Enhancement of paracellular transport of heparin disaccharide across Caco-2 cell monolayers

The enhancement of paracellular transport of heparin disaccharide using several absorption enhancers across Caco-2 cell monolayers was tested. The cytotoxicity of these enhancers was also examined. The enhancing effects by Quillaja saponin, dipotassium glycyrrhizinate, 18beta-glycyrrhetinic acid, sodium caprate and taurine were determined by changes in transepithelial electrical resistance (TEER) and the amount of heparin disaccharide transported across Caco-2 cell monolayers. Among the absorption enhancers, 18beta-glycyrrhetinic acid and taurine decreased TEER and increased the permeability of heparin disaccharide in a dose-dependent and time-dependent manner with little or negligible cytotoxicity. Our results indicate that these absorption enhancers can widen the tight junction, which is a dominant paracellular absorption route of hydrophilic compounds. It is highly possible that these absorption enhancers can be applied as pharmaceutical excipients to improve the transport of macromolecules and hydrophilic drugs having difficulty in permeability across the intestinal epithelium.     

Transport characteristics of isorhamnetin across intestinal Caco-2 cell monolayers and the effects of transporters on it

Flavonoid isorhamnetin occurs in various plants and herbs, and demonstrates various biological effects in humans. This work will clarify the isorhamnetin absorption mechanism using the Caco-2 monolayer cell model. The isorhamnetin transport characteristics at different concentrations, pHs, temperatures, tight junctions and potential transporters were systemically investigated. Isorhamnetin was poorly absorbed by both passive diffusion and active transport mechanisms. Both trans- and paracellular pathways were involved during isorhamnetin transport. Active transport under an ATP-dependent transport mechanism was mediated by the organic anion transporting peptide (OATP); isorhamnetin’s permeability from the apical to the basolateral side significantly decreased after estrone-3-sulfate was added (p < 0.01). Efflux transporters, P-glycoproteins (P-gp), breast cancer resistance proteins (BCRP) and multidrug resistance proteins (MRPs) participated in the isorhamnetin transport process. Among them, the MRPs (especially MRP2) were the main efflux transporters for isorhamnetin; transport from the apical to the basolateral side increased 10.8-fold after adding an MRP inhibitor (MK571). This study details isorhamnetin’s cellular transport and elaborates isorhamnetin’s absorption mechanisms to provide a foundation for further studies.     

Effect of excipients on the stability and transport of recombinant human epidermal growth factor (rhEGF) across Caco-2 cell monolayers

The effect of sixteen excipients on the transport of recombinant human epidermal growth factor (rhEGF) across Caco-2 cell monolayers was examined at 37 degrees C. The apparent apical to basolateral (A-B) permeability (Papp) of 30 microM rhEGF was 8.15 x 10(-7) cm/sec, indicative of a poor level of absorption in the GI tract. The Papp was 1.7- and 6.3-fold greater than the Papp in the basolateral to apical (B-A) direction and the A-B permeability of mannitol, respectively, and decreased dramatically to a negligible level at 4 degrees C, consistent with a receptor mediated transcytosis of rhEGF. The stability of rhEGF was very poor, undergoing more than 85% degradation in 2 h in the transport medium at 37 degrees C. A significant increase in the Papp could be achieved by the addition of certain excipients, as exemplified by 23, 21, 20 and 16-fold increases, in the presence of sodium taurochenodeoxycholate (NaTCDC), sodium taurodeoxycholate (NaTDC), sodium glycodeoxycholate (NaGDC) and sodium laurylsulfate (SLS) (all at a concentration of 1% w/v), respectively. A significant increase in stability could also be achieved by the addition of some of the excipients, as represented by 1% SLS, which nearly completely stabilized the rhEGF. Unfortunately, however, an increase in the Papp of rhEGF could not be achieved without a simultaneous and extensive decrease in the integrity of the cell membranes. Thus, more efficient excipients, that specifically enhance the permeation of rhEGF and do not alter the membrane integrity, should be pursued in order to safely enhance the permeation of rhEGF.     

