2 peptide were added in the inner aqueous phase (INS HA2-W-SLNs) or oil phase of mixed solvents (dichloromethane: methanol 9:1) (INS HA2-O-SLNs). Fluorescent-labeled SLNs were prepared using FITC-labeled insulin (FITC-INS) in the method as mentioned above7, 20-21. Meanwhile, TRITC and FITC were used to fluorescently label INS and HA2 peptide, respectively. The structures of the different SLNs were visualized under confocal laser scanning microscope (CLSM, Live 5 DUO, Carl Zeiss, Jena, Germany). Various SLNs were characterized by Malvern Zetasize NanoZS90. And the size distribution was performed by Nanosight LM10. Transmission Electron Microscope (TEM, Hitachi H-600, Tokyo, Japan) was applied to observe the morphologies of SLNs22. To evaluate the entrapment efficiency (EE %) and drug loading (DL %) of insulin and HA2 peptide19, SLNs were dispersed in pH 2 HCl solutions and centrifuged at 13,000 rpm for 30


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min. The amount of insulin and HA2 peptide in supernatants were quantified by a reverse-phase high performance liquid chromatography (HPLC) (Agilent 1200 series and Alltech, respectively) method and free FITC-INS was quantified by multimode reader10. 2.3. The colloid stability of SLNs The colloidal stability of the SLNs was investigated by incubating SLNs with simulated gastric fluid (SGF, with and without pepsin (0.32%, w/v)) and simulated intestinal fluid (SIF, with and without trypsin (1%, w/v)) at 37 °C23. Furthermore, SGF was pre-neutralized by sodium bicarbonate solution (3%, w/v)24-25. At predetermined time intervals, the particle size of SLNs was determined. TEM was also used to confirm the results. 2.4. The drug and HA2 peptide release of SLNs The in vitro release of insulin and HA2 peptide from SLNs were investigated using the dialysis method26-28. Briefly, 2 mL SLNs were added in dialysis tubing (MWCO: 100 kDa) and then dialyzed against 1000 ml release medium (pH 2, 5.5 and 6.8) at 37 °C. At predetermined time, the concentrations of insulin and HA2 peptide in dialysis tubing were determined by RP-HPLC. The in vitro release in the pH 2 medium neutralized by sodium bicarbonate solution was also performed. 2.5. Cell experiments 2.5.1.

Cell culture and cytotoxicity assay

Human colon carcinoma Caco-2 cells were obtained from Institute of Biochemistry and Cell Biology (Shanghai, China) and cultured as the method described before10, 29. The in vitro cytotoxicity of SLNs was evaluated. In brief, Caco-2 cells were incubated with SLNs for 3 h and were applied to Methylthiazolyl tetrazolium (MTT) assay10. Hank’s balanced salt solution (HBSS) solution treated group was used as control group. 2.5.2.

In vitro cellular uptake

2.5.2.1. Flow cytometry method The cellular uptake of SLNs was quantified by flow cytometry30. In brief, Caco-2 cells were seeded in 12-well plates for 5 days. Then Caco-2 cells were incubated with fluorescence-labeled SLNs (FITC-insulin, 100 µg/mL) at 37°C for 3 h. Afterwards, cells were trypsinized and redispersed in cold PBS, and then analyzed by flow cytometry analysis (CYTOMICS FC500, Beckman Coulter, Miami, FL, USA). 2.5.2.2. The endocytic mechanism of SLNs To investigate the endocytosis mechanism of SLNs, the inhibitors of metabolism (5 mM Sodium azide), clathrin-mediated endocytosis (30 µM chlorpromazine), caveolae-mediated



endocytosis (500 nM filipin) and micropinocytosis (12 µg/ ml amiloride) were incubated with Caco-2 cells at 37°C for 30 min31-32. Afterwards, cells were incubated with SLNs and same amount of inhibitors for 3 h. The internalization was analyzed by flow cytometry. 2.5.2.3. ELISA method ELISA method was selected to test the internalization of insulin with activity in cells. Caco-2 cells were incubated with various SLNs suspensions (insulin, 100 µg/mL) at 37 °C for 3 h. Then, cell extracts containing active insulin were obtained by several freeze-thaw cycles. The concentration of active insulin in supernatants of cell extracts was measured by porcine insulin ELISA kit (R&D System, Inc., MN, USA)33 and calibrated by BCA assay kit (KeyGen Biotech Co., Ltd., Nanjing, China). 2.5.3.

