Size-Dependent Translocation of Nanoemulsions via Oral Delivery

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Size-Dependent Translocation of Nanoemulsions via Oral Delivery Fei Xia, Wufa Fan, Sifan Jiang, Yuhua Ma, Yi Lu, Jianping Qi, Ejaj Ahmad, Xiaochun Dong, Weili Zhao, and Wei Wu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b04916 • Publication Date (Web): 15 Jun 2017 Downloaded from http://pubs.acs.org on June 18, 2017

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ACS Applied Materials & Interfaces

Size-Dependent Translocation of Nanoemulsions via Oral Delivery Fei Xia†, Wufa Fan†, Sifan Jiang†, Yuhua Ma†, Yi Lu†, Jianping Qi†, Ejaj Ahmad†, Xiaochun Dong†, Weili Zhao*,†,‡, Wei Wu*,† †

School of Pharmacy, Fudan University, Key Laboratory of Smart Drug Delivery of

MOE and PLA, Shanghai 201203, China ‡

Key Laboratory for Special Functional Materials of the Ministry of Education, Henan

University, Kaifeng 475001, China ABSTRACT: The in vivo translocation of nanoemulsions was tracked by imaging tools with emphasis on the size effect. To guarantee accurate identification of nanoemulsions in vivo, water-quenching environment-responsive near-infrared fluorescent probes were used to label nanoemulsions. Imaging evidence confirms prominent digestion in gastrointestinal tract and oral absorption of integral nanoemulsions that survive digestion by enteric epithelia in a size-dependent way. In general, reducing particle size leads to slowed in vitro lipolysis and in vivo digestion, prolonged lifetime in small intestine, increased enteric epithelial uptake, and enhanced transportation to various organs. Histological examination reveals pervasive distribution of smaller nanoemulsions (80 nm) into enterocytes and basolateral tissues, whereas bigger ones (550, 1000 nm) primarily adhere to villi surfaces. Following epithelial uptake, nanoemulsions are transported through the lymphatics with a fraction of approximately 3-6%, suggesting considerable contribution of the lymphatic pathway to overall absorption. A majority of absorbed nanoemulsions are found 1 h post administration in livers and lungs. Similar size dependency of cellular uptake and trans-monolayer transport is confirmed in Caco-2 cell lines as well. In conclusion, size-dependent translocation of integral nanoemulsions is confirmed with an absolute bioavailability of at least 6%, envisioning potential applications in oral delivery of labile entities. KEYWORDS: in vivo fate, drug delivery, oral, nanoemulsions, nanoparticles, particle size, environment-responsive

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INTRODUCTION The body has evolved sophisticated defending mechanisms to shield itself from invasions from exogenous substances. These mechanisms allow selective intake of small molecules such as nutrients and bioactive ingredients but expel or destroy relatively large particles crossing the body surface membranes in collaboration with the immune systems. However, there is a transitional region between small molecules and particulates where quasi-particles small enough might be absorbed as well. Actually, nanoparticles of extraordinarily small sizes are able to penetrate the "safeguarding" body surfaces and be translocated to various organs or tissues in the body.1-3 Exploring the in vivo fate of nanoparticles is always of significance to understand toxicological as well as therapeutic potentials of nanoparticles. The oral route is the main entrance of foreign substances to the body. The powerful digestive capacity of the gastrointestinal (GI) tract as well as the presence of enteric epithelial barriers guarantees protection from harmful materials. All substances ingested will be processed extensively in the GI tract and be broken down before absorption. Particles such as pathogens may survive the GI environment but cannot escape the mucosal immune systems and commonly end up in sub-epithelial lymphatic tissues in the Peyer's patches (PPs), which represents the general mechanism of oral mucosal immunization.4,5 Yet a small fraction of particles retained by PPs can be transported into circulation via lymphatics,6 thus creating opportunities for oral delivery of labile biomacromolecules. However, due to limited population of PPs in the enteric epithelia and sub-epithelial retention of particles, total translocation of particulates to circulation is also limited. It is natural to explore alternative pathways to deliver particulates across the intestinal epithelial barriers. Attention is then drawn to the absorptive enterocytic lining because of its enormous gross surface area. Pilot studies with inorganic nanoparticles confirm that nanoparticles of extraordinarily small sizes can be absorbed orally and translocated to various tissues.7,8 The same might happen for biodegradable organic nanoparticles. It is envisaged that breaking through the intestinal epithelial barriers holds tremendous potential for oral delivery of labile biomacromolecules.


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Previous studies only provided inconclusive findings on the in vivo fate of various nanoparticles in the GI tract. Based on observation of trans-monolayer transport behaviors in Caco-2 cell lines, integral nanoparticles are supposed to be able to cross the enterocytic barrier and be transported into circulation via various pathways.9-14 Yet these findings could hardly be translated to interpret the in vivo fate of nanoparticles because of the inadequacy of in vitro cell models to simulate the complex GI conditions. Translocation of nanoparticles to various organs via oral delivery has been investigated in vivo as well. Oral absorption of integral solid lipid nanoparticles (SLNs) were proposed by some researchers based on monitoring fluorescent labels.15,16 However, some others suggest unlikeness of translocation of SLNs into circulation via oral delivery.17,18 This contradiction exposes a fatal drawback of the conventional probes used to label the vehicles. Released or free probes produce the same signals as the vehicles themselves, and might be mistakenly counted into the total number of nanoparticles. The same concern arises when isotopes are used.19 To date, the controversy persists and continuously fuels a demand to clarify the oral fate of various nanoparticles with confirmative evidence. In order to monitor the in vivo fate of nanoparticles accurately, it is crucial to distinguish the vehicles from bulk physiological environment.20 Conventional bioimging protocols commonly use indiscriminative probes,21-23 but suffer from free probe-derived interference.