Effect of cationized gelatins on the paracellular transport of drugs through caco-2 cell monolayers

Cationized gelatins, candidate absorption enhancers, were prepared by addition of ethylenediamine or spermine to gelatin and the effects of the resulting ethylenediaminated gelatin (EG) and sperminated gelatin (SG) on the paracellular transport of 5(6)-carboxyfluorescein (CF), FITC-dextran-4 (FD4), and insulin through caco-2 cell monolayers were examined. The Renkin function was used for characterization of the paracellular pathway and changes in the pore radius (R) and pore occupancy/length ratio (epsilon/L) calculated from the apparent permeability coefficients (P(app)) of CF and FD4 are discussed. Ethylenediaminetetraacetic acid (EDTA) increased the R of the caco-2 cell monolayer and the P(app) of all compounds examined was markedly increased by the addition of EDTA. On the other hand, EG and SG did not increase R and their enhancing effects were not as strong as those of EDTA. The increase in epsilon/L could be the enhancing mechanism for the cationized gelatins. The number of pathways for water-soluble drugs, such as CF and FD4, in the caco-2 monolayers could be increased by the addition of the cationized gelatins. The ratios of the permeability coefficients of insulin (observed/calculated based on the Renkin function) suggest that insulin undergoes enzymatic degradation during transport which is not inhibited by enhancers.     

蜕皮甾酮在Caco-2细胞模型中的摄取和跨膜转运研究

目的以Caco-2细胞单层模型,首次研究了蜕皮甾酮的口服吸收与转运特性。方法采用普通Caco-2细胞模型、表达活性CYP3A4酶的Caco-2细胞模型,分别从AP→BL方向和BL→AP方向研究蜕皮甾酮的摄取和跨膜转运规律。结果蜕皮甾酮在2种模型中的表观渗透系数(Papp)在0.1×10-6～1×10-6cm.s-1之间,药物吸收情况为1%～10%;在4 h的实验过程中,4种浓度蜕皮甾酮的ER值均小于1.5。结论研究表明,蜕皮甾酮主要以被动扩散的方式被细胞摄取和转运,其跨膜转运特性未受到CYP3A4-介导机制的影响。     

High glucose concentration in isotonic media alters Caco-2 cell permeability

Caco-2 cell permeability was evaluated in isotonic media containing high (25mM) or physiological (5.5mM) glucose concentrations. Transepithelial electrical resistance (TEER) and membrane fluidity were measured to assess glucose-induced alterations in physical barrier properties. In parallel, distribution of the actin filament (F-actin) and zonula occludens-1 (ZO-1) proteins was assessed by confocal microscopy. Transepithelial fluxes of mannitol, hydrocortisone, digoxin, and glycyl sarcosine (Gly-Sar) that permeate the intestinal mucosa by various pathways were measured to quantify the effect of glucose-induced changes on Caco-2 cell permeability. High glucose decreased maximum TEER of cell monolayers by 47%, whereas membrane fluidity at the hydrophobic core and lipid/polar head interphase was significantly increased. F-actin distribution in high glucose cells appeared more diffuse while ZO-1 was unchanged. Mannitol and hydrocortisone fluxes across Caco-2 cells cultured in high glucose increased by 65% and 24%, respectively. In addition, high glucose decreased the maximum transport capacity (Vmax) of PepT-1. P-glycoprotein activity, however, was unchanged. In conclusion, high extracellular glucose concentration in isotonic media significantly alters physical barrier properties of Caco-2 cell monolayers, which predominantly affects transepithelial transport of solutes permeating the cell barrier by paracellular and transcellular passive diffusion and facilitated transport mediated by the proton-dependent oligopeptide transporter (PepT-1).     

