Digestive Enzyme Corona Formed in the Gastrointestinal Tract and Its

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Digestive enzyme corona formed in gastrointestinal tract and its impact on epithelial cell uptake of nanoparticles Qiang Peng, Jingying Liu, Ting Zhang, Tian-Xu Zhang, Chao-liang Zhang, and Huiling Mu Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.9b00175 • Publication Date (Web): 20 Mar 2019 Downloaded from http://pubs.acs.org on March 21, 2019

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Digestive enzyme corona formed in gastrointestinal tract and its impact on epithelial cell uptake of nanoparticles Qiang Peng a,b,*, Jingying Liu b, Ting Zhang c, Tian-Xu Zhang a, Chao-Liang Zhang a, Huiling Mu b

a

State Key Laboratory of Oral Diseases, National Clinical Research Center for Oral Diseases, West

China Hospital of Stomatology, Sichuan University, Chengdu 610041, China b Department

of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen,

Copenhagen, DK-2100, Denmark c Department

*

of Pharmacy, West China Hospital, Sichuan University, Chengdu 610041, China.

Corresponding author:

Q. Peng: E-mail: [email protected]; [email protected]

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Abstract The fate of intravenously injected nanoparticles (NPs) is significantly affected by nano-protein interaction and corona formation. However, such interaction between NPs and digestive enzymes occurring in the gastrointestinal tract (GIT) and its impacts on epithelial cell uptake are little known. We synthesized the PHBHHx (Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate))-based cationic NPs (CNPs) and investigated the CNPs-digestive enzymes interaction and its effect on the cellular uptake. The formation of enzyme corona was confirmed by size/zeta potential analysis, morphology, SDSPAGE and enzyme quantification. The cellular uptake of CNPs by Caco-2 cells was significantly reduced upon the formation of enzyme corona. Our findings demonstrate the digestive enzyme corona formation and its inhibited effect on the epithelia cell uptake of CNPs for the first time. Understanding the enzyme corona could offer a new insight into the fate of nanomedicines in the gastrointestinal tract and this understanding would be highly beneficial for guiding future nanomedicine designs.

Keywords: nanomaterials; oral delivery; interfaces; biomacromolecules; protein adsorption; gastrointestinal transit

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1. Introduction Nowadays, several administration routes are available, such as oral administration, intravenous injection, subcutaneous injection and inhalation. Among those, oral administration has been considered as the best route for drug delivery due largely to its great convenience and high patient compliance.1, 2 Nevertheless, this best route is not suitable for many drugs, especially the waterinsoluble, lowly intestinal permeable or/and easily degradable ones. Nanoparticles (NPs) have shown great potentials in facilitating the oral delivery and absorption of various drugs, due to their abilities of improving drugs solubility/dissolution, protecting drugs against enzymatic degradation and enhancing the epithelial cell uptake.3-5 Despite these advantages, one critical issue of NPs-based systems is always ignored, that is the interaction between NPs and digestive enzymes. Recently, the nano-protein interaction and corona formation as well as its effects on the fate of NPs have emerged as a new topic in the area of nanomedicine.6-11 This non-specific interaction is a great challenge for NPs delivery since it changes both physicochemical and biological properties of NPs, such as size, cytotoxicity and biological identity.12-15 More importantly, it changes the receptor recognition and ultimately leads to the loss of targeting capacity and the changes in pharmacokinetic profile and biodistribution.16-19 So far, the reports on nano-serum protein interaction and its various influencing factors, such as material chemistry, NPs size/concentration and individuals have been well documented.20-24 However, the interaction between NPs and digestive enzymes taking place in the gastrointestinal tract (GIT) and its impact on the epithelia cell uptake of NPs has never been reported. On the basis of the verified interaction between NPs and serum proteins, we assume that the orally administered NPs would spontaneously interact with digestive enzymes in the GIT. The formed enzyme corona would also change the physicochemical and biological properties of NPs and ultimately affect the uptake of NPs by intestinal epithelial cells and their transit in the GIT (Scheme 1). As we know, development of oral insulin delivery systems is of great significance and importance. Therefore, we use insulin as the model drug which is loaded in the cationic polymeric NPs (CNPs). The interaction between the CNPs and the representative digestive enzymes (pepsin and pancreatin) and its impact on the epithelial cell uptake of CNPs are investigated for the first time. 3



Scheme 1. Schematic of the formation of digestive enzyme corona in gastrointestinal tract (GIT) and its impact on epithelial cell uptake of cationic nanoparticles.