To

address

this

problem, a

novel

kind of

environment-responsive fluorescent probes24 are employed to label nanoparticles. These dyes emit fluorescence in a dispersed state and form aggregates upon dispersing into aqueous phases, resulting in immediate and absolute fluorescence quenching.24 Therefore, the signals recorded reflect the behaviors of integral nanocarriers, eliminating interference due to free probes. From a broad sense of drug delivery, it is well recognized that particle size plays a very important role in biodistribution and biorecognition in the body.25-30 Apparently, particle size affects intra-GI digestion due to difference in surface areas and trans-epithelial permeation. However, there is no concrete conclusion on the in vivo fate of various nanocarriers for biomedicines to date. Herein, the translocation of a model particle, nanoemulsions (NEs), was studied with emphasis on the effect



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of particle size via oral delivery. NEs are non-equilibrium heterogeneous systems composed of nanoscale droplets of one immiscible liquid well dispersed within another. Due to easy manufacturing, high loading capacity and excellent biocompatibility, NEs have found wide applications as drug carriers, especially for oral delivery.31-33 Despite numerous studies on NE formulations, the in vivo fate as well as underlying mechanisms of NEs are still poorly understood. Generally, after oral administration NEs are firstly degraded by lipases in the GI tract and form mixed micelles with endogenous surfactants such as bile salts and phospholipids before absorption.34,35 Whether integral NEs can be absorbed by enterocytes and transported into circulation is still awaiting validation with sound evidence.

RESULTS AND DISCUSSION The goal of this study is to track the translocation of NEs step by step from the beginning in the GI tract to various organs and tissues by means of collecting imaging evidence. The particle size effect is investigated to explore possibilities of oral absorption of integral NEs as well as using NEs as vehicles to deliver labile entities to circulation. Figure 1 depicts the rationale of identification of NE droplets in vivo. The environment-responsive fluorescent probes used in this study have a BODIPY (P4) or aza-BODIPY (P2) parent structure (Supplementary Figure S1), and exhibit favorable characteristics suitable for in vivo bioimaging including near-infrared emission, excellent chemical stability and high quantum yields.24 This kind of probes are highly lipophilic, and therefore readily dissolve in a variety of organic solvents and can be well dispersed into lipid matrix, either liquid or solid such as in the case of lipid-based nanoparticles, in high entrapment efficiency. The environment-responsiveness is due to the induction of aggregation-caused quenching (ACQ) through π-π stacking of the planar probes upon dispersion into aqueous media. Therefore, they are also called ACQ probes or water-quenching probes. Herein, we incorporate the probes into NEs where they exist in a well-dispersed state and emit fluorescence. Upon biodegradation of NEs, the probe molecules are released, then quench in the ambient aqueous environment of the body. Since water-triggered fluorescence quenching is immediate and absolute, the


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signals observed can be employed to represent NE droplets accurately. The same rationale has worked out in lipid-based nanoparticles24,36 and polymeric micelles37 in our previous studies. To investigate the particle size effect, it is important to prepare NEs with controllable particle sizes and a size distribution as narrow as possible. To this end, a high-pressure homogenization method38 was employed to prepare NEs with sizes less than 550 nm, whereas a membrane emulsification method39 was used to prepare 1000-nm NEs, using Lipophil WL1349 and Solutol® HS15 as the oil and surfactant, respectively. After initial optimization, all NE formulations were prepared at fixed oil/water and surfactant/oil ratios (w/w) of 1:7 and 3:5, respectively, in order to perform parallel comparisons. The particle sizes, polydispersity indexes (PDIs) and Zeta-potentials of NEs are shown in Table 1. The size distribution is quite narrow. Since Solutol® HS15 has a polyethylene glycol (PEG) tail, the NEs prepared using it will be covered with a layer of PEG coating. As a result, the NEs exhibit slightly negative zeta potential, which is typical of surface charges of PEG-coated nanoparticles and is in line with previous reports by other researchers.40,41 Since the zeta potentials measured are nearly neutral42,43 and the difference among various size groups is minor, it is rational to postulate that surface charges might not exert significant effect on the explanation of the size effect in this study. Therefore, we have not carried out an investigation on the effect of zeta potential temporarily. Transmission electron microscopy (TEM) reveals near spherical morphology and similar particle sizes as determined by a nanosizer (Supplementary Figure S2). Either P2 (for live imaging) or P4 (for confocal microscopy) is entrapped with near 100% efficiency due to their high lipophilicity. Before in vitro and in vivo evaluation, the stability of NEs was investigated first. Among various parameters, the fluorescence stability is the most important because leakage of the dyes will result in fluorescence intensity drop and bring about negative interference.24 In case smaller NEs are prone to leakage, aggregation and degradation owing to enlarged specific surface area, we only inspected the stability of 80-nm and 200-nm NEs in this study. It is assumed that if the smaller NEs are stable, the stability of bigger ones can be ensured. For 24 h, little change in fluorescence intensity, particle size and PDI is observed in water and various buffers (Supplementary Figure