Transfer of lipophilic markers from PLGA and polystyrene nanoparticles to caco-2 monolayers mimics particle uptake

Purpose. The objective of this study was to evaluate nanoparticle uptake by the Caco-2 monolayer model in vitro. Special emphasis was placed on the localization and the quantification of the uptake of fluorescently labeled polystyrene and poly(lactic-co-glycolic acid) (PLGA) nanoparticles. Methods. Intracellular fluorescence was localized by fluorescence and confocal laser scanning microscopy. Particle uptake was quantified either directly, by counting internalized nanoparticles after separation from the Caco-2 monolayers, or indirectly, by extraction of the lipophilic fluorescence marker. In vitro release studies of lipophilic markers from nanoparticles were performed in standard buffer systems and buffer systems supplemented with liposomes. Results. Instead of uptake of polystyrene and PLGA nanoparticles by Caco-2 monolayers an efficient transfer of lipophilic fluorescence markers from nanoparticles into Caco-2 cells with subsequent staining of intracellular lipophilic compartments was observed. Whereas in standard buffer no release of fluorescent marker from polystyrene and PLGA nanoparticles was observed, the release studies using liposome dispersions as receiver revealed an efficient transfer of fluorescent marker into the liposome dispersion. Conclusions. The results suggest that the deceptive particle uptake is caused by a collision-induced process facilitating the transfer of lipophilic fluorescent marker by formation of a complex between the nanoparticles and the biomembranes. Diffusion of the marker within this complex into lipophilic compartments of the cell strongly affects quantitative evaluation of particle uptake.     

Nanoemulsion-based delivery system for enhanced oral bioavailability and Caco-2 cell monolayers permeability of berberine hydrochloride

Berberine hydrochloride (BBH) has a variety of pharmacological activities such as antitumor, antimicrobial, anti-inflammation, and reduce irritable bowel syndrome. However, poor stability and low oral bioavailability limited its usage. Herein, an oil-in-water nanoemulsion system of BBH was developed to improve its stability and oral bioavailability. The pseudoternary phase diagrams were constructed for the determination of composition of various nanoemulsions. The nanoemulsions of BBH composed of Labrafil M 1944 CS (oil phase), RH-40 (surfactant), glycerin (co-surfactant), and water (aqueous phase). The O/W nanoemulsion of BBH showed a relative bioavailability of 440.40% compared with unencapsulated BBH and was stable in our 6-month stability study. Further, there was a significant increase in intestinal permeability of BBH as assessed by Caco-2 cell monolayers and a significant reduction in efflux of BBH by the multidrug efflux pump P-glycoprotein. This study confirmed that the nanoemulsion formulation could be used as an alternative oral formulation of BBH to improve its stability, oral bioavailability and permeability.     

仙茅苷在Caco-2细胞模型中跨膜转运特征研究

目的：采用Caco-2细胞Transwell模型研究仙茅苷的跨膜转运机制。方法：建立Caco-2细胞Transwell吸收模型,用聚酯碳酸酯膜培养Caco-2细胞21天,形成致密的单层细胞模型。研究浓度、时间、温度、细胞旁路转运及跨膜转运蛋白（P-糖蛋白,多药耐药蛋白,乳腺癌耐药蛋白）在仙茅苷跨膜转运中的作用。结果：仙茅苷在Caco-2细胞模型中的跨膜转运存在一定的浓度及时间依赖性,细胞旁路转运不参与其转运,仙茅苷的外排转运存在一定的能量依赖性,且由P-gp参与。结论：仙茅苷在Caco-2细胞模型上主要以主动转运方式作跨膜转运,且P-gp参与其跨膜转运。     

LC-MS测定雷公藤甲素及其在Caco-2细胞上的转运特征

目的：建立体外模拟体内肠道细胞的Caco-2细胞Transwell模型,以此研究雷公藤甲素在Caco-2细胞模型上的跨膜转运特征。方法：采用聚酯碳酸酯膜连续培养Caco-2细胞21天,形成致密的单层细胞模型。然后对影响雷公藤甲素在Caco-2细胞模型上转运特征的因素包括浓度、时间及跨膜转运蛋白（P-糖蛋白,多药耐药蛋白,乳腺癌耐药蛋白）进行考察;同时采用LC-MS对溶液中的雷公藤甲素的含量进行测定。结果：雷公藤甲素主要以主动转运的方式进行吸收,且随着时间和药物浓度的增加,转运量明显增加。结论：雷公藤甲素在Caco-2细胞上转运存在一定的浓度及时间依赖性,且P-gp介导雷公藤甲素在Caco-2细胞上转运。     