2. Materials and methods 2.1. Materials Porcine insulin powder was supplied by Wanbang Biopharmaceuticals (Xuzhou, China). Phospholipid (soybean lecithin of injection grade containing >70% phosphatidylcholine) was purchased from Shanghai Tai-wei pharmaceutical Co. Ltd. (Shanghai, China). Octadecylamine, pepsin, pancreatin, coumarin 6 and DMSO were purchased from Sigma-Aldrich (St. Louis, USA). Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx, Mw = 270 kD) containing 15 mol% 3hydroxyhexanoate (3HHx) was kindly donated by Lukang Group (Jining, China). Poloxamer188 (F68) was kindly provided by BASF (China) Co. Ltd. (Shanghai, China). Caco-2 cell line was obtained from American Type Culture Collection (ATCC, VA, USA).

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2.2. Preparation of cationic NPs (CNPs) PHBHHx, a biodegradable and biocompatible biopolyester, was used to prepare the CNPs.25, 26 Briefly, 20 mg PHBHHx was dissolved in 0.5 ml chloroform containing 0.6 mg/ml octadecylamine. The resultant organic solution was mixed with 10 ml aqueous solution containing 0.5% (w/v) F68 followed with probe sonication for 30 seconds at a power of 60%. After sonication, an emulsion was obtained. The CNPs suspension was obtained after the chloroform in the emulsion was removed by rotation evaporation at room temperature for 20 min. When preparing insulin-loaded CNPs, the insulin-phospholipid complex (Ins-PLC) was prepared in advance.27, 28 The Ins-PLC (equivalent to 1 mg insulin) was co-dissolved with 20 mg PHBHHx and the Ins-PLC-CNPs were prepared as above. In addition, a fluorescent molecule coumarin 6-loaded CNPs (C6-CNPs, equivalent to 10 μg/ml of C6) were also prepared as above.

2.3. Size and zeta potential (ZP) measurement The hydrodynamic size and ZP of CNPs were measured using Dynamic Light Scattering (DLS) and Electrophoretic Light Scattering (ELS) technologies (Zetasizer Nano ZS90, Malvern Instruments Ltd, Malvern, UK). The CNPs suspension was diluted by 10-fold using distilled water before size measurement. The particle size was presented as intensity distribution. The size distribution was presented by polydispersity index (PDI).

2.4. Encapsulation efficiency (EE) of Ins-PLC-CNPs The EE of Ins-PLC-CNPs was quantified using HPLC. Briefly, 0.5 ml of Ins-PLC-CNPs was added to the ultrafiltration tubes (30 kD, Millipore) followed by centrifugation (4500 rpm, 10 min). The filtrate was removed and another 0.5 ml Ins-PLC-CNPs was added again. After centrifugation, an aliquot of 20 µl filtrate was injected into HPLC (Thermo Scientific UltiMate 3000 HPLC) to determine the amount of free insulin (Wf). The total amount of insulin (Wt) was calculated according to the added amount. The EE was then calculated by the following equation: EE = (Wt - Wf) / Wt × 100%. In the HPLC analysis, the stationary phase Acclatim RSLC 120 C18 (2.1 × 100 mm, 2.2 µm) 5



was used. The mobile phase consisted of phase A (acetonitrile: water: trifluoroacetic acid = 5: 95: 0.1) and phase B (acetonitrile: water: trifluoroacetic acid = 95: 5: 0.1). The gradient elution at a flow rate of 0.3 ml/min with phase A changing from 25% to 40% was used. The signal was recorded at 214 nm.

2.5. Scanning electron microscopy (SEM) The morphology of CNPs was examined by SEM (InsPECT F, FEI, Netherlands). The NPs suspension was 100-fold diluted with distilled water, one drop of which was placed on a clean glass sheet. After air-drying, the specimen was coated with gold for SEM.

2.6. In vitro release The in vitro release of insulin from Ins-PLC-CNPs was investigated in simulated gastric fluid containing no enzyme (SGF-), simulated intestinal fluid containing no enzyme (SIF-) and cell culture medium DMEM (without serum). Briefly, 0.05 ml Ins-PLC-CNPs was mixed with 0.5 ml of SGF-, SIF- or DMEM and then shaken in a horizontal shaker (100 rpm, 37 oC). At fixed time intervals, the specimens were centrifuged (12 krcf, 5 min) and the insulin remaining in the pellets was quantified using HPLC. The released insulin amount was calculated by the difference between the total and the remaining insulin. The release profile was presented by the percentage of cumulative insulin release.

