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Accumulation of transferrin in Caco-2 cells: A possible mechanism of intestinal transferrin absorption
Abstract
Transferrin receptor (TfR)-mediated endocytosis and transcytosis in enterocyte-like Caco-2 cells was investigated in order to elucidate the transport mechanism of orally administered Tf-fusion proteins. Cellular uptake and pulse chase studies were performed in Caco-2, MCF-7 and bladder carcinoma (5637) cells using 125I-labeled Tf (125I-Tf). Co-localization studies of Rab 11 and FITC-Tf endocytosed at either the apical or basolateral membrane were performed in polarized Caco-2 cells grown on Transwells, using confocal laser scanning microscopy (LSM510, Zeiss). Unlike in MCF-7 or 5637 cells, where rapid recycling of Tf was observed, a significant amount of endocytosed 125I-Tf accumulated in Caco-2 cells. This accumulation was especially noticeable with the internalization of 125I-Tf from the apical membrane of polarized Caco-2 cells. Confocal microscopy studies showed that apically, but not basolaterally, endocytosed FITC-Tf was delivered to a Rab11-positive compartment. Our results suggest that a significant amount of apically endocytosed Tf in intestinal epithelial cells is transported to a Rab11-positive compartment, possibly a late endosomal and slow recycling compartment. The Rab11-positive compartment may control the release of apically internalized Tf for either slow recycling to apical membrane or processing to transcytotic compartments.
... Several permeability data obtained with this model even showed a strong correlation with in vivo results in humans, in particular for passive transport [94,95]. This simple and low-cost approach has also been used to study the active transport of macromolecules such as insulin [96,97], vitamin B12 [98], or transferrin [99]. However, obtaining primary cell cultures from intestinal tissues is still challenging, mostly because of the short life span of enterocytes, the dependency of cells on the extracellular matrix, and their incapacity to form an organized polarized monolayer of epithelial cells [83]. ...
... Several permeability data obtained with this model even showed a strong correlation with in vivo results in humans, in particular for passive transport [94,95]. This simple and low-cost approach has also been used to study the active transport of macromolecules such as insulin [96,97], vitamin B12 [98], or transferrin [99]. ...
... Several permeability data obtained with this model even showed a strong correlation with in vivo results in humans, in particular for passive transport [94,95]. This simple and low-cost approach has also been used to study the active transport of macromolecules such as insulin [96,97], vitamin B12 [98], or transferrin [99]. In summary, although this model is simple, widely used, cheap, commercially available, and the data obtained with this approach are very informative for a preliminary screening, complementary studies using more complex models are needed. ...
Recombinant biological molecules are at the cutting-edge of biomedical research thanks to the significant progress made in biotechnology and a better understanding of subcellular processes implicated in several diseases. Given their ability to induce a potent response, these molecules are becoming the drugs of choice for multiple pathologies. However, unlike conventional drugs which are mostly ingested, the majority of biologics are currently administered parenterally. Therefore, to improve their limited bioavailability when delivered orally, the scientific community has devoted tremendous efforts to develop accurate cell- and tissue-based models that allow for the determination of their capacity to cross the intestinal mucosa. Furthermore, several promising approaches have been imagined to enhance the intestinal permeability and stability of recombinant biological molecules. This review summarizes the main physiological barriers to the oral delivery of biologics. Several preclinical in vitro and ex vivo models currently used to assess permeability are also presented. Finally, the multiple strategies explored to address the challenges of administering biotherapeutics orally are described.
... In this intestinal segment it is thought to mediate apical internalization of specific proteins, such as the intrinsic factor-vitamin B12 complex [15] or transferrin (Tf) [16]. In addition, apical Tf uptake and transcytosis has been linked to a Tf receptor in vitro [11,17,18]. In the kidney, apical receptor-mediated-endocytosis (RME) of proteins and peptides has been well characterized. ...
... Previous reports have indicated that MT and PC [8,9] and other proteins, such as HIV or immunoglobulins [3,18,44], may be taken up by the intestine in intact form. Moreover, transcytosis of Tf has been demonstrated in Caco-2 cells [11,17,45]. We have previously demonstrated that MT and Tf are ligands of r24p3-R/hNGAL-R [24], therefore, we investigated whether hNGAL-R-mediated uptake of PC 3 , MT and Tf is associated with transcytosis of these proteins in colon-like Caco-2 BBE cells. ...
... So far, in vivo studies have provided limited evidence for expression of the megalin/ cubilin/amnionless receptor complex in the terminal ileum [13,14] (reviewed in 15, which plays a role in apical endocytosis of he intrinsic factor-vitamin B12 complex and possibly Tf [16]. Moreover, the Tf receptor may mediate apical uptake and transcytosis of Tf though, in contrast to in vitro studies [11,17,18], evidence for apical expression of the Tf receptor in vivo is sparse [18]. Here we focussed on the possibility that endo-/transcytosis of proteins, such as Tf, but also MT and PC 3 , may also be mediated by the r24p3-R/ hNGAL-R in the intestine. ...
The lipocalin 2//NGAL/24p3 receptor (NGAL-R/24p3-R) is expressed in rodent distal nephron where it mediates protein endocytosis. The mechanisms of apical endocytosis and transcytosis of proteins and peptides in the intestine are poorly understood. In the present study, the expression and localization of rodent 24p3-R (r24p3-R) and human NGAL-R (hNGAL-R) was investigated in intestinal segments by immunofluorescence and confocal laser scanning microscopy, immunohistochemistry and immunoblotting. r24p3-R/hNGAL-R was also studied in human Caco-2 BBE cells and CHO cells transiently transfected with r24p3-R by immunofluorescence microscopy, RT-PCR and immunoblotting of plasma membrane enriched vesicles (PM). To assay function, endocytosis/transcytosis of putative ligands phytochelatin (PC3), metallothionein (MT) and transferrin (Tf) was assayed by measuring internalization of fluorescence-labelled ligands in Caco-2 BBE cells grown on plastic or as monolayers on Transwell inserts. The binding affinity of Alexa 488-PC3 to colon-like Caco-2 BBE PM was quantified by microscale thermophoresis (MST). r24p3-R/hNGAL-R expression was detected apically in all intestinal segments but showed the highest expression in ileum and colon. Colon-like, but not duodenum-like, Caco-2 BBE cells expressed hNGAL-R on their surface. Colon-like Caco-2 BBE cells or r24p3-R transfected CHO cells internalized fluorescence-labelled PC3 or MT with half-maximal saturation at submicromolar concentrations. Uptake of PC3 and MT (0.7 µM) by Caco-2 BBE cells was partially blocked by hNGAL (500 pM) and an EC 50 of 18.6 ± 12.2 nM was determined for binding of Alexa 488-PC3 to PM vesicles by MST. Transwell experiments showed rapid (0.5-2 h) apical uptake and basolateral delivery of fluorescent PC3/MT/Tf (0.7 µM). Apical uptake of ligands was significantly blocked by 500 pM hNGAL. hNGAL-R dependent uptake was more prominent with MT but transcytosis efficiency was reduced compared to PC3 and Tf. Hence, r24p3-R/hNGAL-R may represent a high-affinity multi-ligand receptor for apical internalization and transcytosis of intact proteins/peptides by the lower intestine.