The intracellular endo-lysosomal trafficking of SLNs

2.5.3.1. The colocalization of SLNs in endosomes and lysosomes The localization of SLNs with different endosomes (early endosome and late endosome) was analyzed using immunofluorescence staining10. Briefly, cells were then incubated with SLNs at 37 °C for 1 h. After incubation time, cells were rinsed by PBS, fixed with 4 % paraformaldehyde for 15 min, permeabilized by 1 % Triton X-100 for 10 min and blocked with 5 % normal goat serum for 1 h. Then primary rabbit anti-Rab5 and mouse anti-Rab7 antibodies were added separately at 4 °C overnight. The secondary antibodies 594-labeled Goat Anti-Rabbit and Anti-Mouse IgG were incubated with cells at 37 °C for 2 h. Additionally, to observe the localization of SLNs with lysosomes, Caco-2 cells were incubated with 50 nM Lyso-Tracker probe at 37 °C for 30 min. DAPI was used to stain cell nuclei before CLSM observation10. 2.5.3.2. The hemolysis assay To determine the pH-sensitive amphiphilic endosomal escape of various SLNs, hemolytic assay was used as previous studies34-35. Briefly, red blood cells (RBCs) obtained from Sprague-Dawley (SD) rats were diluted 1:50 in PBS at pH 7.4, pH 6.5, and pH 5.5. Then INS SLNs, INS HA2-W-SLNs, INS HA2-O-SLNs, HA2 (HA2, 30 µg/ml) and Triton X-100 (1%, w/v) were incubated with the RBC solutions at 37 °C for 2 h. Multimode reader was applied to detect the absorbance of supernatant at 540 nm. 1% Triton X-100 was used as the reference for 100% hemolytic activity. 2.5.4. The transepithelial transport study The transepithelial transport study of tested samples was investigated on the Caco-2 monolayers seeded on Transwell inserts36. Cell monolayers were first equilibrated at 37 °C by HBSS. After equilibration, the cell monolayers were incubated with INS SLNs, INS


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HA2-W-SLNs, INS HA2-O-SLNs and free insulin (insulin, 100 µg/mL) for 6 h at 37 °C. The amount of permeated insulin in the basolateral chambers was measured by ELISA kit. The transepithelial electrical resistant (TEER) values were measured before and after incubation to explore the integrity of cell monolayer. 2.6. The ex vivo ligated intestinal loop assay The permeation of insulin in intestinal loop was evaluated by ex vivo ligated intestinal loop assay37. The animal study protocol was approved by the Institutional Animal Care and Use Committee of Sichuan University. Male SD rats (220–250 g) were sacrificed. Then 2 cm sections of the duodenum, jejunum, ileum were obtained, carefully washed and ligated at both end. Then the suspensions of SLNs (pH 5.5 or pH 6.8) were injected into the loops. And the sections were incubated with pH 7.4 medium at 37°C for 2 h. The amount of permeated insulin in medium was determined by ELISA kits. 2.7. Pharmacological and pharmacokinetics studies The pharmacological and pharmacokinetics studies of various SLNs were performed on diabetic rats38. The male SD rats (220-250 g) were injected by streptozotocin (70 mg/kg) solution to induce diabetes. Rats with the fasting blood glucose above 16 mM were diabetic models. Prior to the experiments, rats were fasted overnight with the supplement of water. Before oral administration of SLNs, 0.5 ml sodium bicarbonate solution (3%, w/v) was orally administrated to neutralize the gastric acid. Then, free insulin solution (50 IU/kg), INS SLNs (50 IU/kg), INS HA2-W-SLNs and INS HA2-O-SLNs (50 IU/kg) were intragastrically administrated. And insulin solution (5 IU/kg) was subcutaneously administrated. At different time intervals, blood glucose levels of rats were determined by a glucose meter. Meanwhile, plasma insulin levels were measured by ELISA kit. The pharmacological availability (PA %) and relative bioavailability (FR %) to subcutaneous injection were calculated as following formulas:

PA(%) =

‫ܥܣܣ‬௢௥௔௟ × ‫݁ݏ݋ܦ‬௦.௖. × 100 ‫ܥܣܣ‬௦.௖. × ‫݁ݏ݋ܦ‬௢௥௔௟

FR(%) =

‫ܥܷܣ‬௢௥௔௟ × ‫݁ݏ݋ܦ‬௦.௖. × 100 ‫ܥܷܣ‬௦.௖. × ‫݁ݏ݋ܦ‬௢௥௔௟

2.8. Statistical analyses All experiments were performed in triplicate unless otherwise stated and presented as mean ± standard deviation. Statistical analyses of the data were conducted with SPSS program 16.0 and two-tail Student's t test. Statistical significance was defined when P < 0.05. 3. Result and discussions 3.1. Preparation and characterization of SLNs



SLNs were prepared by W/O/W double emulsion strategy, which has been commonly used to loaded hydrophobic and hydrophilic macromolecules18, 39. The primary W/O emulsion was firstly obtained and the emulsion was then emulsified in a continuous aqueous phase to form the double emulsion, which contained insulin in the inner aqueous phase, lipid matrix in oil phase and pluronic F68 as emulsifier in the outer aqueous phase. Finally, the evaporation of the solvent from the W/O/W emulsion allowed the formation of SLNs40. The combined use of soybean lecithin, tripalmitin and stearic acid has been previously proven to increase the crystallinity of nanoparticles, which could prevent the leakage of the incorporated insulin (Figure S1)18. Meanwhile, sodium cholate41, an amphipathic biosurfactant, was introduced to encapsulate hydrophilic insulin in internal aqueous phase to further improve the entrapment efficiency and provide the sufficient protection for drugs via the hydrophobic effect between the amphipathic biosurfactant and peptides18, 42. The formulation and preparation processes were optimized (Table S1). HA2 peptide contained SLNs were prepared by loading HA2 peptide in internal aqueous phase or oil phase (INS HA2-W-SLNs or INS HA2-O-SLNs) (Figure 1A).For preparation of INS HA2-O-SLNs, mixed organic solvent was used to increase the solubility of HA2 peptide, achieving the successful entrapment of HA2 peptide mainly in outer lipid shell. The characterizations of the obtained SLNs were presented in Table 1. Small PDI index (under 0.3) indicated the stability of various SLNs in terms of narrow size distribution. And the mean particle sizes of the formulated SLNs were in a range of 150 nm to 170 nm (Table 1, Figure 1B). Excitingly, The optimal SLNs exhibited high insulin entrapment efficiency (over 97 %) and drug loading efficiency (over 7%), indicating that introducing appropriate noncovalent interactions (hydrophobic effect, hydrogen bonding, π-effects, etc.) and preparation methods could efficiently solve the contradiction between the hydrophobic lipid matrix and hydrophilic proteins42-44. Meanwhile, both INS HA2-W-SLNs and INS HA2-O-SLNs showed high HA2 peptide entrapment efficiency (over 75%), demonstrating the capacity of the vehicles encapsulating both hydrophobic and hydrophilic agents. Insulin loaded SLNs (INS SLNs) were negatively surface charged (-9.2 mV), while insulin and HA2 peptide loaded SLNs (INS HA2-O-SLNs, INS HA2-W-SLNs) showed even more negative surface charge (-16.1 mV, -13.1 mV) due to the existence of anionic HA2 peptide45. The different surface charges between INS HA2-O-SLNs and INS HA2-W-SLNs suggested that HA2 peptide was mainly installed in different layers of the particles. In addition, the confocal images of TRITC-insulin and FITC-HA2 peptide dual-loaded SLNs also confirmed the results (Figure S2). Moreover, the preparation technique avoided the high cost of grafting HA2 peptide either with drugs46 or nanocarriers47. The characterizations of the obtained FITC-insulin loaded SLNs were shown in Table 1, which was similar to that of insulin-loaded SLNs. The TEM images showed that the morphology of all SLNs was spherical in shape (Figure 1C).