2.7. Formation and characterization of Ins-PLC-CNPs-digestive enzyme complex The simulated gastric fluid (SGF) containing pepsin or not was presented as SGF+ or SGF-, and the simulated intestinal fluid (SIF) containing pancreatin or not was presented as SIF+ or SIF-. These simulated fluids were prepared according to a previous report. 29 SGF- consisted of 0.2% NaCl (w/v) and HCl (qs) with pH 1.2, and SGF+ contained extra 0.32% pepsin (w/v). SIF- consisted of 0.68% KH2PO4 (w/v) and NaOH (qs) with pH 6.8, and SIF+ contained extra 1% pancreatin (w/v). Ins-PLC-CNPs-digestive enzyme complex was formed by incubating Ins-PLC-CNPs with enzyme solutions. Briefly, Ins-PLC-CNPs was added to SGF+ or SIF+ (volume ratio 1:10) and shaken in a horizontal shaker (100 rpm, 37 oC). At fixed time intervals, the size and ZP of the complex Ins-PLC6


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CNP-Pepsin (Ins-PLC-CNP-Pep) and Ins-PLC-CNP-Pancreatin (Ins-PLC-CNP-Pan) were measured. The size and ZP of Ins-PLC-CNPs in SGF- or SIF- were also measured. The morphology of these complexes was examined by atomic force microscopy (AFM, Model SPM9600, Shimadzu, Japan) according to the previous method.20, 30 Briefly, the complex suspension obtained by incubation for 10 min was centrifuged (250 rcf, 3 min) to get rid of any impurities. The supernatant was collected and further centrifuged (10 krcf, 3 min). The precipitant was washed with 1 mM HCl for Ins-PLC-CNP-Pep and distilled water for Ins-PLC-CNP-Pan for three times. One drop of each resultant suspension was placed on a freshly exfoliated mica sheet. After air-drying, the specimen was examined by AFM using the mode of phase imaging.

2.8. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) The self-assembled Ins-PLC-CNP-Pep and Ins-PLC-CNP-Pan were separated by centrifugation (10 krcf, 5 min), followed by washing for 3 times with SGF- or SIF- to get rid of the softly bonded enzymes. The hard bonded enzymes were separated by 12% SDS-PAGE and the enzyme bands were stained by Coomassie Brilliant Blue.31

2.9. Quantification of adsorbed enzyme The adsorbed enzyme on CNPs was quantified by BCA assay. Briefly, Ins-PLC-CNPs were incubated with SGF+ or SIF+ (100 rpm, 37 oC). At fixed time intervals, the suspension was centrifuged (10 krcf, 5 min) and the non-adsorbed enzyme in the supernatant was determined by BCA assay. The adsorbed enzyme amount was calculated by the difference between the total and the nonadsorbed amount. The enzyme adsorption kinetics of Ins-PLC-CNPs was presented by the adsorbed enzyme amount against the incubation time. In addition, the enzyme adsorption capacity of Ins-PLCCNPs with varied concentrations was also investigated. Briefly, Ins-PLC-CNPs with final concentrations of 0.1, 0.4 and 1.0 mg/ml were incubated with SGF+ or SIF+ for 30 min (100 rpm, 37 oC).

The followings were the same as above.

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2.10. Chemical stability of Ins-PLC-CNPs The chemical stability of insulin loaded in Ins-PLC-CNPs was examined in SGF and SIF. In order to accurately detect the remained insulin, the volume ratio of Ins-PLC-CNPs to SGF/SIF was set at 1:1. For enzyme-free media, 50 µl Ins-PLC-CNPs were mixed with 50 µl SGF- or SIF- and shaken at 37 oC (100 rpm). At predetermined time intervals (0, 10, 20 and 30 min), the remained insulin was quantified by HPLC. For enzyme-contained media, 50 µl Ins-PLC-CNPs were mixed with 50 µl SGF+ or SIF+ and pre-incubated at 37 oC for 5 min to deplete the free insulin and the one adsorbed on particle surface. After pre-incubation, the mixture was shaken at 37 oC (100 rpm). At fixed time intervals (0, 10, 30, 60 and 120 min), the remained insulin was quantified by HPLC. In addition, the chemical stability of insulin solution was also examined in SGF and SIF.