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S3a,b,c & Figure S4a,b,c), indicating stable and integral NE structures, without obvious leakage of the dyes or soaking by water, which are factors that might lead to unwanted quenching of the dyes.24 However, in simulated GI fluids, as much as 5-15% decrease in fluorescence intensity is detected after 24 h (Supplementary Figure S3d,e,f & Figure S4d,e,f), indicating gradual abrasion of the NE matrix in the presence of bile salts and phospholipids, a process that might take place in the GI tract as well. Taking together, it is confirmed that NEs stay stable in various media, and the dyes can be securely embedded in the lipid matrix, which ensures the accuracy of follow-up in vitro and in vivo investigations. The size dependency of lipolysis was first investigated using either in vitro lipolysis or in vivo live imaging protocols. In vitro lipolysis monitored by a conventional alkaline compensation method44,45 show very quick degradation of a majority of NEs within 20 min for all size groups (Figure 2a). Comparing different particle sizes, negative correlation exists between NE sizes and degradation rate with the smallest NEs showing the slowest lipolysis rate. This finding nevertheless contradicts previous findings that smaller NEs merit faster digestion owing to significantly enhanced specific surface area,46,47 or particle size has little to do with lipolysis rate of submicron emulsions because of the rapid GI digestion rate.48,49 The underlying mechanisms are yet to be explored. Obviously, it cannot be explained by the lack of sensitivity of extremely small NEs to lipases because the profiles clearly show near complete lipolysis for all size groups. However, the relatively large population of smaller NEs may contribute to the delayed lipolysis because lipases might be saturated temporarily during the process of lipolysis. Following similar procedures, the performance of dye-loaded NEs was studied by monitoring fluorescence in simulated media containing bile salts and phospholipids. Despite the accordance in the overall trends with alkaline consumption results, the behaviors indicated by fluorescence intensity deviate in details especially for 80-nm NEs. The most obvious difference lies in the retention of approximately 20% fluorescence for larger NEs and 50% for 80-nm NEs after completion of lipolysis (Figure 2b). This might be ascribed to the fluorescence rekindling effect as a result of formation of surfactant-based micelles.24 Moreover, smaller ones seem to be more efficient in transformation from NEs to mixed micelles. Seemingly, the in vitro


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simulation results might compromise the accuracy of identification of NEs in vivo, for which instant and absolute quenching is demanded. However, we should note that in vivo situation of digestion of lipid-based nanoparticles is different from that of in vitro lipolysis. Although in vitro lipolysis is frequently used to simulate in vivo lipid biodegradation, it is an accelerated process. As shown in this study (Figure 2), NEs can be degraded near completely in vitro within 1 h, whereas it is 24 h in vivo (Figure 3a). For the in vitro lipolysis, the probes are released simultaneously along with the lipolysates in large amount, and there are plenty of chances for them to be embedded into newly formed mixed micelles. However, in vivo digestion takes place much slowly, and released probes are able to be dispersed quickly or removed through absorption or excretion. Although re-partitioning of the probes into mixed micelles might happen, the rekindling effect are supposed not to be strong enough to cause significant interference with in vivo live imaging due to dilution by many folds.24 The transport of NEs in the GI tract was first tracked by live imaging in rats (Figure 3a). Faint fluorescence is observed in the abdominal region 2 and 4 h post administration of various controls, i. e. quenched P2 solution (control-1), a physical mixture of quenched P2 with NEs (control-2) and a lipolysate of 200-nm NEs from in vitro lipolysis (control-3), used to simulate the in vivo fate of the dyes. These signals are negligible in comparison with the test groups and will not impose significant interference on detection of NEs, which in part clears up the concerns with fluorescence retention after in vitro lipolysis. As for P2-NEs, the fluorescence signals slightly increase in the first 2 h as a result of the initial dispersing of NE droplets in stomach after gavage administration.18 Then the fluorescence declines gradually and vanishes after 12 h. There is no visually discernable difference between each two size groups. However, quantification of fluorescence reveals difference between different size groups (Figure 3b). Although no significant difference in resident time is observed between two adjacent size groups, the 80-nm group show apparently extended resident time, compared to 1000-nm group. The in vivo digestion profiles can be well fitted to first-order kinetics (Figure 3c) with t1/2s of4.51 ± 0.31, 4.37 ± 0.19, 3.92 ± 0.13 and 3.24± 0.51 h for 80-nm, 200-nm, 550-nm and 1000-nm NEs, respectively. There is significant difference (P < 0.05) between each two size groups