Effects of superporous hydrogels on paracellular drug permeability and cytotoxicity studies in Caco-2 cell monolayers

The aim of this study was to evaluate the effect of superporous hydrogel (SPH) and SPH composite (SPHC) as permeation enhancers for peptide drug delivery on Caco-2 cell monolayers. Moreover, the cytotoxic effects of these polymers were also studied using trypan blue test, MTT assay and propidium iodide staining. Transepithelial electrical resistance (TEER) studies revealed that both SPH and SPHC polymers were able to decrease TEER values to about 40% of initial values, indicating the ability of these polymers to open tight junctions. Recovery studies of TEER showed that the effects of polymers on Caco-2 cell monolayers were reversible, indicating viability of the cells after incubation with polymers. Both polymers were able to enhance the transport of the hydrophilic marker 14C-mannitol up to 2.7 and 3.8-fold in comparison to the control group. The cumulative transport of fluorescein isothiocyanate labelled dextrans with a molecular weight of 4400 Da (FD4) and 19600 Da (FD20) was enhanced by SPH and SPHC polymers by opening of tight junctions; however, this enhancement was inversely proportional to the molecular weight of marker compounds. Cytotoxicity studies confirmed that the transport enhancing properties of SPH and SPHC polymers were not caused by damage of the Caco-2 cell monolayers. The cells were able to exclude trypan blue as well as propidium iodide after incubation with SPH and SPHC polymers. MTT assay showed that the number of viable cells was higher than 95% after incubation with SPH and SPHC polymers. This indicates that the mitochondrial metabolic activities of the cells were preserved after application of the polymers.     

Oleic acid decreases BCRP mediated efflux of mitoxantrone in Caco-2 cell monolayers

Breast cancer resistance protein (BCRP) efflux restricts intestinal absorption of substances like heterocyclic amines, mycotoxins and certain human and veterinary drugs. Fat rich meals seem to increase absorption of drugs which are BCRP substrates or inhibitors. We therefore hypothesize that absorption of toxicants normally effluxed by BCRP are increased by fatty acids in food. Transport across and accumulation of ³H-Mitoxantrone (MXR) in Caco-2 cell monolayers were measured after 60 min exposure to emulsions of ³H-MXR (1 μM) and oleic acid (0.5–5 mM). In addition, BCRP gene expression (RT-PCR) and the amount of BCRP protein (Western blot) were measured in oleic acid exposed Caco-2 cells. Oleic acid increased transport of MXR in a concentration dependent manner and 2 mM oleic acid or higher increased accumulation of MXR in cells, without any signs of cytotoxicity. Gene expression of BCRP was increased after exposure to oleic acid for 6 h, but the amount of BCRP protein was not increased. In conclusion, oleic acid clearly induced BCRP gene expression and reduced BCRP mediated efflux, although the amount of BCRP in cells was not affected. Consequently, effects of fatty acids on BCRP mediated efflux are important to consider in risk assessment of toxicants in food.     