2.11. Cellular uptake Human colon carcinoma Caco-2 cells were used for uptake study according to a previous report.32 Briefly, Caco-2 cells were cultured in DMEM (containing 25 mM glucose, 15% (v/v) fetal bovine serum, 1% (v/v) non-essential amino acid, 2 mM L-glutamine, 100 Ul/ml penicillin and 100 µg/ml streptomycin) at 37 oC and 5% CO2 and the culture medium was changed every other day. The confluent cells were digested with 0.25% trypsin/0.02% EDTA and seeded in 6-well culture plates at a density of 2.5×105 cells/well. After culturing for 12 h, the culture medium was removed and the cells were washed with PBS (pH 7.2) for three times. Fluorescent C6-CNPs and C6-CNPs-Pan (the free and soft-binding pancreatin was removed by centrifugation at 10 krcf for 5 min) were prepared and diluted to 90 or 180 ng/ml (equivalent to C6 concentration) with DMED containing 15% FBS or not. The cells were treated with the above C6-CNPs and C6-CNPs-Pan at 37 oC for 30 min. The cells treated with blank DMEM served as control. Subsequently, the medium was removed and the cells were washed with cold PBS and digested for flow cytometry assay (Cytomics FC 500, Beckman Conlter, USA). The cellular uptake was quantified by the mean fluorescent intensity. In addition, after washing with cold PBS, the cells were fixed with 4% paraformaldehyde and the cellular uptake was also observed by fluorescent microscope. 8


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2.12. Statistics All the experiments were performed in triplicate and the data were presented as mean ± s.d. (standard deviation). The one-way analysis of variance was used to compare the difference between groups, which was considered as statistically significant if the P value was less than 0.05.

3. Results and discussion 3.1. Characterization of Ins-PLC-CNPs

Figure 1. Characterization of Ins-PLC-CNPs. A) Typical size distribution by intensity; B) Typical zeta potential distribution; C) Morphology examined by SEM. Scale bar: 3 µm.

The Ins-PLC-CNPs had a mean hydrodynamic size of 268.6 nm, a positive zeta potential of 26.5 mV and a polydispersity index (PDI) of 0.259. The typical size distribution by intensity and zeta 9



potential distribution of Ins-PLC-CNPs are shown in Figure 1AB. The encapsulation efficiency (EE) of Ins-PLC-CNPs determined by HPLC was up to 90.84%, which is comparable with the high EE of the negatively charged NPs due to the formation of insulin-phospholipid complex (Ins-PLC) 28. The increased lipophilicity of insulin after formation of Ins-PLC enhances its affinity to the hydrophobic material PHBHHx and thus improves its incorporation in PHBHHx-based NPs. In this work, the organic solvents DMSO, acetic acid and chloroform were used for preparing Ins-PLC and NPs, and were eventually removed by lyophilization or rotation evaporation. Our previous reports have shown that the use of these organic solvents would not affect the activity of insulin.25, 28, 33 The morphology of Ins-PLC-CNPs examined by SEM is shown in Figure 1C. Ins-PLC-CNPs are small in size and spherical in shape. The particle size distribution observed from SEM image is narrow and comparable with the PDI measured by DLS.

3.2. In vitro release of Ins-PLC-CNPs According to our experience in the release speed of payload from PHBHHx-based NPs 28, the insulin release from Ins-PLC-CNPs would be quite slow. Therefore, the in vitro release study was conducted for 8 days although the transit time of Ins-PLC-CNPs in GIT would not be so long. The in vitro release was conducted in SGF- (pH 1.2), SIF- (pH 6.8) and DMEM (pH 7.4), respectively. As shown in Figure 2, all release profiles can be divided into the initial burst release stage (the first 8 h) and the sustained release stage (from 8 h to the end). But the release process is varied depending on the release medium. In detail, Ins-PLC-CNPs showed the fastest and the most release in SGF- (the cumulative release within 8 days was up to 78%), with the highest initial burst release (31%). In contrast, the release in SIF- and DMEM was obviously slower and less (24% and 45% were released at day 8 in SIF- and DMEM, respectively), with the lower initial burst release (~21%). It is noteworthy that the low release in SGF- and SIF- in the first 2 h is beneficial to protecting insulin from enzymatic degradation. Due to the possible degradation of insulin by pepsin and pancreatin, it is hard to accurately quantify the release of insulin in enzyme-containing SGF+ or SIF+. It is assumed

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that the release of insulin in enzyme-free media is similar to the release in SGF+ and SIF+ since the protease would have no effects on the polyester PHBHHx, the main composition of CNPs.

Figure 2. In vitro release of Ins-PLC-CNPs in SGF- (simulated gastric fluid without pepsin) and SIF(simulated intestinal fluid without pancreatin) as well as in cell culture medium DMEM. The insert is the magnification of the release profiles in the first 8 h. Data are presented as mean ± sd (n=3).