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except between 80 and 200 nm, or 550 and 1000 nm. Very good correlation is established between in vitro lipolysis and in vivo degradation profiles (Figure 3d,e,f,g). The live imaging results somehow confirm that smaller NEs have longer lifetime in the GI tract. Ex vivo imaging after excision of the whole GI segments reveals clearer real-time translocation of NEs along the GI tract (Figure 4a). Unlike in vivo live images, quantification of ex vivo images clearly reveals the size-dependent transport of NEs in the whole GI tract with 80-nm NEs showing the longest residence time (Figure 4b,c,d,e). This disparity might be explained by the limitations of live imaging in quantification. Since fluorescence can only weakly penetrate the biological tissues, quantification of fluorescence by live imaging is not accurate enough (semi-quantitative) although parallel comparison can be performed. Therefore, the intricate differences between size groups might not be identifiable by live imaging. That is exactly why we choose to dissect gastrointestinal segments for more accurate quantification. Similar to SLNs,18 NEs of all size groups reside in stomach in a declining tendency for as long as 8 h, but without significant difference. The fluorescence in small intestine increases gradually following gastric emptying, peaks at 2-4 h, then declines gradually until 24 h due to degradation, absorption and excretion of NEs. There is little difference between bigger NEs (200, 550 and 1000 nm), whereas the residence of smaller NEs (80 nm) is relatively longer. For all groups, the signals in the colon segment were quite low, indirectly suggesting that most NEs are degraded or absorbed in small intestine. In spite of the good correlation between in vitro lipolysis and in vivo digestion, the lifetime of NEs in the GI tract is almost ten times longer than in vitro. It suggests that the virtual digestive conditions in the GI tract may differ significantly from the in vitro simulated conditions. The negative correlation between particle size and in vivo resident time is in coincidence with the case of in vitro lipolysis. Again, the prolonged residence of smaller NEs might be explained by their huge population that might saturate the lipases available for lipolysis. In addition, smaller NEs have more chances to diffuse into the mucosal lining as well as various microscopic structures in GI tract, where less lipases reside, and survive lipolysis for longer times. To obtain evidence of oral absorption of integral NE droplets, the easiest way is to


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scan various organs such as liver, lung, spleen and kidney, or tissues by simple fluorescence imaging after excision. As shown in Figure 5a, the quenched P2 control group exhibits no fluorescent signals in all organs and tissues, excluding interference due to fluorescence re-illumination. Fluorescence appears in liver 2 h post administration for 80, 200 and 550 nm groups and 4 h for 1000 nm (Figure 5a), respectively, and quantification of total fluorescence indicates higher accumulation of 80-nm NEs than any other group (Figure 5b,c,d,e). Distribution to lung can be observed after 8 h for all size groups, whereas only faint signals are found in spleen and kidney at all time points. Of all size groups, biodistribution of NEs follows the order of liver >>lung >spleen≈Kidney. Figure 5f shows the fluorescence intensity in blood vs. time plot after oral administration of P2-NEs. Of note is that the blood-borne fluorescence intensity is about one thousand times less than that in the organs at all time points. Signals of 80-nm NEs appear in the blood after 2 h, much faster than bigger ones, and peak at about 8 h. The absorption rate of NEs decreases with the increase of particle size. Signals of 550-nm and 1000-nm NEs peak at around 12 h. To achieve a rough estimation of the distribution percentage, the fluorescence intensity in the stomach at 0.5 h after gavage administration is taken as the total fluorescence dose administered, and the percent distribution of fluorescence in various organs is calculated based on this value (Supplementary Figure S5a,b,c,d). For 80-nm NEs at 4 h, a maximum of 18.0±4.9% of administered dose can be found in liver, for 200-nm NEs it is 15.8±2.8% at 8 h, and for 550-nm and 1000-nm NEs it is about 10% at 12 h and 18 h, respectively. Figure 5g shows the cumulative transport percentage of P2-NEs via the lymphatic route in rats. Of all size groups, the lymphatic transportation begins within the first 2 h, continues at a certain rate until 12 h, and slows down until no more increase at 24-36 h. It is notable that smaller NEs have higher lymphatic transport percentage: 80 nm, 5.94 ± 1.02%; 200 nm, 5.14 ± 0.88%; 550 nm, 3.03 ± 0.33%; 1000 nm, 3.33 ± 0.31%. Taking together the biodistribution and pharmacokinetic results, it is evident that integral NE droplets have been absorbed via oral delivery, and have been



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transported through the systemic circulation to various organs, with distinctive size dependency. In general, smaller NEs are absorbed and distributed to organs more quickly and in more amount. NEs mainly accumulate in liver as well as in lung and spleen but in less amount due to the phagocytic uptake by macrophages in the reticuloendothelial system (RES). Yet the absolute bioavailability of NEs droplets still cannot be calculated accurately though we managed to carry out a rough estimation (Supplementary Figure S5). The real-time distribution percentages of NEs in various organs do not stand for the absolute bioavailability of NE droplets at all. On the other hand, the quantification of cumulative lymphatic transportation is quite accurate because it is based on a standard calibration method, which has been validated for linearity and accuracy. There are two possible pathways for lymphatic transportation, i.e., the “M cell → sub-epithelial lymphatics in PPs → circulation” pathway and the “enterocytes → cellular interstitial fluid → lymphatics → circulation” pathway. To date, it is impossible to discriminate between the two pathways. If the gross oral bioavailability of NEs can be accurately calculated in the future and found higher than the overall lymphatic transportation amount, it is supposed that there are pathways other than the lymphatics, e.g., the portal pathway, for the entry of NEs into the circulation. To explore the exact mechanisms at the intestinal mucosal surfaces, retention of NEs in small intestine was first studied by in situ perfusion (Figure 6a,b). Comparing different particle sizes, it is evident that smaller NEs have stronger fluorescence than bigger ones, which indicates stronger association of NEs to the intestinal lining. This result partly explains the longer residence time of smaller NEs in live imaging. To further explore the interaction of NEs with intestinal epithelia, the jejunum segments were sliced and observe by confocal laser scanning microscopy (CLSM). As shown in Figure 6c, all groups give P4 signal (red), indicating the presence of integral NEs, and there is a negative correlation between particle size and fluorescence intensity. Noticeably, very strong co-localized signals of P4/DAPI (purple) at the basolateral side in addition to villi surfaces for 80-nm NEs confirm extensive uptake of integral NEs by the enterocytes. 200-nm NEs also show absorption of integral NEs but in less quantity. Remarkably, co-localization signals in both 550 nm and 1000 nm groups are found locating mostly to the villi surfaces, and only faint signals are