... We have previously reported that the amount of endocytosed hTF increases slowly and is accumulated linearly in enterocyte-like Caco-2 cells, but reaches a plateau rapidly in most other cell lines including HeLa cells due to the rapid recycling and release [29]. To investigate if rhTF's endocytosis is similar to that of hTF, the endocytosis kinetics of rhTF and hTF were assayed in HeLa and Caco-2 cells. ...
... This result is consistent with our previous finding in a competitive immunoassay that rhTF is equivalent to baby hamster kidney (BHK) cell derived recombinant N-His hTF in its ability to bind to the soluble portion of the TFR [15]. It is also in good agreement with other reports that hTF's internalization increases linearly with time in enterocyte-like Caco-2 cells due to this type of cells' unique accumulation of TF [29]. These results indicate that rhTF is similar to hTF in its ability of binding to human TFR, and then being endocytosed through TFR-mediated pathway. ...
Background
Transferrin (TF) plays a critical physiological role in cellular iron delivery via the transferrin receptor (TFR)-mediated endocytosis pathway in nearly all eukaryotic organisms. Human serum TF (hTF) is extensively used as an iron-delivery vehicle in various mammalian cell cultures for production of therapeutic proteins, and is also being explored for use as a drug carrier to treat a number of diseases by employing its unique TFR-mediated endocytosis pathway. With the increasing concerns over the risk of transmission of infectious pathogenic agents of human plasma-derived TF, recombinant hTF is preferred to use for these applications. Here, we carry out comparative studies of the TFR binding, TFR-mediated endocytosis and cellular iron delivery of recombinant hTF from rice (rhTF), and evaluate its suitability for biopharmaceutical applications.
Result
Through a TFR competition binding affinity assay with HeLa human cervic carcinoma cells (CCL-2) and Caco-2 human colon carcinoma cells (HTB-37), we show that rhTF competes similarly as hTF to bind TFR, and both the TFR binding capacity and dissociation constant of rhTF are comparable to that of hTF. The endocytosis assay confirms that rhTF behaves similarly as hTF in the slow accumulation in enterocyte-like Caco-2 cells and the rapid recycling pathway in HeLa cells. The pulse-chase assay of rhTF in Caco-2 and HeLa cells further illustrates that rice-derived rhTF possesses the similar endocytosis and intracellular processing compared to hTF. The cell culture assays show that rhTF is functionally similar to hTF in the delivery of iron to two diverse mammalian cell lines, HL-60 human promyelocytic leukemia cells (CCL-240) and murine hybridoma cells derived from a Sp2/0-Ag14 myeloma fusion partner (HB-72), for supporting their proliferation, differentiation, and physiological function of antibody production.
Conclusion
The functional similarity between rice derived rhTF and native hTF in their cellular iron delivery, TFR binding, and TFR-mediated endocytosis and intracellular processing support that rice-derived rhTF can be used as a safe and animal-free alternative to serum hTF for bioprocessing and biopharmaceutical applications.
... Their contribution to nutrient uptake is well established, notably in the small intestine 21 . Examples are fat-soluble vitamins 22 , the iron transporter transferrin 23,24 , and the glucose clearance hormone insulin 25 . All these are internalized by endocytosis from the apical membrane that faces the intestinal lumen, and then released after intracellular trafficking on the opposing basolateral side to reach the bloodstream. ...
A host of endocytic pathways exist at the surface of eukaryotic cells, which lead to the internalization of the bulk of membranes along with membrane proteins, signaling receptors, growth factors, and other cargoes (Smith et al. 2017). For decades, the clathrin-mediated pathway has been the major well characterized endocytic process where clathrin polymerizes along with the associated adaptor proteins to include ligand-bound receptors, leading to membrane bending, membrane scission, and endocytosis (Smith et al. 2017). Recently, multiple alternative mechanisms have been uncovered which facilitate the endocytic uptake of cargo molecules and membrane receptors even in the absence of clathrin machinery (Mayor et al.2014). A model of endocytosis that doesn’t require clathrin but rather sugar-binding galectins and glycolipids has been proposed by my host laboratory (Lakshminarayan et al. 2014). Galectins constitute a family of beta-galactoside–binding lectins, which to date consists of 15 members in mammals. Galectins are broadly distributed in a variety of cells and tissues (Leffler et al. 2004). They are translocated from the cytosol to the extracellular space by a process of non-classical secretion (Hughes 1999). Glycosphingolipids (GSLs) are ubiquitous membrane constituents that are subdivided in neutral or acidic fractions. The term GSLs applies to compounds that contain at least one monosaccharide and a ceramide. Of note, the enzyme UDP-glucose ceramide glucosyltransferase (Ugcg) catalyzes the initial step for the biosynthesis of glycosylceramide-based GSLs.Our current working model, which involves glycolipids and lectins, was termed the GL-Lect hypothesis (Johannes et al. 2016). It is backed up by experimental data as described in Ref. (Lakshminarayan et al. 2014) and can be described as follows:i) Monomeric Gal3 binds to glycoproteinsii) Gal3 then starts to oligomerizeiii) Oligomerized Gal3 has the capacity to bind to glycosphingolipids and this may induce clustering of GSLsiv) Gal3-GSL cluster are inducing the invagination of the plasma membrane to generate tubular endocytic pits from which clathrin-independent carriers (CLICs, which are pre-early endosomes) are generated.Oligomeric Gal3 is indeed able to bind to GSLs and to induce membrane deformation (Lakshminarayan et al. 2014) in a similar way the pathogenic lectin Shiga toxin-B subunit (STxB) does. Therefore, both processes could be summarized under the same hypothesis, the GL-Lect hypothesis, where GL stands for the glycosphingolipids (Gb3 for STxB and gangliosides for Gal3) and Lect summarizes the lectins (STxB, Gal3 and possibly others as well).Understanding if this clathrin-independent but Gal3-dependent internalization mechanism is conserved not only in vitro model systems but in vivo is a main challenge in the field of trafficking.We characterized for the first time that in the gut a new mechanism facilitates endocytic uptake of cargo. This mechanism is driven by Galectin3 and operates in intestinal enterocytes for transcytosis like process and is glycosphingolipid dependent. Indeed, we have found that the lactotransferrin (LTF), a Gal3 cargo that we have identified by Mass spec, strongly required Gal3 and GSLs for its efficient endocytosis and its transcytosis like distribution pattern, respectively. Based on these findings in mouse intestinal epithelium, we established a functional in vivo model system where the newly proposed endocytic mechanism termed in our lab as GL-Lect, was physiologically investigated.