Generally, the drug loaded by NPs includes two parts: one is adsorbed on the surface of NPs and contributes to the initial burst release; the other is located in the inner space of NPs and contributes to the sustained release. Therefore, the burst release in the first 8 h is attributed to the insulin adsorbed on CNPs surface and the sustained release thereafter is resulted from the insulin loaded in the inner space of CNPs. In this work, the pH of release medium played an important role in Ins-PLC-CNPs release. Insulin has an isoelectric point of 5.35~5.45 and thus can be positively or negatively charged in the media with different pH values.34 When dispersed in SGF- (pH 1.2), the insulin adsorbed on Ins-PLCCNPs surface was positively charged due to the strong acidic environment. Meanwhile, Ins-PLCCNPs itself was also highly positively charged in SGF- (24 mV). As a result of a strong electrostatic repulsive force, the insulin adsorbed on surface was rapidly desorbed, leading to the significantly high burst release of Ins-PLC-CNPs in SGF- (31%). When dispersed in SIF- or DMEM with neutral pH, 11



insulin was negatively charged and Ins-PLC-CNPs itself was almost neutral (4 mV). The absence of electrostatic repulsive force led to a significantly lower burst release (21%). In addition to the burst release, the highest cumulative release in SGF- would also be attributed to the low pH of SGF-. It is known that the acids would accelerate the degradation of polyesters via catalysis of hydrolysis of the ester bonds.35, 36 Therefore, the degradation of PHBHHx would be accelerated in SGF- due to the H+catalyzed hydrolysis and thus led the faster release of insulin from the inner space of CNPs.

3.3. Size and zeta potential (ZP) change upon enzyme adsorption The transit time of a drug in the GIT is usually influenced by many factors, such as dosage form, pathological status, gastrointestinal physiology and food. It is estimated that the gastric emptying time and the small intestinal transit time of a liquid drug is 0.5~2 h and 2~6 h, respectively.37 Herein, in order to simply simulate the transit of CNPs suspension in GIT, the total incubation time was set as 30 and 120 min in SGF and SIF, respectively. Also, pepsin and pancreatin were used as the typical enzymes although there are many other enzymes in the GIT. When incubating Ins-PLC-CNPs in SGF(pH 1.2, pepsin free), there was no substantial change in size or ZP (Figure 3A). However, an obvious size increase and a slight ZP increase could be observed when incubating in SGF+ (pH 1.2, pepsin contained) due to the adsorption of pepsin on Ins-PLC-CNPs (Figure 3B). The particle size increased to 382.7, 345.9 and 366.0 nm at 10, 20 and 30 min, respectively. The ZP increased from 26.5 mV to a stable value of 33 mV throughout the experiment. In SIF- (pH 6.8, pancreatin free), particle size had no change but ZP decreased to ~4 mV due to the neutral buffer environment of SIF- (Figure 3C). The most obvious changes in both size and ZP could be found when incubating Ins-PLC-CNPs in SIF+ (pH 6.8, pancreatin contained). As shown in Figure 3D, the particle size remarkably increased in SIF+ with a little fluctuation at different time intervals. Moreover, the size increase in SIF+ is more remarkable than that in SGF+. Interestingly, the ZP of Ins-PLC-CNPs converted from positive to negative charge after incubation in SIF+, suggesting that a large amount of acidic pancreatic enzyme (such as pancreatic lipase) were adsorbed on Ins-PLC-CNPs.

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Figure 3. The change of hydrodynamic size and zeta potential (ZP) of Ins-PLC-CNPs when incubating in simulated gastrointestinal fluids A) SGF-; B) SGF+; C) SIF- and D) SIF+. The symbol “-” means the fluids contain no enzyme; SGF+ and SIF+ means the fluid contains pepsin and pancreatin, respectively. Data are presented as mean ± sd (n=3). Statistical significance: * p < 0.05, ** p < 0.01, *** p < 0.001.

As we know, the interaction of NPs with proteins is a dynamic process, in which, protein adsorption and desorption occurred simultaneously.38, 39 In addition, some associated protein molecules are tightly adsorbed (hard corona) and others are adsorbed loose (soft corona).12 All of these can lead to the fluctuation of hydrodynamic size even if the amount of associated protein is the same. The size fluctuation was also observed in our previous works.12, 21 Thus, the larger hydrodynamic size of CNPs-protein complex at certain time points cannot definitely indicate more protein adsorption. Nevertheless, the significant size increase of Ins-PLC-CNPs upon incubation in SGF+ and SIF+ can definitely indicate the enzyme adsorption since the size has little change in SGF- or SIF- (Figure 3). In 13



other words, Ins-PLC-CNPs would become much larger in size after oral administration due to enzyme adsorption. These changes provide a kind of evidence for the nonspecific interaction of InsPLC-CNPs with digestive enzymes.

3.4. Morphology change of Ins-PLC-CNPs upon enzyme adsorption

Figure 4. AFM images of A) Ins-PLC-CNPs-pepsin complex and B) Ins-PLC-CNPs-pancreatin complex. Scale bar: 1 µm. The enzyme corona is clearly presented surrounding the particles.