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observed at the basolateral side, which implies that only small NEs can be taken up efficiently by enterocytes and be transported to circulatory system. However, this proposition seems to be paradoxical because ex vivo imaging results crystal-clearly confirm translocation of larger NEs (550, 1000 nm) to livers and lungs. To clarify this issue, we should pay close attention to the fact that NEs, either large or small, undergo digestion by lipases in the GI tract. It seems that in the ex vivo imaging test, the observed translocation of bigger NEs into circulation virtually reflects the translocation of their smaller NE counterparts due to gradual reduction in particle size by lipolysis. A plausible explanation for the apparent size dependency lies in the difference in the population of NEs of different sizes. At a fixed dose of total lipids, reduction in particle size leads to exponential increase in total NE population and thereby enhanced amount of trans-epithelial transport of NE droplets. Since the lipases have been removed in an in situ perfusion study, translocation of bigger NEs can hardly be observed. In addition, there is a fraction of smaller particles in in the “bigger” population (Supplementary Figure S2d), which might also contribute to the absorption and translocation to various organs. However, the effect might be negligible for the “bigger”-size group because the translocation of NEs to the basolateral side is hardly discernable. The interaction of NEs with intestinal epithelia was further studied in Caco-2 cell models. Figure 7a,b shows the fluorescent images as well as quantification results of cellular uptake of P4-NEs in Caco-2 and Caco-2/HT29-MTX cell models. The control group incubated with quenched P4 solution gives almost no P4 signals, excluding the interference due to fluorescence rekindling. As observed in both cell models, the particle size of NEs shows significant impact on cellular uptake. The fluorescence intensity of 80-nm P4-NE group is much higher than other bigger size groups, indicating that smaller NEs can be internalized in more amount no matter whether there is a layer of mucus or not. Comparing the two cell models, the fluorescence intensity in Caco-2 cells is slightly higher than that in Caco-2/HT29-MTX cells. CLSM were used to locate the precise location of P4-NEs using Z-axis scanning mode from basolateral (BL) to apical (AP) side of cell monolayer. As shown in Figure7c, the fluorescence intensity of P4 signal (Red) decreases with the increase of particle size. In Caco-2 models, most P4 signals are in



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the same horizontal plane with cell nucleus or at BL side, indicating internalization of integral NEs by the cells. In comparison with Caco-2 models, the P4 signal in Caco-2/HT29-MTX models locates more to the AP side, which represents surface adhesion. The results prove that mucus layer presents as a barrier to the penetration of NEs. The cellular uptake mechanism of nanoemulsion has been studied by Gao et al in Caco-2 cell lines, and a clathrin-mediated endocytosis was proposed for the internalization of nanoemulsions by enterocytes.50 Figure 8 shows the trans-monolayer transport of P4-NEs in four cell models. P4 signals detected at the BL side confirms transport of intact P4-NEs across the cell monolayers. The size effect is also notable in this case. In Caco-2 cell models, the P4 signals of all size groups appear at 1 h and increases as a function of time. The cumulative transport of 80-nm NEs at 4 h is about 1%. In comparison, the fluorescence of 80-nm and 200-nm NEs is much lower in Caco-2/HT29-MTX cell models, and no signals of 550-nm and 1000-nm NEs can be detected during the first 2 h, which suggests that the mucus layer retards the penetration and transport of NEs significantly, which is more remarkable for bigger NEs. When Raji cells are incorporated, the total trans-monolayer transport of NEs is nearly doubled for all size groups in either Caco-2/Raji or Caco-2/HT29-MTX/Raji cell lines. Combining the CLSM imaging results of both histological examination of intestinal segment and trans-monolayers transport, it is confirmed that integral NE particles of less than 200 nm are able to penetrate the enteric epithelia. Size dependency is obvious with smaller ones achieving more and quicker oral absorption. The M-cell pathway possibly plays a role but needs further validation with both imaging and quantitative evidence.