... The Tf was required to generate the Tf-TfR complex, the target of CRT, and therefore, the presence of Tf increased the effectiveness of TMC-CRT NPs to facilitate cellular insulin uptake. The transport of nanoparticles via TfR is mediated in apical-tobasolateral direction (Lim et al., 2007;Norouziyan et al., 2008;Liu et al., 2018). This study demonstrates that a judiciously selected ligand can reverse the antagonistic effect of endogenous ligands in receptor-mediated cellular uptake of exogenous therapeutic compounds. ...
Subcutaneous and inhaled insulins are associated with needle phobia, lipohypertrophy, lipodystrophy, and cough in diabetes treatment. Oral nanoinsulin has been developed, reaping the physiologic benefits of peroral administration. This review profiles intestinal receptors exploitable in targeted delivery of oral nanoinsulin. Intestinal receptor targeting improves oral insulin bioavailability and sustains blood glucose-lowering response. Nonetheless, these studies are conducted in small animal models with no optimization of insulin dose, targeting ligand type and content, and physicochemical and molecular biologic characteristics of nanoparticles against the in vivo/clinical diabetes responses as a function of the intestinal receptor population characteristics with diabetes progression. The interactive effects between nanoinsulin and antidiabetic drugs on intestinal receptors, including their up-/downregulation, are uncertain. Sweet taste receptors upregulate SGLT-1, and both have an undefined role as new intestinal targets of nanoinsulin. Receptor targeting of oral nanoinsulin represents a viable approach that is relatively green, requiring an in-depth development of the relationship between receptors and their pathophysiological profiles with physicochemical attributes of the oral nanoinsulin. SIGNIFICANCE STATEMENT: Intestinal receptor targeting of oral nanoinsulin improves its bioavailability with sustained blood glucose-lowering response. Exploring new intestinal receptor and tailoring the design of oral nanoinsulin to the pathophysiological state of diabetic patients is imperative to raise the insulin performance to a comparable level as the injection products.
... Their contribution to nutrient uptake is well established, notably in the small intestine 22 . Examples are fat-soluble vitamins 23 , the iron transporter transferrin 24,25 , and the glucose-clearance hormone insulin 26 . All these are internalized by endocytosis from the apical membrane that faces the intestinal lumen and then released after intracellular trafficking on the opposing basolateral side to reach the bloodstream. ...
The GlycoLipid-Lectin (GL-Lect) hypothesis provides a conceptual framework to explain how endocytic pits are built in processes of clathrin-independent endocytosis. According to this hypothesis, oligomeric cellular or pathogenic lectins interact with glycosylated plasma membrane lipids in a way such as to drive the formation of tubular endocytic pits that then detach to generate clathrin-independent endocytic carriers for the cellular uptake of cellular or pathogenic products. This process operates in a complementary manner to the conventional clathrin pathway for biological function linked to cell polarity. Up to date, the premises of the GL-Lect hypothesis have been based on model membrane and cell culture experiments. It has therefore become urgent to extend its exploration to complex organisms. In the current protocol, we describe methods to study the endocytosis and transcytosis of a key driver of the GL-Lect mechanism, the cellular galectin-3, and of one of its cargoes, lactotransferrin, in enterocytes of the intact jejunum of mice. In a step-by-step manner, we present the generation of fluorescent endocytic ligands, tissue preparation for cellular uptake measurements, binding and internalization assays, tissue fixation and preparation for sectioning, light and electron microscopical observations, and quantification of data by image processing. Pitfalls are discussed to optimize the chances of success with the described methods.
... In order to figure out whether this man-made ligand of TfR could also be used as a carrier for drug delivery like hTf, the transcytosis efficiency of HG1-9 across epithelium barrier was evaluated. Caco-2 cells are enterocyte-like cells and often used as the intestinal epithelial cells to investigate the intracellular processing of internalized Tf [29]. Thus, an epithelium model was established by growing Caco-2 cell monolayer in a transwell culture plate. ...
General cancer-targeted ligands that can deliver drugs to cells have been given considerable attention. In this paper, a high-affinity DNA aptamer (HG1) generally binding to human tumor cells was evolved by cell-SELEX, and was further optimized to have 35 deoxynucleotides (HG1-9). Aptamer HG1-9 could be taken up by live cells, and its target protein on a cell was identified to be human transferrin receptor (TfR). As a man-made ligand of TfR, aptamer HG1-9 was demonstrated to bind at the same site of human TfR as transferrin with comparable binding affinity, and was proved to cross the epithelium barrier through transferrin receptor-mediated transcytosis. These results suggest that aptamer HG1-9 holds potential as a promising ligand to develop general cancer-targeted diagnostics and therapeutics.
... Their contribution to nutrient uptake is well established, notably in the small intestine 22 . Examples are fat-soluble vitamins 23 , the iron transporter transferrin 24,25 , and the glucose-clearance hormone insulin 26 . All these are internalized by endocytosis from the apical membrane that faces the intestinal lumen and then released after intracellular trafficking on the opposing basolateral side to reach the bloodstream. ...