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In addition to size and ZP, enzyme adsorption would also lead to a significant morphology change of Ins-PLC-CNPs. The original Ins-PLC-CNPs presented a clean and clear surface (Figure 1C). Interestingly, the Ins-PLC-CNPs-enzyme complexes look like the fried eggs under AFM. As shown in Figure 4A, Ins-PLC-CNPs were tightly associated with pepsin upon incubation in SGF+, forming a clear pepsin corona. Also, the pancreatin corona surrounding Ins-PLC-CNPs after incubation in SIF+ can be clearly observed from Figure 4B. These AFM images provide the visualized evidence for the interaction between Ins-PLC-CNPs and enzymes.

3.5. SDS-PAGE In order to further confirm the adsorption of pepsin and pancreatin on Ins-PLC-CNPs, we used 12% SDS-PAGE to separate the associated enzymes. As shown in Figure 5A, pepsin and Ins-PLC-CNPsPep samples almost show the same bands. In contrast, pancreatin and Ins-PLC-CNPs-Pan samples present quite different bands (Figure 5B). In the pancreatin solution (Lane: Pan), the abundance of the protein bands a, b and d is the highest and band c shows a significantly lower abundance. Interestingly, Ins-PLC-CNPs hardly adsorbed the enzymes a or b but adsorbed the enzyme d and selectively accumulated the enzyme c (Lane: 10, 30, 60 and 120). These results further confirm the interaction between Ins-PLC-CNPs and digestive enzymes and indicate the selective adsorption of enzyme. SDS-PAGE is a commonly used tool to analyze the proteins adsorbed on NPs. In consistent with our previous reports,20, 21 the SDS-PAGE results in this work (Figure 5) support the SEM observations in Figure 4 and provide more evidence for the association of pepsin and pancreatin with Ins-PLCCNPs. Moreover, Ins-PLC-CNPs selectively accumulated a certain enzyme (band c) though its content in the original pancreatin solution is relatively low. In other words, Ins-PLC-CNPs can be used to condense this low abundant enzyme from pancreatin. It is assumed that enzyme c might be an acidic enzyme (for example, pancreatic lipase, whose isoelectric point is around 5.540 ), which is negatively charged in neutral pH solution and the strong electrostatic attraction force significantly enhance its association with the positively charged Ins-PLC-CNPs. 15



Figure 5. 12% SDS-PAGE and Coomassie Brilliant Blue staining of the enzymes adsorbed on InsPLC-CNPs. A) The enzyme bands of Ins-PLC-CNPs-pepsin complex obtained at 10, 20 and 30 min post-incubation (Lane: 10, 20, 30), pepsin solution (Lane: Pep) and plain Ins-PLC-CNPs (Lane: NP). B)

The enzyme bands of Ins-PLC-CNPs-pancreatin complex obtained at 10, 30, 60 and 120 min

post-incubation (Lane: 10, 30, 60, 120), plain Ins-PLC-CNPs (Lane: NP) and pancreatin solution (Lane: Pan). Arrows a-d indicate four enzyme bands of pancreatin.

3.6. Enzyme adsorption kinetics and adsorption capacity The spontaneous adsorption of pepsin and pancreatin on Ins-PLC-CNPs has been proved as above. Herein, we quantified the adsorbed enzyme and plotted the adsorption kinetics of Ins-PLC-CNPs at 0.1 mg/ml. The amount of adsorbed pepsin for 0.03 ml Ins-PLC-CNPs was 17.10 µg at 10 min, 18.5 16