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Table 1. Characterization of various insulin-loaded SLNs and FITC-insulin-loaded SLNs prepared by the optimized formulation. Data shown as mean ± SD (n = 3). Samples

Size(nm)

INS SLN

PDI

Zeta (mV)

EE Insulin (%)

EE HA2 (%)

DL (%)

171.2±1.6

0.23±0.02

-9.2±0.2

97.93±0.13

-

8.03±0.19

INS HA2-W-SLN

148.8±4.2

0.17 ±0.02

-13.1±1.3

98.90±0.35

75.97±0.65

7.23±0.26

INS HA2-O-SLN

161.6±3.7

0.25±0.03

-16.1±0.5

98.16±0.80

92.67±1.11

7.52±0.31

FITC-INS SLN

172.4±1.5

0.22±0.03

-8.4±1.1

96.60±0.33

-

7.91±0.15

FITC-INS HA2-W-SLN

157.8±3.1

0.24±0.02

-11.0±0.3

97.64±0.15

78.64±0..91

7.42±0.26

FITC-INS HA2-O-SLN

160.2±2.0

0.26±0.03

-14.6±0.9

97.60±0.26

90.83±0.76

7.71±0.24

3.2. The colloid stability of SLNs For orally delivered NPs, it is of importance to overcome the harsh environment of GIT before NPs intruding the epithelia cells. Thus, the evaluation of the colloid stability of SLNs in simulated GI fluid (SGF and SIF) is essential. The aggregation of SLNs was observed in SGF, whereas SLNs remained unchanged in neutralized SGF (Figure 2A-B, Figure S3A-B). Various SLNs presented no significant aggregation or disintegration in SIF with or without trypsin (1%, w/v) (Figure 2C, Figure S3C). Additionally, regular and smooth morphology of particles was observed when SLNs were dispersed in enzyme-containing neutralized SGF and SIF (Figure 2D), suggesting that the colloidal stability of SLNs would not be affected by the main proteases in GIT. However, SLNs in SGF could be observed aggregation and morphological change (Figure 2D (b)). In the present study, before oral administration of SLNs, sodium bicarbonate solution was intragastrically administrated to neutralize the gastric acid following previous reports24-25. In clinical setting, enteric-coated gelatin capsule or tablets could also be utilized to prevent the exposure of SLNs in acidic gastric fluids.



Figure 1. (A) Schematic diagram of SLNs. (B) The size distribution of INS SLNs (a), INS HA2-W-SLNs (b) and INS HA2-O-SLNs (c) performed by Malvern NanoSight LM10 system. (C) TEM images of INS SLNs (a), INS HA2-W-SLNs (b) and INS HA2-O-SLNs (c). Scale bar: 100 nm.


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Figure 2. Variation in particle size of insulin-loaded SLNs following incubation in (A) SGF with 0.32% pepsin, (B) neutralized SGF with 0.32% pepsin, (C) SIF with 1% trypsin at predetermined time intervals (mean ± S.D., n=3). * p < 0.05 versus INS SLNs at 0 h; # p < 0.05 versus INS HA2-W-SLNs at 0 h; & p < 0.05 versus INS HA2-O-SLNs at 0 h. (D) TEM images of INS HA2-O-SLNs (a), INS HA2-O-SLNs in SGF with 0.32% pepsin (b), INS HA2-O-SLNs in neutralized SGF with 0.32% pepsin (c) and INS HA2-O-SLNs in SIF with 1% trypsin (d). Scale bar: 100 nm.