Glycoproteins and glycolipids at the plasma membrane contribute to a range of functions from growth factor signaling to cell adhesion and migration. Glycoconjugates undergo endocytic trafficking. According to the glycolipid-lectin (GL-Lect) hypothesis, the construction of tubular endocytic pits is driven in a glycosphingolipid-dependent manner by sugar-binding proteins of the galectin family. Here, we provide evidence for a function of the GL-Lect mechanism in transcytosis across enterocytes in the mouse intestine. We show that galectin-3 (Gal3) and its newly identified binding partner lactotransferrin are transported in a glycosphingolipid-dependent manner from the apical to the basolateral membrane. Transcytosis of lactotransferrin is perturbed in Gal3 knockout mice and can be rescued by exogenous Gal3. Inside enterocytes, Gal3 is localized to hallmark structures of the GL-Lect mechanism, termed clathrin-independent carriers. These data pioneer the existence of GL-Lect endocytosis in vivo and strongly suggest that polarized trafficking across the intestinal barrier relies on this mechanism.
... Thus, modifying drugs or drugs-loaded nanoparticles by coupling a special targeting molecule could be an efficient way to enhance the intestinal epithelial cell permeability of drugs. Some investigators have used the targeting ligands such as folic acid [57,58], vitamin B12 [59][60][61][62], transferring [63][64][65], lectins [66,67] to facilitate macromolecule drugs absorption. In this way, the affinity between drugs or drugs-loaded nanoparticles and intestinal epithelial cells is strengthened. ...
Heparins show great anticoagulant effect with few side effects, and are administered by subcutaneous or intravenous route in clinics. To improve patient compliance, oral administration is an alternative route. Nonetheless, oral administration of heparins still faces enormous challenges due to the multiple obstacles. This review briefly analyzes a series of barriers ranging from poorly physicochemical properties of heparins, to harsh biological barriers including gastrointestinal degradation and pre-systemic metabolism. Moreover, several approaches have been developed to overcome these obstacles, such as improving stability of heparins in the gastrointestinal tract, enhancing the intestinal epithelia permeability and facilitating lymphatic delivery of heparins. Overall, this review aims to provide insights concerning advanced delivery strategies facilitating oral absorption of heparins.
... Transferrin receptor (TfR)-mediated endocytosis and transcytosis in intestinal Caco-2 cells was previously investigated using Tf-fusion proteins. It was shown that apically-endocytosed Tf in Caco-2 cells is transported to a Rab11-positive compartment, which may control its release to follow either slow recycling to apical membrane or progress to transcytotic compartments [26]. Shah and Shen studied the transport of Tf-conjugated insulin across Caco-2 cell monolayers and reported that transport of the complex, which was mediated via the TfR, was increased by 5-to 15-fold compared to free insulin [16]. ...
The aim of this study was to probe whether the transferrin (Tf) transport pathway can be exploited for intestinal delivery of nanoparticles. Tf was adsorbed on 100 nm model polystyrene nanoparticles (NP), followed by size characterisation of these systems. Cell uptake of Tf and Tf-adsorbed NP was investigated in intestinal epithelial Caco-2 cells cultured on multi-well plates and as differentiated polarised monolayers. Tf-NP demonstrated a remarkably higher cell uptake compared to unmodified NP in both non-polarised (5-fold) and polarised cell monolayers (16-fold difference). Application of soluble Tf significantly attenuated the uptake of Tf-NP. Notably, Tf-NP displayed remarkably higher rate (23-fold) of epithelial transport across Caco-2 monolayers compared to unmodified NP. This study therefore strongly suggests that the Tf transport pathway should be considered as a candidate biological transport route for orally-administered nanomedicines and drugs with poor oral bioavailability.
... Physiologically relevant intestinal epithelial cell models are needed in order to characterize cell-material interactions. Of the various intestinal endothelial models, human adenocarcinoma Caco-2 cells are particularly suitable [35] as, when cultured under specific and well-established conditions [36], they spontaneously differentiate, expressing several morphological and biochemical characteristics of small intestinal enterocytes [37], including the TfR [38]. The last stage in the characterization of a mucoadhesive material is its performance in the most realistic and adverse environment, namely in vivo, where rats are frequently used as the in vivo model for mucoadhesive tests [39,40]. ...
The development of mucoadhesive materials is of great interest and is also a major challenge. Being adsorption sites, mucosae are suitable targets for drug delivery, but as defensive barriers they are complex biological surfaces to interact with, mainly due to their protective mucus layer. As such, first- and second-generation mucoadhesives focused on material-mucus interactions, whereas the third generation of mucoadhesives introduced structural motifs that are able to interact with the cells beneath the mucus layer. The combination of different prerequisites (water solubility, soft gel formation at body temperature and able to interact with the mucus) in a single molecule is easily achieved using elastin-like recombinamers (ELRs) given their multiple block design. Moreover, we have been able to introduce a short amino-acid sequence known as T7 that is able to bind to transferrin receptors in the epithelial cell layer. The T7 sequence enhances the cell-binding properties of the mucoadhesive ELR (MELR), as demonstrated using a Caco-2 epithelial cell model. In vivo experiments confirmed the mucoadhesive properties found in vitro.
Statement of Significance
The development of a mucoadhesive material is a major challenge. Mucosae are suitable targets for drug delivery, but as defense barriers, they are complex surfaces to interact with. In this work we report the first ELR that combines different functional blocks, in a single molecule, which provide it with the properties of soft-gel forming at body temperature and being able of efficiently adhering to the mucus layer of mucosas, as well as to the underlying epithelial cell layer, as demonstrated in vitro and in vivo.
The rationally designed materials presented in this work sets the basis for developing ELR-based, mucosa-directed drug delivery systems, which could improve patient’s compliance, enhancing drug retention at the mucosal site.
... TfR-mediated transcytosis has been shown to be an effective approach for the delivery of proteins and peptides across the intestinal epithelium (Widera et al., 2003). However, the limitation of this approach lies in the low rate of apical-to-basolateral transcytosis for TfR (Lim et al., 2007). ...