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µg at 20 min and increased to 36.8 µg at 30 min, indicating the rapid and significant pepsin adsorption on Ins-PLC-CNPs (Figure 6A). In comparison with pepsin, pancreatin showed a much stronger affinity to Ins-PLC-CNPs. The amount of adsorbed pancreatin for 0.03 ml Ins-PLC-CNPs was 151.1 µg at 10 min and increased to197 µg at 60 min and maintained at this level thereafter. One important reason for this result is that a large amount of acidic pancreatic enzymes (such as pancreatic lipase) which were negatively charged in SIF (pH 6.8) were adsorbed on Ins-PLC-CNPs due to the electrostatic attraction force. In contrast, pepsin (isoelectric point: 1.0~2.5) was almost neutral in SGF (pH 1.2) and thus had little electrostatic attraction interaction with Ins-PLC-CNPs. Another reason is the larger quantity of enzyme in SIF+ (1%, w/v) than pepsin in SGF+ (0.32%, w/v). It is interesting to find that the size of Ins-PLC-CNPs-pepsin was smaller at 30 min post-incubation than that at 10 min (Figure 3B) but the adsorbed pepsin was more at 30 min (Figure 6A). Similarly, the size of Ins-PLC-CNPs-pancreatin was smaller at 120 min than that at 10 min (Figure 3D) but more pancreatin was adsorbed at 120 min (Figure 6A). The uncorrelation between size and associated protein amount of NP-protein complex were also observed in our previous works.12, 21 We have stated above that a certain amount of protein can form both soft corona (which leads to a larger size) and hard corona (which leads to a relatively smaller size). Furthermore, protein corona formation is a dynamic process, in which the soft corona can convert to the hard one.9 Hence, the larger hydrodynamic size of CNP-protein complex at certain time points cannot definitely imply more protein adsorption. Generally, NPs with higher concentrations would adsorb more total amount of enzymes. But this situation is different from adsorption capacity. The enzyme adsorption capacity of Ins-PLC-CNPs indicates the enzyme amount adsorbed on every milligram of Ins-PLC-CNPs (enzyme adsorption capacity = adsorbed enzyme amount (µg) / added CNPs amount (mg)) and relates tightly to the exposure concentration of NPs. As shown in Figure 6B, the Ins-PLC-CNPs with lower concentrations present the significantly higher adsorption capacity for both pepsin and pancreatin, which is consistent with the result reported previously.20, 21 This result implies that at a lower Ins-PLC-CNPs

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concentration, the enzyme amount adsorbed on each individual particle surface would be more and thus may have more significant impacts on gastrointestinal transit of Ins-PLC-CNPs.

Figure 6. Quantification of pepsin and pancreatin adsorption on Ins-PLC-CNPs. A) Enzyme adsorption kinetics of Ins-PLC-CNPs (0.1 mg/ml); B) Enzyme adsorption capacity of Ins-PLC-CNPs at 0.1, 0.4 and 1.0 mg/ml. Data are presented as mean ± sd (n=3). Statistical significance: * p < 0.05, ** p < 0.01, *** p < 0.001. 18


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3.7. Chemical stability of Ins-PLC-CNPs

Figure 7. Chemical stability of insulin solution (Ins-Sol) and Ins-PLC-CNPs in simulated gastrointestinal fluids A) SGF-; B) SGF+; C) SIF- and D) SIF+. The symbol “-” means the fluids contain no enzyme; SGF+ and SIF+ means the fluid contains pepsin and pancreatin, respectively. Data are presented as mean ± sd (n=3).

The enzyme-mediated chemical degradation of insulin in GIT is one of the greatest challenges for oral insulin.41 Herein, we examined the protective effect of CNPs on the insulin loaded in Ins-PLCCNPs upon enzyme adsorption. In order to eliminate the influence of varied burst release of Ins-PLCCNPs in different media (Figure 2) and to accurately evaluate the protective effect of CNPs on the insulin loaded in the particle core, we depleted the free and adsorbed insulin by pre-incubation of Ins19



PLC-CNPs with enzyme for 5 min. As shown in Figure 7, both insulin solution and Ins-PLC-CNPs are stable in enzyme-free media SGF- and SIF- (Figure 7AC). In pepsin-contained SGF+, however, insulin solution was totally degraded within 10 min, which is consistent with the published work.42 In contrast, 93% Ins-PLC-CNPs still remained at 30 min (Figure 7B). Insulin solution was also totally degraded in pancreatin-contained SIF+ within 10 min and 80% Ins-PLC-CNPs still remained at 120 min (Figure 7D). These results clearly indicate the high chemical stability of Ins-PLC-CNPs in the presence of pepsin and pancreatin. Interestingly, more insulin of Ins-PLC-CNPs was degraded in SIF+ than in SGF+. There might be two main reasons for this result: 1) Pancreatin (1%, w/v) is more powerful than pepsin (0.32%, w/v) in degrading insulin. 2) Formation of Ins-PLC-CNPs-Pep or Ins-PLC-CNPs-Pan complex may reduce the activity of the enzymes,8 but the corona of Ins-PLC-CNPs-Pan would consist of more amount of the acidic enzyme (such as pancreatic lipase, isoelectric point ~5.5) than the alkaline enzyme (such as trypsin, isoelectric point ~10.5) due to the differential electrostatic interaction. Thus, more amount of the alkaline enzyme would be in a free state and maintain a high activity of degrading insulin.