Caco-2细胞模型比较黄芩苷和黄芩苷滴丸在小肠的吸收

目的：比较黄芩苷和黄芩苷滴丸在小肠的吸收。方法：采用Caco-2细胞单层模型研究黄芩苷和黄芩苷滴丸由绒毛面到基底面的跨膜转运过程。通过测定黄芩苷和黄芩苷滴丸在Caco-2细胞模型的转运百分率及表观渗透系数（Papp）,比较二者的跨膜转运能力。结果：在细胞转运实验中,135min时,黄芩苷和黄芩苷滴丸的转运百分率分别为1.00%和6.25%,Papp分别为（0.664±0.103）×10-6cm·s-1和（4.462±1.10）×10-6cm·s-1,黄芩苷滴丸的Papp是黄芩苷的6倍。在跨膜转运180min内,黄芩苷滴丸的转运百分率与时间成正比。结论：将黄芩苷制成滴丸可能提高黄芩苷在小肠的吸收。     

Enhanced permeability of the antimicrobial agent 2,5-bis(4-amidinophenyl)furan across Caco-2 cell monolayers via its methylamidoidme prodrug

Purpose. DB75 [2,5-bis(4-amidinophenyl)furan] is a promising antimicrobial agent although it has poor oral potency. In contrast, its novel prodrug, 2,5-bis(4-amidinophenyl)furan-bis-O-methyl- amidoxime (DB289), has excellent oral potency. The mechanisms of transport of DB289 and DB75 across intestinal epithelium have been investigated in these studies to understand differences in their oral potency. Methods. Caco-2 cell monolayers were used as an in vitro model to examine the mechanisms of transport of DB289 and DB75. Samples collected from the transport studies were quantified using high-performance liquid chromatography with ultraviolet and fluorescence detection. Results. A low permeability coefficient (3.8 × 10^–7 cm/s for transport in apical [AP] to basolateral [BL] direction) and high sensitivity to extracellular Ca²⁺ suggest that AP to BL transport of DB75 across Caco-2 cell monolayers occurs predominantly via a paracellular route. DB289 has an 85-fold higher transport rate (322.0 × 10^–7 cm/s for transport in the AP to BL direction) across Caco-2 monolayers than that of DB75. This, with its insensitivity to extracellular Ca²⁺ indicates that AP to BL transport of DB289 across Caco-2 cell monolayers occurs predominantly via a transcellular route. Conclusions. DB75 is transported across Caco-2 cell monolayers predominantly via paracellular pathways, whereas the prodrug DB289 is transported via transcellular pathways. This could account for the much higher oral activity of DB289 over DB75.     

白术内酯类在人源Caco-2细胞单层模型中的肠吸收研究(英文)

采用人源Caco-2细胞单层模型,研究了倍半萜内酯类的白术内酯Ⅰ、Ⅱ和Ⅲ的肠渗透性。研究了它们从顶端到基底侧以及从基底侧到顶端的双向渗透性。HPLC法测定其浓度。白术内酯Ⅰ、Ⅱ、Ⅲ的P_app值皆在10^-5cm/s水平,表明其具有高肠渗透率和良好吸收的特性。它们的双向转运具有时间和浓度依赖性,提示其吸收转运的主要机制为被动扩散。此外,白术内酯Ⅰ的吸收转运可能部分存在主动转运机制。     

Effect of self-microemulsifying drug delivery systems containing Labrasol on tight junctions in Caco-2 cells

The aim of this study was to investigate the effect of two novel self-microemulsifying drug delivery systems (SMEDDS) containing Labrasol with different dilutions on tight junctions. Changes in barrier properties of Caco-2 cell monolayers, including transepithelial electrical resistance (TEER) and permeability to the paracellular marker, i.e., mannitol, were assessed in response to dilutions and surfactants contents within formulations. The cytotoxicity of SMEDDS and the effect of surfactants on Caco-2 cells were evaluated by the MTT. Changes in subcellular localization of the tight junction proteins, ZO-1 and F-actin, were examined by confocal laser scanning microscopy. Results demonstrated that negatively charged SMEDDS with different dilutions had no effect on the TEER, but significantly increased the permeability of mannitol. In contrast, the positively charged formulation showed a dilution-dependent reduction in TEER. A corresponding increase in mannitol permeability of up to 29.4-fold to 64.7-fold greater than the control was also observed across the monolayer. Labrasol with the concentration of 0.1 and 1% was shown to increase the permeability of mannitol by 4.6-fold and 33.8-fold, respectively. The mechanism of opening of tight junctions was found to involve F-actin-related changes and redistribution of ZO-1.     