Non-parenteral delivery of drugs using nanotechnology-based delivery systems is a promising non-invasive way to achieve effective local or systemic drug delivery. The efficacy of drugs administered non-parenterally is limited by their ability to cross biological barriers, and epithelial tissues particularly present challenges. Polymeric micelles can achieve transepithelial drug delivery because of their ability to be internalized into cells and/or cross epithelial barriers, thereby delivering drugs either locally or systematically following non-parenteral administration. This review discusses the particular characteristics of various epithelial barriers and assesses their potential as non-parenteral routes of delivery. The material characteristics of polymeric micelles (e.g., size, surface charge, and surface decoration) and of unimers dissociated from polymeric micelles determine their interactions (non-specific and/or specific) with mucus and epithelial cells as well as their intracellular fate. This paper outlines the mechanisms governing the major modes of internalization of polymeric micelles into epithelial cells, with an emphasis on specific recent examples of the transport of drug-loaded polymeric micelles across epithelial barriers.
Therapeutic release from hydrogels is traditionally controlled by encapsulation within nanoparticles; however, this strategy is limited for the release of proteins due to poor efficiency and denaturation. To overcome this problem, we designed an encapsulation-free release platform where negatively charged proteins are adsorbed to the exterior of transiently cationic nanoparticles, thus allowing the nanoparticles to be formulated separately from the proteins. Release is then governed by the change in nanoparticle surface charge from positive to neutral. To achieve this, we synthesized eight zwitterionic poly(lactide-block-carboxybetaine) copolymer derivatives and formulated them into nanoparticles with differing surface chemistry. The nanoparticles were colloidally stable and lost positive charge at rates dependent on the hydrolytic stability of their surface ester groups. The nanoparticles (NPs) were dispersed in a physically cross-linked hyaluronan-based hydrogel with one of three negatively charged proteins (transferrin, panitumumab, or granulocyte-macrophage colony-stimulating factor) to assess their ability to control release. For all three proteins, dispersing NPs within the gels resulted in significant attenuation of release, with the extent modulated by the hydrolytic stability of the surface groups. Release was rapid from fast-hydrolyzing ester groups, reduced with slow-hydrolyzing bulky ester groups, and very slow with nonhydrolyzing amide groups. When positively charged lysozyme was loaded into the nanocomposite gel, there was no significant attenuation of release compared to gel alone. These data demonstrate that electrostatic interactions between the protein and NP are the primary driver of protein release from the hydrogel. All released proteins retained bioactivity as determined with in vitro cell assays. This release strategy shows tremendous versatility and provides a promising new platform for controlled release of anionic protein therapeutics.
Conjugation of proteins to drug-loaded polymeric structures is an attractive strategy for facilitating target-specific drug delivery for a variety of clinical needs. Polymers currently available for conjugation to proteins generally have limited chemical versatility for subsequent drug loading. Many polymers that do have chemical functionality useful for drug loading are often insoluble in water, making it difficult to synthesize functional protein-polymer conjugates for targeted drug delivery. In this work, we demonstrate that reactive, azlactone-functionalized polymers can be grafted to proteins, conjugated to a small molecule fluorophore, and subsequently internalized into cells in a receptor-specific manner. Poly(2-vinyl-4,4-dimethylazlactone) (PVDMA) synthesized using reversible addition‐fragmentation chain transfer (RAFT) polymerization was modified post-polymerization with substoichiometric equivalents of triethylene glycol monomethyl ether (mTEG) to yield reactive water-soluble, azlactone-functionalized copolymers. These reactive polymers were then conjugated to proteins holo-transferrin and ovotransferrin. Protein gel analysis verified successful conjugation of proteins to polymer, and protein-polymer conjugates were subsequently purified from unreacted proteins and polymers using size exclusion chromatography. Internalization experiments using a breast cancer cell line that overexpresses the transferrin receptor on its surface showed that the holo-transferrin-polymer conjugate was successfully internalized by cells in a mechanism consistent with receptor-mediated endocytosis. Internalization of protein-polymer conjugate demonstrated that the protein ligand maintained its overall structure and function following conjugation to polymer. Our approach to protein-polymer conjugate synthesis offers a simple, tailorable strategy for preparing bioconjugates of interest for a broad range of biomedical applications.
Compartmentalisation is a precondition for the development of life, allowing concentration gradients to be maintained, facilitating selective transport of molecules, functional polarisation, protection of cells and tissues. Consequently, organisms have evolved highly sophisticated structures and mechanisms that allow compartmentalisation to be maintained and controlled in a highly regulated fashion.
Under normal conditions these compartmentalising structures are essential building blocks of life, their smooth functioning being central to our health. However, the same effectiveness that is a bonus under physiological conditions means the same structures may become considerable barriers to the pharmacotherapy of diseases, as access of drugs to the sites of disease may be severely restricted. This chapter describes the architecture, organisation, and function of key barriers that therapeutic nanoparticles may encounter for the most important routes of drug administration. The epithelial barriers (skin, mucosa of the airways, and gastrointestinal tract) and endothelial barriers share many commonalities as they all share key design elements that have evolved to support compartmentalisation.
Aim: To investigate the uptake of vitamin D 3 imbed by self-assembled sodium alginate nanoparticles labeled by FITC (sSAN-VD 3-FITC), and the influential factors with Caco-2 cells. And to study the transmembrane transport of it with Caco-2 cell absorption model. Methods: Laser scanning confocal microscope (LSCM) was utilized to exam the uptake of SSAN-VD 3-FITC absorbed by Caco-2 cells. The effect of drug incubation time on the uptake was analyzed. Caco-2 cell absorption model was used to study the transmembrane transport of sSAN-VD 3-FITC. The apparent permeability coefficient (Papp) was calculated and the fluorescence intensity was delected to quantitatively assay the influence of drug incubation time on the transmembrane transport. Results: It took only 1h for the appearance of sSAN-VD 3-FITC fluorescence particle diameter in Caco-2 cells. It was brighter when incubated for 4 h. The fluorescence intensity and the accum transport quantity of sSAN-VD 3-FITC group were higher than those of the control group (P < 0. 01), and they were higher while the time was longer (P < 0. 05). Papp was fairly stabilized, but it was smaller while the time was longer. Conclusions: sSAN-VD 3 can be uptaken by Caco-2 cells, and can also do transmembrane transport with Caco-2 cell absorption model in a time-dependent manner. It indicates that sSAN-VD 3 can be absorbed into blood via gastrointestinal tract pathway.