CONCLUSIONS For a better comparison of the size effect, NEs with particle sizes of approximately 80, 200, 550 and 1000 nm and a narrow size distribution were successfully prepared. By labeling with novel environment-responsive near-infrared fluorescent probes with water-quenching properties, only integral NEs are identifiable after oral administration, and any interference due to free probes can be eliminated. In vitro


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fluorescent stability excludes possible errors that might arise due to leakage of the dyes. Inclusion of various controls to simulate the in vivo fate of the dyes throughout the whole study helps justify the accuracy of identification of NEs in vivo. Apparent size dependency is observed for lipolysis both in simulated media and in vivo. Reducing the particle size tends to slow the overall lipolysis rate and result in extended residence in the GI tract. The whole journey of NEs starting from the GI tract to various organs was tracked with imaging evidence. Smaller size (80, 200 nm) groups show comparable retention in small intestine, which is significantly higher than bigger size groups (550, 1000 nm). Histological examination using CLSM reveals co-localized signals of 80-nm NEs in intestinal tissues, confirming pervasive permeation of NEs into enterocytes and basolateral tissues. As for 200-nm NEs, the particles can be translocated to enterocytes but less to basolateral tissues. As for bigger ones (550, 1000 nm), NEs cannot permeate into enterocytes and are only found on villi surfaces. NEs can be traced in liver 2 h post oral administration with smaller NEs showing faster transportation in more amount. Recovery of NEs from mesentery lymph shows 3-6% total transportation via the lymphatic route. The size dependency was further confirmed using in vitro Caco-2 cell models. There is negative correlation between particle size and cellular uptake/trans-monolayer transport. Taking together all evidence, it is proposed that smaller NEs can be absorbed through the enterocytes, indicating potentials of exploiting NEs as vehicles to delivery labile entities to the circulatory system.

EXPERIMENTAL SECTION Materials. Labrafac Lipophil WL1349 (Gattefossé Co., Cedex, France); Solutol® HS15 (BASF Co., Shanghai, China); lecithin (Lipoid GmbH Company, Ludwigshafen, Germany); sodium taurocholate (Tokyo Chemical Industry Co., Ltd, Tokyo, Japan); lipase from porcine pancreas, Trisma® maleate, 4-Bromophenylboronic acid (Sigma-Aldrich Co., Shanghai, China); FaSSIF, FeSSIF and FaSSGF Powder (Biorelevant.com, Croydon, UK); Dulbecco modified Eagle’s minimal essential medium (DMEM), Roswell Park Memorial Institute-1640 medium (RPMI 1640), fetal bovine serum (FBS) (Gibco Invitrogen Co., Carlsbad, USA); MEM nonessential amino



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acids, penicillin–streptomycin solution, Hank’s balanced salt solution buffer (HBSS), and phosphate buffered saline (PBS) with or without calcium and magnesium (Beijing Solarbio Science & Technology co., Ltd.); 4,6-diamidino-2-phenylindole (DAPI) (Yeasen Bio-tech Co., Ltd., Shanghai, China); human colorectal adenocarcinoma Caco-2 cells and human Burkitt’s lymphoma Raji cells (Cell Bank of Chinese Academy of Sciences, Shanghai, China); human colorectal adenocarcinoma HT29-MTX cells (China Center for Type Culture Collection, Wuhan, China). Deionized water was prepared by using a Milli-Q system from Millipore (Billerica, USA). The ACQ fluorescent probes P2 (λabs/λem=708/732) and P4 (λabs/λem=651/662) were synthesized in our lab according to previous procedures;51,52 All other reagents were of at least analytical grade and purchased from local distributors. Male Sprague Dawley (SD) rats, weighing 200 ± 20 g, were obtained from Shanghai Laboratory Animal Center (Shanghai, China). All animal experimental procedures were reviewed by the Institutional Animal Care and Use Committee at School of Pharmacy, Fudan University. Preparation and characterization of P2/P4-labeled NEs. The oil-in-water type NEs of different sizes were prepared by either a high-pressure homogenization method38 or a membrane emulsification method,39 using Labrafac Lipophil WL1349 as the oil phase and Solutol® HS15 as the emulsifier. A coarse emulsion was formed by homogenization using a high-shear homogenizer (Scientz Biotechnology Co., Ltd, China) after pouring the oil phase (5 g Lipophil WL1349; 200 µg P2 or 20 µg P4) into the aqueous phase (3 g Solutol® HS15 in 35 mL deionized water). Then the NEs (80 and 200 nm) were prepared through high-pressure homogenization of the coarse emulsion at a pressure of 1000 bar for 3 min or 100 bar for 2 min, respectively. The NEs of bigger sizes (550 and 1000 nm) were prepared by a membrane emulsification method using 0.8-μm and 1.2-μm membrane tube, respectively, using the same formulation as smaller ones. The particle size, PDI and Zeta-potential of NEs were measured by a Zetasizer Nano (Malvern Instruments, Malvern, UK) at room temperature. The morphology of NEs was characterized by a JEM-1230 TEM (JEOL, Tokyo, Japan). Stability of fluorescently labeled NEs. The in vitro stability was evaluated by