3.3. The drug and HA2 peptide release of SLNs The in vitro release profiles of insulin and HA2 peptide in pH 2 buffer is shown in Figure S4. Approximately 40% insulin and 65% HA2 peptide released from INS HA2-O-SLNs within 2 h. While in neutralized medium, both HA2 peptide and insulin showed sustained release from SLNs. Moreover, the release rate of HA2 peptide from INS HA2-W-SLNs was significantly lower than that from INS HA2-O-SLNs. Then, we studied insulin and HA2 peptide release in pH 5.5 and pH 6.8 medium, which mimicked the pH environments in the acidic endosomes48 and small intestine18, respectively. INS HA2-O-SLNs and INS HA2-W-SLNs showed different trends of release for insulin and HA2 peptide. The release profile of insulin from INS HA2-W-SLNs was more sustained compared with that of INS HA2-O-SLNs and INS SLNs. After incubation for 12 h, approximately 55% and 35% encapsulated insulin were released from the INS HA2-W-SLNs in pH 6.8 and pH 5.5 buffer respectively, which was significantly lower compared with that from the INS SLNs and INS HA2-O-SLNs (~70% and ~50%, p < 0.05) (Figure 3A and B).



Interestingly, the release at pH 5.5 was lower than that at pH 6.8 for all of the samples. As insulin (isoelectric point = 5.3) in media at pH 5.5 was dominantly in nonionic form, the solubility at pH 5.5 was much lower than that at pH 6.8. Therefore, the decrease of release rate at pH 5.5 might be due to the low solubility of insulin49-50. Additionally, a much faster release rate of HA2 peptide located in external oil phase (INS HA2-O-SLNs) was also observed compared with that of HA2 peptide located in internal aqueous phase (INS HA2-W-SLNs) at pH 5.5 (Figure 3C), which was similar with the insulin release because of the interaction of HA2 peptide and insulin in aqueous phase. Similar phenomenon was also observed in pH 6.8 (Figure 3D). This result demonstrated that spatial location for HA2 peptide could greatly influence the release rate. Therefore, with a much faster release rate of HA2 peptide loaded in external oil phase, instead of internal aqueous phase, INS HA2-O-SLNs was expected to acquire a desirable ability of rapid endosomal escape.

Figure 3. Accumulation release profiles of insulin against PBS at (A) pH 5.5 and (B) pH 6.8 (mean ± S.D., n=3). * p < 0.05 versus INS HA2-W-SLNs of INS HA2 SLNs; # p < 0.05 versus INS HA2-W-SLNs of INS HA2-O-SLNs. Accumulation release profiles of HA2 peptide against PBS at (C) pH 5.5 and (D) pH 6.8 (mean ± S.D., n=3). ^ p < 0.05 versus INS HA2-W-SLNs of INS HA2-O-SLNs. 3.4. Cell experiments 3.4.1.

Caco-2 cell viability study

Caco-2 cells were used as the cell model for the cell experiments due to the advantage of


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perfectly mimicking the enterocytes of intestinal gut. The biocompatibility of SLNs was evaluated by MTT assay. As the viability of both insulin and FITC-labeled insulin encapsulated in SLNs was range from 80% to 120%, all SLNs at test concentration exhibited no cytotoxicity (Figure S5). 3.4.2.

Cellular uptake

To investigate whether the addition of HA2 peptide could affect cellular uptake of SLNs, flow cytometry was utilized to quantify the amount of fluorescence-labeled SLNs internalized into cells. Interestingly, as shown in figure 4A, the detected fluorescent intensity showed no significant difference among all samples, indicating that the endocytosis was not be influenced by addition of HA2 peptide. Furthermore, the endocytic mechanism of various SLNs was evaluated. All tested formulations exhibited obvious reduction of cellular uptake in the presence of sodium azide, chlorpromazine and amiloride (Figure 4B), suggesting that clathrin-mediated endocytosis was the endocytic mechanism of SLNs involved. Meanwhile, the retained active insulin after cellular internalization was determined by ELISA assay. The activity of proteins with fragile structures was easily disrupted under harsh conditions51-52. During the transport processes, the internalized insulin might lose biological activity due to the complex environment within cells33. ELISA method used in our study proved that insulin contained in all samples could retain partly biological activity after cellular internalization. More excitingly, INS HA2-O-SLNs displayed the greatest insulin activity (2.64 folds, P < 0.05) compared with INS SLNs and INS HA2-W-SLNs, which exhibited similar amounts of active insulin in cells (Figure 4C). These results indicated that HA2 peptide with the appropriate application played a crucial role in protecting biological activity of insulin though it did not elevate the uptake of SLNs. 3.4.3.