Our recent studies have demonstrated the potential of cell-penetrating peptides (CPPs) to significantly stimulate the intestinal absorption of therapeutic peptides and proteins. This study examined the mechanisms underlying the intestinal epithelial uptake and permeation of CPPs and their contribution to the enhanced absorption of insulin. Fluorescein-tagged octaarginine (R8) and penetratin were used as the promising CPPs, and in vitro uptake and permeation assays were conducted using Caco-2 cell monolayer. The assay conducted under low temperature conditions revealed that energy-dependent pathways are not involved in d-form arginines (d-R8) uptake or its stimulatory effect on insulin uptake. The Km value (3.82 μM), calculated from the dose dependence of d-R8 uptake, suggested that a part of the d-R8 uptake was saturated at the functional concentration (60 μM d-R8). An analysis based on the binding parameters of insulin and d-R8 also showed an increase in the uptake clearance of the insulin/d-R8 complex, even at a saturated concentration of d-R8, implying that this complex is taken up by Caco-2 cells via pathways that differ from those that take up unbound d-R8. Thus, this study suggests that CPPs such as oligoarginines stimulate the intestinal epithelial transport of peptide and protein drugs via energy-independent unsaturable internalization. © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci.
Transcellular transport is essential for transmucosal and plasma-to-tissue drug delivery by nanoparticles, whereas its fundamental pathways have not been fully clarified. In this study, an in-depth investigation was conducted into the intracellular itinerary and the transcytosis pathway of wheat germ agglutinin-functionalized nanoparticles (WGA-NP) with various polymer architectures in the Caco-2 cell model. GFP-Rabs, Rab4, Rab5, Rab7, Rab11, GTPases served as key regulators of vesicular transport, and their mutants were transfected to Caco-2 cells respectively to determine the cellular itinerary of WGA-NP and the role of Rabs therein. Transcytosis inhibition experiments indicated that transcellular transport of WGA-NP (PEG(3000)-PLA(40000) formulation) happened in a cytoskeleton-dependent manner and majorly by means of clathrin-mediated mechanism. Intracellular transport, especially the endolysosome pathway was found largely contribute to the transcytosis of WGA-NP. WGA-NP with shorter surface PEG length (2000) resulted in higher cellular association and more colocalization with the clathrin-mediated transport pathway, while that with longer surface PEG length (5000) avoided the clathrin-mediated transport pathway but achieved higher transcytosis after 4 h incubation. WGA-NP with PLGA as the core materials obtained elevated lysosome escape and enhanced transcytosis after 2 h incubation. These findings provided important evidence for the role of polymer architectures in modulating cellular transport of functionalized nanocarriers, and would be helpful in improving carrier design to enhance drug delivery.
Several viruses can pass the maternal-fetal barrier, and cause diseases of the fetus or the newborn. Recently, however, it became obvious, that viruses may invade fetal cells and organs through different routes without acute consequences. Spermatozoa, seminal fluid and lymphocytes in the sperm may transfer viruses into the human zygotes. Viruses were shown to be integrated into human chromosomes and transferred into fetal tissues. The regular maternal-fetal transport of maternal cells has also been discovered. This transport might implicate that lymphotropic viruses can be released into the fetal organs following cellular invasion. It has been shown that many viruses may replicate in human trophoblasts and syncytiotrophoblast cells thus passing the barrier of the maternal-fetal interface. The transport of viral immunocomplexes had also been suggested, and the possibility has been put forward that even anti-idiotypes mimicking viral epitopes might be transferred by natural mechanisms into the fetal plasma, in spite of the selective mechanisms of apical to basolateral transcytosis in syncytiotrophoblast and basolateral to apical transcytosis in fetal capillary endothelium. The mechanisms of maternal-fetal transcytosis seem to be different of those observed in differentiated cells and tissue cultures. Membrane fusion and lipid rafts of high cholesterol content are probably the main requirements of fetal transcytosis. The long term presence of viruses in fetal tissues and their interactions with the fetal immune system might result in post partum consequences as far as increased risk of the development of malignancies and chronic pathologic conditions are discussed.
Two endosome populations involved in recycling of membranes and receptors to the plasma membrane have been described, the early and the recycling endosome. However, this distinction is mainly based on the flow of cargo molecules and the spatial distribution of these membranes within the cell. To get insights into the membrane organization of the recycling pathway, we have studied Rab4, Rab5, and Rab11, three regulatory components of the transport machinery. Following transferrin as cargo molecule and GFP-tagged Rab proteins we could show that cargo moves through distinct domains on endosomes. These domains are occupied by different Rab proteins, revealing compartmentalization within the same continuous membrane. Endosomes are comprised of multiple combinations of Rab4, Rab5, and Rab11 domains that are dynamic but do not significantly intermix over time. Three major populations were observed: one that contains only Rab5, a second with Rab4 and Rab5, and a third containing Rab4 and Rab11. These membrane domains display differential pharmacological sensitivity, reflecting their biochemical and functional diversity. We propose that endosomes are organized as a mosaic of different Rab domains created through the recruitment of specific effector proteins, which cooperatively act to generate a restricted environment on the membrane.
Recycling of endocytosed membrane proteins involves passage through early endosomes and recycling endosomes. Previously, we demonstrated a role for clathrin-coated vesicles in transferrin receptor recycling. These clathrin-coated vesicles are formed from recycling endosomes in a process that was inhibited in dynamin-1(G273D)-overexpressing cells. Here we show a second transferrin recycling pathway, which requires phosphatidylinositol 3-kinase activity. Two unrelated phosphatidylinositol 3-kinase inhibitors, LY294002 and wortmannin, retained endocytosed transferrin in early endosomes but did not affect transfer through recycling endosomes. The inhibitory effects of LY294002 and dynamin-1(G273D) on transferrin recycling were additive. In combination with brefeldin A, a drug that prevents the formation of clathrin-coated buds at recycling endosomes, LY294002 inhibited transferrin recycling synergistically. Collectively, these data indicate two distinct recycling pathways. One pathway involves transfer from early endosomes to recycling endosomes, from where clathrin/dynamin-coated vesicles provide for further transport, whereas the other route bypasses recycling endosomes and requires phosphatidylinositol 3-kinase activity.