9种中药提取物细胞毒性及其与人结肠腺癌细胞系单层细胞旁通透性的相关性研究

目的了解9种中药提取物对Caco-2细胞毒性与对其细胞单层细胞-细胞间通透性的相关性。方法用MTT法测定9种中药提取物24 h及48 h的细胞毒,并计算IC50值。采用TranswellTM培养板构建Caco-2细胞单层,细胞电阻仪测定受试物对其TEER值的影响,找到影响细胞单层紧密连接的临界剂量,以此剂量与细胞毒参数行相关分析。结果 24、48 h细胞毒参数(IC50)与临界剂量(最大无毒剂量、最小有毒剂量)相关系数均>0.84,相关性检验P值均<0.01。最大无毒剂量与IC50的比值列阵显示,《小品方》甘草饮最小,三七醇提物最大。结论 9种中药提取物对Caco-2细胞毒与该细胞单层细胞旁通透性高度相关,采用细胞毒参数来预估中药提取物在Caco-2单层细胞模型供侧起始最大浓度具可行性。     

Characterization of the transport of uracil across Caco-2 and LLC-PK1 cell monolayers

Purpose. The purpose of this study was to characterize the transport of uracil, a pyrimidine nucleobase, in Caco-2 and LLC-PK₁ cells. Methods. Caco-2 and LLC-PK₁ cells were grown to confluency on a permeable polycarbonate membrane insert to permit transport and uptake experiments after the loading of uracil on either the apical or basolateral side. Results. The vectorial transport of uracil in both directions was saturable with comparable K_m and V_max in Caco-2 cell monolayers, probably because of a Na⁺-independent transport system located on the basolateral membrane. In LLC-PK₁ cell monolayers, two distinct transport systems, namely a Na⁺-dependent and a Na⁺-independent, were functional in the apical to basolateral (A-B) transport of uracil. The first system was saturable with a K_m value of 3.67 ± 0.40 M, a V_max of 11.31 ± 0.91 pmol/cm²/min, and a Na⁺:uracil coupling stoichiometry of 1.28 ± 0.20. The second system was Na⁺ independent and satuable with a low affinity (K_m, 50.37 ± 9.61 M) and V_max (16.01 ± 4.48 pmol/cm²/min). The two transport systems appeared to be located on the apical membrane. Conclusion. The mechanism of uracil transport differs depending on cell lines; a Na⁺-independent system on the basolateral membrane in Caco-2 cells and both Na⁺-dependent and Na⁺-independent systems on the apical membrane in LLC-PK₁ cells seem to be responsible for the difference.     

黄连素对P-糖蛋白底物在Caco-2和L-MDR1细胞跨膜转运的影响

目的研究黄连素(berberine,Ber)对P-糖蛋白底物环孢素(cyclosporine A,CsA)和地高辛在Caco-2和L-MDR1细胞跨膜转运的影响。方法以Caco-2、L-MDR1细胞为模型,在50μmol·L-1～5mmol·L-1Ber作用后,测定地高辛和CsA跨膜转运的转运率和表观渗透系数。结果Ber在50μmol·L-1～5mmol·L-1范围内可剂量依赖性地降低地高辛在Caco-2和L-MDR1细胞单层底端(B侧)至顶端(A侧)的转运。对于CsA在Caco-2细胞的转运,50μmol·L-1～5mmol·L-1的Ber不但剂量依赖性地降低CsA从B→A方向的转运,而且也明显增加其在A→B方向的转运。在Caco-2和L-MDR1细胞,Ber抑制地高辛B→A方向转运的IC50值分别为1.44mmol·L-1和1.24mmol·L-1。Ber抑制CsA在Caco-2细胞转运的IC50值为607μmol·L-1。结论Ber和CsA相互作用的机制可能涉及P-gp功能的抑制和饱和。