The endosomal escape of SLNs

SLNs have been confirmed to involve in clathrin-mediated endocytosis. After the sequential transport through early and late endosomes, SLNs transported through an intracellular endolysosomal pathway will enable the biotherapeutics delivered to unique enzymatic degradative compartments named lysosomes53. Rab GTPases play a vital role on vesicular transport in endolysosomal pathway54. Rab5 and Rab7 are expressed in early endosomes and late endosomes, respectively55-56. As expected, the colocalization of FITC-labeled INS SLNs, INS HA2-W-SLNs and INS HA2-O-SLNs with early endosome compartments (Rab5) was observed (Figure 4D), suggesting that all formulations were involved in early endosomes. It was reported that after endocytosis, most nanoparticles would first be transported to early endosomes and then they could be trafficked to other organelles57-58. Interestingly, INS HA2-O-SLNs, showing faster release of HA2 peptide, displayed less colocalization with late endosomes and lysosomes. While INS SLNs and INS



HA2-W-SLNs significantly overlaid with the late endosomes and lysosomes. Thus, the results indicated the crucial role of HA2 peptide on the endosomal escape of cargoes by destabilizing the membrane of endosomes. For INS HA2-W-SLNs, however, HA2 peptide and insulin would be exposed in late endosomes or lysosomes simultaneously after degradation of the lipid materials19, 59, resulting in the entrapment and degradation of drugs in lysosomes. In accordance with the in vitro cellular uptake assays, INS HA2-O-SLNS demonstrated the highest efficiency of endosomal escape by the interactions between HA2 and membrane of late endosomes, protecting insulin from the transport to lysosomes. Meanwhile, the mechanism of endosomal escape of SLNs was further investigated using hemolytic assay34-35. It was demonstrated that the relative hemolytic activity of INS HA2-O-SLNs increased with the acidification of maturing endosomes (Figure 5A). The hemolysis of INS HA2-O-SLNs is 15.53% and 52.61% at pH 6.5 and pH 5.5, respectively, suggesting the excellent ability of INS HA2-O-SLNs to interact with late endosomes. While INS HA2-O-SLNs exhibited low hemolytic activity (2.85%) at pH 7.4, indicating the low toxicity on cells. In contrast, INS HA2-W-SLNs presented relative low pH-sensitive amphiphilic endosomal escape. The hemolytic activity of INS SLNs did not increase with the acidification of endosomes. This result was consistent with the intracellular trafficking of various SLNs observed under CLSM, and INS HA2-O-SLNs were confirmed to display the pH-sensitive amphiphilic endosomal escape.


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Figure 4. (A) Flow cytometry quantification of internalization of FITC-labeled INS SLNs, INS HA2-W-SLNs and INS HA2-O-SLNs (mean ± S.D., n=3). (B) The endocytic mechanism of SLNs in term of energy dependence, clathrin-mediated endocytosis, caveolae-mediated endocytosis and micropinocytosis. * p < 0.05 versus control group of INS SLNs; & p < 0.05 versus control group of INS HA2-W-SLNs; # p < 0.05 versus control group of INS HA2-O-SLNs. (C) ELISA quantification of INS SLNs, INS HA2-W-SLNs and INS HA2-O-SLNs on Caco-2 cells at tested insulin concentration (mean ± S.D., n=3).** P < 0.01 vs INS HA2-O-SLNs. (D) CLSM images of SLNs (green) and Caco-2 cells stained with early endosome (red), late endosome (red). (E) CLSM images of SLNs (green) and Caco-2 cells stained with lysosomes (red). Yellow color denotes the overlay of colocalization of red and green fluorescence. DAPI staining of nuclei (blue).