Permanent cell culture lines derived from human breast cancer tissue are important experimental models in the study of human breast cancer cell proliferation. In the present work, pimozide, thioridazine, W-13, and W-12 were shown to inhibit MCF-7 human breast cancer cell growth. The 50% inhibition concentration values determined in two proliferation assays, [3H]thymidine incorporation and cell number, were in close agreement for each compound tested. The order of potency for growth inhibition in the presence of 2% stripped calf serum was pimozide (Ki 2 microM) greater than thioridazine (Ki 5 microM) greater than W-13 (Ki 15 microM) greater than W-12 (Ki 39 microM). Similar concentrations of these compounds blocked estradiol-induced growth of MCF-7 cells, but estrogen receptor (ER) interactions do not seem to be involved. Pimozide and thioridazine had no effect on the estradiol binding properties of the MCF-7 ER, nor did pimozide interfere with the induction of progesterone receptors by estradiol. Furthermore, pimozide also inhibited incorporation of [3H]thymidine into MCF-7 cells stimulated by polypeptide hormones in serum-free medium. The Ki for pimozide in serum-free medium alone, 0.46 microM, was similar to that determined in the presence of insulin (0.42 microM), insulin-like growth factor I (0.54 microM), and epidermal growth factor (0.43 microM). The effects of pimozide on breast cancer cell growth were not limited to the MCF-7 cell line. Pimozide also blocked cell growth and [3H]thymidine incorporation into the ER-positive T47D and ZR75-1B human breast cancer cell lines and the ER-negative human breast cancer cell line, MDA-MB-231. Although numerous mechanisms of action of pimozide and thioridazine have been identified, both drugs are calmodulin antagonists at drug concentrations that inhibit breast cancer cell growth in vitro. Inhibition of MCF-7 cell growth by the selective calmodulin antagonists W-13 and W-12 is consistent with a role for calmodulin antagonism in the broad growth-inhibitory properties of pimozide. We conclude that pimozide and thioridazine may be useful in the control of estradiol- and polypeptide hormone-induced growth of ER-positive and ER-negative human breast tumors.
A specific polyclonal antibody was used to investigate the subcellular distribution of the small GTPase, rab11p, in the neuroendocrine cell line, PC12. We took advantage of a previously described pulse-chase protocol based on sulfation to examine the distribution of rab11 along the secretory pathway. Using the rab11 antiserum, but not serum depleted of rab11 antibodies, we were able to specifically immunoisolate markers of the constitutive and the regulated secretory pathway in the trans-Golgi network (TGN) as well as after their exit from this compartment (constitutive secretory vesicles, immature, and mature secretory granules). We therefore conclude that rab11p is associated with the TGN and with TGN-derived vesicles of both the constitutive and the regulated secretory pathway in PC12 cells.
The mechanism by which a novel major histocompatibility complex class I protein, HFE, regulates iron uptake into the body
is not known. HFE is the product of the gene that is mutated in >80% of hereditary hemochromatosis patients. It was recently
found to coprecipitate with the transferrin receptor (Feder, J. N., Penny, D. M., Irrinki, A., Lee, V. K., Lebron, J. A.,
Watson, N., Tsuchihashi, Z., Sigal, E., Bjorkman, P. J., and Schatzman, R. C. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 1472–1477; Parkkila, S., Waheed, A., Britton, R. S., Bacon, B. R., Zhou, X. Y., Tomatsu, S., Fleming, R.E., and Sly,
W. S. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 13198–13202) and to decrease the affinity of transferrin for the transferrin receptor (Feder et al.). In this study, HeLa cells were transfected with HFE under the control of the tetracycline-repressible promoter. We demonstrate
that HFE and the transferrin receptor are capable of associating with each other within 30 min of their synthesis with pulse-chase
experiments. HFE and the transferrin receptor co-immunoprecipitate throughout the biosynthetic pathway. Excess HFE is rapidly
degraded, whereas the HFE-transferrin receptor complex is stable. Immunofluorescence experiments indicate that they also endocytose
into transferrin-positive compartments. Combined, these results suggest a role for the transferrin receptor in HFE trafficking.
Cells expressing HFE have modestly increased levels of transferrin receptor and drastically reduced levels of ferritin. These
results implicate HFE further in the modulation of iron levels in the cell.
A single chain Fv antibody-streptavidin fusion protein was expressed and purified from bacterial inclusion bodies following cloning of the genes encoding the variable region of the heavy chain and light chain of the murine OX26 monoclonal antibody to the rat transferrin receptor. The latter undergoes receptor mediated transcytosis through the brain capillary endothelial wall in vivo, which makes up the blood-brain barrier (BBB); therefore, the OX26 monoclonal antibody and its single chain Fv analog may act as brain drug delivery vectors in vivo. Attachment of biotinylated drugs to the antibody vector is facilitated by production of the streptavidin fusion protein. The bi-functionality of the OX26 single chain Fv antibody-streptavidin fusion protein was retained, as the product both bound biotin and the rat transferrin receptor in vitro and in vivo, based on pharmacokinetic and brain uptake analyses in anesthetized rats. The attachment of biotin-polyethyleneglycol-fluorescein to the OX26 single chain Fv antibody-streptavidin fusion protein resulted in illumination of isolated rat brain capillaries in confocal fluorescent microscopy. In conclusion, these studies demonstrate that genetically engineered single chain Fv antibody-streptavidin fusion proteins may be used for non-invasive neurotherapeutic delivery to the brain using endogenous BBB transport systems such as the transferrin receptor.