3.8. Cellular uptake The high chemical stability of Ins-PLC-CNPs can ensure the majority of loaded insulin intact in the upper GIT. Thus, the oral absorption of Ins-PLC-CNPs is mainly dependent on the small intestinal epithelial cell uptake. Herein, we evaluated the influence of nano-enzyme complex formation on cellular uptake of CNPs in a Caco-2 cell model, which has been widely used to simulate the small intestinal epithelial cells for investigating the cellular uptake and oral absorption.32, 43 Considering the dynamic contact of nanosuspension with intestinal epithelial cells in GIT, treatment for 30 min might be quite enough for the static incubation in vitro. A fluorescent probe (coumarin-6, C6) was incorporated in CNPs and the fluorescent signal in cells was detected by flow cytometry and microscopy. In the absence of FBS, cellular uptake of C6-CNPs was 2-fold enhanced when C6 concentration increased from 90 to 180 ng/ml, indicating a concentration-dependent uptake manner (Figure 8A). As expected, cell uptake of C6-CNPs-Pan was significantly lower than that of C6-CNPs 20


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at both concentrations, indicating the inhibited effect of pancreatin corona on epithelial cellular uptake. Interestingly, cell uptake in each group was further and significantly reduced in the presence of FBS in the culture medium. These results were further confirmed by fluorescent micrographs of Caco-2 cells (Figure 8B). The pancreatin corona-caused inhibited cellular uptake is consistent with the albumin corona-caused reduced phagocytosis.12

Figure 8. Cellular uptake of C6-CNPs and C6-CNPs-Pan (90 and 180 ng/ml, equivalent to C6) by Caco-2 cells upon incubation in the media containing FBS or not for 30 min. A) Quantitative analysis 21



by flow cytometry. Symbols “-” and “+” indicate the absence and presence of FBS in the medium, respectively. Data are presented as mean ± sd (n=3). Statistical significance: b, d and **: p < 0.01; a, c and ***: p < 0.001. B) Microscopic fluorescent graphs of Caco-2 cells. Scale bar: 100 µm.

The above results clearly indicate that the formation of CNPs-Pan complex can significantly inhibit the epithelial cell uptake of CNPs by caco-2 cells. We exclude the effect of CNPs-Pep on cell uptake because the adsorbed pepsin in stomach will be rapidly degraded due to the presence of various proteases in the small intestine. It is assumed that the substantial size increase upon nano-enzyme interaction (Figure 3) is a reason for the reduced uptake since it is reported that the small size could facilitate the cell uptake of nanoparticles.44 Another possible reason could be attributed to the pancreatin adsorbed on particle surface, which may decrease the cell uptake activities and lead to the reduced cell uptake. In addition, the pancreatin corona may render CNPs stealthy from cells because pancreatin itself would be hardly taken up and may change the receptor recognition of cells.14, 19 Therefore, the oral absorption of NPs in small intestine may be probably inhibited owing to the nanoenzyme interaction and corona formation, even though the loaded drug has a high chemical stability in GIT. On the other hand, however, this result implies that the oral absorption of NPs can be regulated by modifying the physicochemical properties of NPs and controlling the digestive enzyme adsorption.

4. Conclusions Oral delivery of drugs using NPs as the carriers is considered to be useful for improving the oral bioavailability. However, a critical issue of nano-protein interaction in GIT is ignored. In this work, we demonstrated the spontaneous interaction of CNPs with digestive enzymes for the first time. The enzyme adsorption and corona formation was confirmed by the changes of CNPs in particle size, zeta potential and morphology, as well as SDS-PAGE and enzyme quantitative analysis. Most importantly, the cellular uptake of CNPs by Caco-2 cells was significantly inhibited due to the formation of enzyme corona. Our findings have significant implications for the design and development of NPsbased oral drug delivery systems. First, the nano-enzyme interaction may influence the digestive 22


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function. Second, the absorption of NPs in the small intestine will be inhibited by the formation of enzyme corona. Third, the inhibited absorption of NPs in the upper GIT may provide a novel approach for colon targeted delivery. In addition, the enzyme corona-caused inhibited cellular uptake of NPs may help explain the reason for the in vitro/in vivo uncorrelation in oral absorption of NPs. This present work would also inspire the further investigations involving bile salts, mucus and even ex vivo and in vivo studies which can provide more insights into the impact of enzyme corona on the transit and oral absorption of NPs in the GIT. In addition, the interaction of the other NPs (such as liposome and anionic NPs) with enzymes is a potential direction for future works.

Author Information Corresponding author *E-mail: [email protected]; [email protected] ORCID Qiang Peng: 0000-0002-2268-3178 Notes The authors declare no competing financial interest.

Acknowledgements This work was supported by the National Natural Science Foundation of China (No. 81402860), the Excellent Young Scientist Foundation of Sichuan University (No. 2016SCU04A02) and the Huohuaku Foundation of Sichuan University (No. 2018SCUH0083). We thank Liying Hao for her kind help in AFM.

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Digestive Enzyme Corona Formed in the Gastrointestinal Tract and Its

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