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monitoring the particle size, PDI and fluorescence intensity of P2/P4-NEs in various aqueous and bio-relevant media at 37°C for 24 h using a water-bath shaker. The test media include deionized water, buffers of different pHs (1.2-hydrochloric acid; 4.5-acetate; 6.8- and 7.4-phosphate), simulated gastric fluid (SGF), simulated intestinal fluid (SIF), fasted-state simulated gastric fluid (FaSSGF), fasted-state simulated intestinal fluid (FaSSIF) and fed-state simulated intestinal fluid (FeSSIF). All the media were prepared according to previously reported procedures.24 See Supplementary Table S1 for the compositions of various bio-relevant media. In vitro lipolysis. In vitro lipolysis of P2-NEs was conducted following previous procedures in FaSSIF with a pH of 7.5 ± 0.05. 53 Briefly, add 3 mL P2-NEs to 18 mL FaSSIF pre-heated to 37°C and mix homogeneously. Then, add 2 mL porcine pancreatic lipase dispersion (100 mg/mL) to initiate lipolysis. During the 1.5-h reaction process, the mixture was stirred at 600 rpm, keeping temperature at 37 ± 0.5°C, and a pH-stat titrator (TitraLab® 854, Radiometer Analytical) was used to maintain the pH at 7.5 by neutralizing the fatty acid produced by lipolysis with 0.2 M NaOH solution. The consumption volume of NaOH was recorded by the machine. Meanwhile, lipolysis was also monitored by measuring the fluorescence intensity as described before.24 Live imaging in rats. Test groups include P2-NEs of different sizes (80, 200, 550 and 1000 nm) with various control groups, i. e. quenched P2 solution (control-1), a physical mixture of quenched P2 with NEs (control-2) and a lipolysate of 200-nm NEs from in vitro lipolysis (control-3) (n = 3). The concentration of the dye is carefully modulated to the same level as the test groups (0.0004%, w/v). The hair on the abdominal side was removed by depilatory cream to reduce hair-derived auto-fluorescence. Blank images were captured before administration, then 1 mL samples were given to each rat by gavage. P2 signals were captured post administration using IVIS Spectrum Live Imaging System (Perkin Elmer, USA). The rats were narcotized by an on-line gas anesthetizing system using isoflurane during the whole imaging process. To locate NEs in the GI tract, the rats were sacrificed at time intervals and the whole GI tract was dissected and visualized by IVIS imaging system. The



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fluorescence intensity of different GI segments was quantified and compared following previous procedures.18,24 Biodistribution and pharmacokinetics. To investigate the biodistribution of NEs after oral administration, main organs such as liver, lung, spleen and kidneys were excised immediately after sacrificing the animals at time intervals, and the fluorescent signals were recorded by IVIS system and quantified. Pharmacokinetics of P2-NEs were also studied by monitoring the fluorescent signals in blood. Briefly, 100 μL blood samples were withdrawn from eye socket vein and instilled into the wells of black 96-well plates at time points after gavage administration of 1mL P2-NEs. Then, blood-borne P2 signal was monitored by IVIS imaging system. In situ retention and enterocytic uptake study. In situ single-pass intestinal perfusion experiment were carried out to investigate the retention behavior and membrane permeability of NEs in the GI tract according to previously procedures.24 Briefly, jejunum segment (15 to 25 cm downward from the pylorus) was first cannulated to form a loop, then the NEs dispersion was perfused at one direction through the intestinal loop. Effluent was collected and assayed for fluorescence intensity until an equilibrium was reached. Subsequently, this jejunum segment was excised and observed by the IVIS imaging system after removal of lumen content by air flow. The intestinal tissues were fixated, dehydrated, cut into small rings and frozen in Optimal Cutting Temperature compound (OCT). The frozen tissues were cut into 10 μm-thick slices using a Leica Microm CM3050S cryostat (Leica Inc., Germany) and stained with DAPI. Tissue sections were observed by CLSM. Transport via mesenteric lymphatics. Lymphatic transportation of P2-NEs was evaluated by sampling via lymphatic cannulation after oral administration. SD rats weighing 300-350 g were anesthetized by intra-peritoneal injection of 10% chloral hydrate solution (4 mL/kg). Then, the animals were incised and the mesenteric lymphatic duct was cannulated as described previously.54,55 Lymph samples were collected continuously and cumulative P2 fluorescence was measured using the IVIS live imaging system until 36 h post oral administration.


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Cellular uptake and trans-monolayer transport. Cell culturing was carried out exactly according to previous procedures.18 Four cell monolayer models (Caco-2, Caco-2/HT29-MTX, Caco-2/Raji, Caco-2/HT29-MTX/Raji) were established to assess the interaction of NEs with the cell monolayers following reported procedures.18,56 Caco-2 and Caco-2/HT29-MTX co-culture monolayers were used for cellular uptake investigations.18 Cells in exponential phase were seeded into black 96-well plate or glass bottom cell culture dishes in a density of 5 × 104 cells per cm2. After culturing for 14 d, the cell model was ready for cellular uptake study. P4-NEs (200 μL) of different sizes were diluted by HBSS, added into plate wells or glass cell culture dishes and incubated for 2 h. Then the cells were washed with PBS for fluorescence determination by IVIS imaging system and stained by DAPI for CLSM observation, respectively. Caco-2 and Caco-2/HT29-MTX (7:3) mixed cells were seeded onto the AP side of Millicell-CM insert cell culture plates and cultured for 21 d. Raji cells were then added to the BL side and co-cultured for another 4-5 d until the trans-epithelial electrical resistance (TEER) value declined obviously, indicating successful infiltration of Raji cells. After establishment of the cell models, 400 μL P4-NEs of different sizes were diluted by HBSS and added into the AP side of the insert compartments, while the BL side was filled with 600 μL HBSS. After incubation of 4 h at 37°C, 200 μL samples were withdrawn from the BL side at 1, 2 and 4 h for fluorescence intensity measurements.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/******. Compositions of various simulated bio-relevant media (Table S1); Chemical structures of the two probes used to label NEs (Figure S1); TEM photographs of NEs of different particle sizes (Figure S2); Stability of fluorescence, particle size and PDI of 80-nm (Figure S3) and 200-nm (Figure S4) NEs upon storage in various buffers and bio-relevant media; Estimation of NE distribution percentages in various organs