Page 16 of 25

Figure 5. (A) The relative hemolysis efficiency of 1% triton X-100, various insulin loaded SLNs and HA2 peptide at pH 7.4, 6.5 and 5.5, and PBS as control group. * P < 0.05 versus pH 7.4;

#

P < 0.05 versus pH 6.5. (B) The transepithelial transport of insulin and insulin

loaded SLNs. * P < 0.05 versus INS;

#

P< 0.05 versus INS SLNs;

&

P < 0.05 versus INS

HA2-W-SLNs. (C) TEER value of Caco-2 cell monolayers before and after incubation of insulin or SLNs. (D) The permeated amount of transported insulin in ligated intestinal loop assay. * P < 0.05 versus INS SLNs; # P< 0.05 versus INS HA2-W-SLNs;

&

P< 0.05 versus

INS HA2-O-SLNs in duodenum at pH 6.8. 3.4.4.

The transepithelial transport study

INS HA2-O-SLNs have been confirmed to protect insulin from lysosomal degradation by successful endosomal escape. The permeation of insulin through Caco-2 cell monolayer was further investigated. As shown in Figure 5B, after loaded into SLNs, insulin showed the enhanced transepithelial transport efficiency (P < 0.05). Of note, insulin of INS HA2-O-SLNs exhibited the highest accumulated amount in basolateral side, which was respectively 4.30-fold (P < 0.05) and 2.19-fold (P < 0.05) higher than that of free insulin and INS SLNs. By contrast, insulin of INS HA2-W-SLNs merely presented slight increase of transepithelial transport efficiency than that of free insulin and INS SLNs (P < 0.05). Thus, INS HA2-O-SLNs were confirmed to promote the transepithelial transport, which might be due to the protection of insulin during the intracellular trafficking. Meanwhile, TEER value


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remained unchanged after incubation of various SLNs (Figure 5C), indicating that the monolayer remained intact during the transcellular transport of insulin. 3.5. Ex vivo ligated intestinal loop assay The oral intestinal absorption of INS HA2-O-SLNs was further investigated by the ex vivo ligated intestinal loop assay (Figure 5D)37. Consistent with the in vitro behavior of SLNs, INS HA2-O-SLNs showed 1.48-fold higher intestinal transport than INS SLNs and INS HA2-W-SLNs in jejunum. Interestingly, the absorption of SLNs in jejunum was much higher than that in duodenum and ileum, which might be due to the more dense jejunum villi distribution in small intestine60. Moreover, INS HA2-O-SLNs dispersed in pH 5.5 enhanced insulin transport through the duodenum, while no significant increase was observed in INS SLNs or INS HA2-W-SLNs in the same condition. The result suggested that INS HA2-O-SLNs could potentially enhance the oral absorption of insulin in duodenum. However, SLNs might not have enough retention time in duodenum on animal models. The enhanced duodenal absorption of INS HA2-O-SLNs may be weaken by intestinal flow, rapid dilution as well as spreading61.

Figure 6. (A) Blood glucose level (% of initial) and (B) Serum insulin level of rats following administration of various formulations (mean±SD，n=5).* P < 0.05 versus insulin solution. # P < 0.05 versus INS SLNs. & P < 0.05 versus INS HA2-W-SLNs.

3.6. Pharmacological and pharmacokinetics studies The in vivo pharmacological and pharmacokinetics effect of various SLNs were determined on diabetic rats. As shown in figure 6A, subcutaneously injected insulin (5 IU/kg) represented remarkable hypoglycemia effect and the blood glucose level decreased to the minimum at 2 h, while oral administration of insulin (50 IU/kg) failed to reduce the blood glucose level. Oral administration of INS SLNs, INS HA2-W-INS and INS HA2-O-SLNs generated significant