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(Figure S5) (PDF)

AUTHOR INFORMATION Corresponding Authors *W.W. tel: +86-21-51980084; fax: +86-21-51980084; e-mail: [email protected]. *W.Z. tel: +86-21-51980111; fax: +86-21-51980111; e-mail: [email protected]. Author Contributions The manuscript was written through contributions of all authors. W.W., F.X. and W.Z. conceived the project; F.X., W.F., S.J. and Y.M. completed the experiments together; F.X., Y.L., J.Q., E.A. and W.W. analyzed the results; X.D and W.Z. synthesized the dyes; and F.X. and W.W. wrote the manuscript. Notes The authors declare no competing ﬁnancial interest.

ACKNOWLEDGEMENTS This study was financially supported by National Natural Science Foundation of China (81573363, 81690263, 21372063), Shanghai Commission of Science and Technology (14JC1490300), and National Key Basic Research Program (2015CB931800).

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Table 1 Particle sizes, PDI and Zeta-potential of NEs (mean ± SD, n = 3) 80 nm

200 nm

550 nm

1000 nm

Particle size (nm)

80.56±3.25 206.23±2.25 530.17±11.16 1014.77±39.29

PDI

0.11±0.01

0.20±0.01

0.16±0.02

0.15±0.05

Zeta-potential (mV)

-3.80±0.28

-3.73±0.74

-3.46±0.53

-5.05±0.72

Figure 1. Rationale of identification of integral NEs by labeling with water-quenching environment-responsive near-infrared fluorescent probes.

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ACS Paragon Plus Environment


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Figure 2. In vitro lipolysis vs. time plots monitored by a conventional alkaline compensation method (a) or by measuring fluorescence intensity (ARE) of P2-NEs (b). The NEs are digested by lipases to generate fatty acids, which are then neutralized by continuous alkaline compensation using a titrator while maintaining a constant pH of 7.5. The lipolysis percentages at each time point are calculated as percent residual alkaline to consume by setting the maximum consumption to 100%, or as percent residual fluorescence by setting the initial fluorescence to 100%.

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Figure 3. In vivo live images captured using an IVIS live imaging system (a) and quantification plots (ARE vs. time) (b) in rats after gavage administration of P2-NEs of different sizes and various controls (quenched P2 solution, Control-1; physical mixture of quenched P2 with blank NEs, Control-2; lipolysate of 200-nm NEs, Control-3); fitting ARE vs. time plots to first-order kinetics (c); point-to-point linear correlation between in vitro lipolysis and in vivo live imaging of P2-Nes of different particle sizes: 80 nm (d), 200 nm (e), 550 nm (f), 1000 nm (g).

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Figure 4. Ex vivo imaging of the whole dissected GI tract after gavage administration of P2-NEs (a); quantification of total fluorescence of GI segments (stomach, intestine, colon) for P2-NEs of different particle sizes: 80 nm (b), 200 nm (c), 550 nm (d), 1000 nm (e). The ex vivo images of the dissected gastrointestinal tissues are captured using an IVIS live imaging system after sacrificing the animals (n = 3).

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Figure 5. Ex vivo imaging (a) and fluorescent quantification of dissected organs after gavage administration of P2-NEs of different sizes indicate distribution of integral NE droplets to various organs: 80 nm (b), 200 nm (c), 550 nm (d), 1000 nm (e); pharmacokinetics in blood presented as total radiant efficiency of blood-borne fluorescence vs. time (f); cumulative transport percentage vs. through lymphatics (g). For evaluation of lymphatic transportation, lymph is withdrawn via mesentery cannulation and cumulative lymphatic transportation is calculated by summing up the total amount of nanoparticles in lymph collected in each period. 29



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Figure 6. Live images (a) and fluorescent quantification (b) of ex vivo jejunum segments after one-direction in situ perfusion with P2-NEs; CLSM images of frozen section of jejunum segments (c). DAPI (blue) and P4 (red) signals stand for the cell nuclei and NEs, respectively, whereas co-localization of both signals (pink) highlights infiltration of NEs into the enteric tissues, especially for 80-nm NEs.

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Figure 7. Live images (a) and quantification results (b) of uptake of P4-NEs by Caco-2 and Caco-2+HT29-MTX cell lines; x-y, x-z and y-z view of CLSM images of cell monolayers visualize the internalization of P4-NEs (c). The arrows highlight the P4 signals at the AP sides in Caco-2+HT29-MTX models. The cells are cultured in wells, incubated with NEs and imaged after removing excessive NE dispersion. The fluorescence of the wells is measured as total radiant efficiency, which stands for total cellular uptake.

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Figure 8. Cumulative transport vs. time of P4-NEs across Caco-2 (a), Caco-2+HT29-MTX (b), Caco-2+Raji (c) and Caco-2+HT29-MTX+Raji (d) cell monolayers. NE dispersion is instilled to the AP side of the trans-well insert compartment and the cumulative transport amount is measured by monitoring NE-associated fluorescence in the BL side.

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Table of content image 111x64mm (300 x 300 DPI)


Size-Dependent Translocation of Nanoemulsions via Oral Delivery

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