Page 18 of 25

hypoglycemic response (p < 0.05), indicating that bioactivity of insulin encapsulated in SLNs was protected against various pH environment and enzymatic digestion. Excitingly, INS HA2-O-SLNs presented the most excellent hypoglycemic effect with 37% blood glucose decrease at 3 h. The pharmacological availability (PA%) of different samples related to subcutaneous injection was shown in Table 2. INS HA2-O-SLNs demonstrated PA% of 7.32%, which was 4 folds and 1.7 folds higher than free insulin solution and INS HA2-W-SLNs. The serum insulin concentration was shown in Figure 6B. Compared with subcutaneously administrated free insulin, SLNs administration by gavage showed a relatively slower increase in serum insulin concentration. At the dose of 50 IU/kg, INS HA2-O-SLNs showed a significant higher relative bioavailability of 5.47%, which was 3.2-fold higher than free insulin. Therefore, the in vivo-in vitro correlation was confirmed. Taken previous results together, HA2 peptide in INS HA2-O-SLNs could be appropriately released and destabilize the membrane of endosomes to achieve the endosomal escape of insulin. The designed delivery system showed the great potency as a promising strategy to overcome degradation of insulin in lysosomes and further enhanced the oral bioavailability of insulin. Table 2. Pharmacological and pharmacokinetic parameters of insulin formulations in diabetic rat model. Samples

Dose (IU/kg)

AUC (mIU ·h/L)

PA (%)

F (%)

SC insulin

5

278.1±69.2

-

100

oral insulin

50

47.3±6.1

1.83

1.70

INS SLN

50

96.5±10.5

3.40

3.47

INS HA2-W-SLN

50

96.6±23.2

4.33

3.47

INS HA2-O-SLN

50

152.1±12.0

7.32

5.47

AUC: area under the plasma concentration–time curve; PA%: pharmacological bioavailability; F%: relative bioavailability. 4. Conclusion In summary, we rationally developed the functional nanovehicles for the efficient oral absorption of biomacromolecules. The optimized SLNs (INS HA2-O-SLNs) were comprised of a lipid shell containing endosomal escape agent (HA2 peptides) and an aqueous core containing insulin. HA2 peptide in external oil phase (INS HA2-O-SLNs) was demonstrated to possess a much faster release compared with that of HA2 in external aqueous phase (INS HA2-W-SLNs). When exposed to acidic endosomes, the released HA2 peptide helped cargoes escape from degradation in lysosomal pathway by rapidly disrupting the late endosomal


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membranes. In vitro cell experiments confirmed that INS HA2-O-SLNs could retain the highest biological activity of internalized insulin. As demonstrated in the intracellular trafficking of SLNs, INS HA2-O-SLNs displayed much less distribution in late endosomes and lysosomes. Meanwhile, INS HA2-O-SLNs exhibited the highest transepithelial transport efficiency, with 2.19 folds (P < 0.05) and 1.72 folds (P < 0.05) higher intestinal transport as compared to INS SLNs and INS HA2-W-SLNs. Further, the ex vivo ligated intestinal loop assay demonstrated that insulin from INS HA2-O-SLNs exhibited the highest permeation in various regions of intestines. Finally, the developed SLNs were confirmed to increase the serum insulin concentration and generate an excellent hypoglycemic response, suggesting the importance of endosomal escape for orally delivered proteins and peptides. The excellent in vitro-in vivo correlation confirmed the great potency for SLNs with endosomal escape function, and the strategy could be further applied in the oral delivery of other biomacromolecules. Associated Content Supporting Information Physicochemical characterization of SLNs, CLSM images of HA2-O-SLNs and HA2-W-SLNs, accumulation release profiles of insulin and HA2 in pH 2; Caco-2 cell viability treated with various SLNs by MTT assays. Author information *E-mail: [email protected]. Tel/Fax: +86 2885501617. West China School of Pharmacy, Sichuan University, No. 17, Section 3, Southern Renmin Road, Chengdu 610041, China. Notes The authors declare no competing financial interest. Acknowledgement The authors 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).



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TOC


Novel Solid Lipid Nanoparticle with Endosomal Escape Function for

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