Two endosome populations involved in recycling of membranes and receptors to the plasma membrane have been described, the early and the recycling endosome. However, this distinction is mainly based on the flow of cargo molecules and the spatial distribution of these membranes within the cell. To get insights into the membrane organization of the recycling pathway, we have studied Rab4, Rab5, and Rab11, three regulatory components of the transport machinery. Following transferrin as cargo molecule and GFP-tagged Rab proteins we could show that cargo moves through distinct domains on endosomes. These domains are occupied by different Rab proteins, revealing compartmentalization within the same continuous membrane. Endosomes are comprised of multiple combinations of Rab4, Rab5, and Rab11 domains that are dynamic but do not significantly intermix over time. Three major populations were observed: one that contains only Rab5, a second with Rab4 and Rab5, and a third containing Rab4 and Rab11. These membrane domains display differential pharmacological sensitivity, reflecting their biochemical and functional diversity. We propose that endosomes are organized as a mosaic of different Rab domains created through the recruitment of specific effector proteins, which cooperatively act to generate a restricted environment on the membrane.
An expression construct harboring granulocyte colony-stimulating factor (G-CSF)-transferrin (Tf) fusion protein (G-CSF-Tf) was engineered by fusing human cDNAs encoding G-CSF and Tf to explore the feasibility of using Tf as a carrier moiety for oral delivery of therapeutic proteins. The recombinant protein, G-CSF-Tf, was harvested from protein-free, conditioned medium of transfected HEK293 cells. The in vitro studies demonstrated that the purified G-CSF-Tf fusion protein possesses the activity of both Tf receptor (TfR) binding in Caco-2 cells and G-CSF-dependent stimulation of NFS-60 cell proliferation. Subcutaneous administration of G-CSF-Tf fusion protein to BDF1 mice demonstrated a pharmacological effect comparable to the commercial G-CSF on the increase of absolute neutrophil counts (ANC). However, the fusion protein elicited a significant increase in ANC upon oral administration to BDF1 mice, whereas G-CSF had no effect. This study also showed that orally administered G-CSF-Tf elicits a sustained myelopoietic effect up to 3 days, whereas the s.c. administered G-CSF or G-CSF-Tf lasts only 1 day. Furthermore, coadministration of free Tf abolished the increase of ANC by orally delivered G-CSF-Tf, suggesting that the recombinant protein is absorbed via a TfR-mediated process in the gastrointestinal tract. Taken together, we conclude that the Tf-based recombinant fusion protein technology represents a promising approach for future development of orally effective peptide and protein drugs.
• myelopoiesis
• oral delivery
• protein drug
Peptides and recombinant proteins such as neurotrophins, enzymes and monoclonal antibodies, have not been developed as new drugs for the brain because these large molecule drugs do not cross the brain capillary wall, which forms the blood-brain barrier (BBB) in vivo. A new solution to the brain drug delivery problem is the genetic engineering of recombinant fusion proteins. The therapeutic peptide or protein drug is fused to a molecular Trojan horse, which is a second peptide or peptidomimetic monoclonal antibody that binds a specific receptor on the BBB. The Trojan horse enables receptor-mediated delivery of the fusion protein across the BBB so that the protein drug can enter the brain and exert the desired pharmacological effect.
Experiments demonstrating the existence of receptors for iron-saturated transferrin on both B and T lymphoblastoid cell lines of human origin are described. Binding of 125I-labeled transferrin is rapid, saturable and reversible. It can be specifically inhibited by unlabeled transferrin but not by other proteins. The number of receptors on T cell lines determined by Scatchard analysis is almost double the number on B cell lines but the binding affinities are equal. The putative transferrin receptor can be removed from the cell by the proteolytic enzymes papain and trypsin, and is re-expressed during overnight incubation at 37 degrees C. Resynthesis is inhibited by puromycin. The receptor can be solubilized by deoxycholate, and retains transferrin binding capacity when non-covalently attached to an amphipathic matrix consisting of deoxycholate-coupled poly(L-lysyl) Agarose.
A variety of ligands and macromolecules enter cells by receptor-mediated endocytosis. Ligands bind to their receptors on the cell surface and ligand-receptor complexes are localized in specialized regions of the plasma membrane called coated pits. Coated pits invaginate and give rise to intracellular coated vesicles containing ligand-receptor complexes which are thus internalized. Transferrin, a major serum glycoprotein which transports iron into cells, enters cells by this pathway. It binds to its receptor on the cell surface, transferrin-receptor complexes cluster in coated pits and are internalized in coated vesicles. Coated vesicles then lose their clathrin coat and fuse with endosomes, an organelle with an internal pH of about 5-5.5. Most ligands dissociate from their receptors in endosomes and they finally end up in lysosomes where they are degraded, while their receptors remain bound to membrane structures and recycle to the cell surface. Transferrin has a different fate: in endosomes iron dissociates from transferrin but apotransferrin remains bound to its receptor because of its high affinity for the receptor at acid pH. Apotransferrin thus recycles back to the plasma membrane still bound to its receptor. When the ligand-receptor complex reaches the plasma membrane or a compartment at neutral pH, apotransferrin dissociates from its receptor with a half-life of 18 s because of its low affinity for its receptor at neutral pH. The receptor is then ready for a new cycle of internalization, while apotransferrin enters the circulation, reloads iron in the appropriate organs and is ready for a new cycle of iron transport.
Human colonic carcinoma Caco-2 cells grown in vitro form epithelial layers of highly polarized cells. Unlike colonic adsorptive cells they possess a mucosal membrane with very limited ionic conductance, even after exposure to aldosterone. When grown on filters, Caco-2 cells were sensitive to various secretagogues; these included 10(-5) M dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP) and 10(-10) M vasoactive intestinal peptide, both of which, added serosally, enhanced the short-circuit current. The same applied to mucosal forskolin. Caco-2 cell sensitivity to serosal epinephrine was lower. Ion substitutions and 22Na-36Cl flux measurements indicated the possibility of secretagogue-dependent chloride secretion. Measurements on cells grown on Petri dishes and exposed to 1 mM DBcAMP for 1 h enabled detection of more profound modifications. Sustained 20-mV cell depolarization and a large reduction in the relative electrical resistance of the mucosal membrane were concomitant with a sizable decrease in 36Cl accumulation. These results suggest that Caco-2 cells, which to some extent resemble colonic crypt cells, possess the cAMP-dependent mucosal chloride conductance characteristic of secretory cells.
Detailed studies of the parameters which effect the trace iodination of IgG with 125I, at levels of 1 atom/4000–10,000 moles IgG, using chloramine-T, are described. The following observations were noted: (1) The reactions, (a) the formation of ‘active iodine’ (H2OI+) and, (b) the incorporation of ‘active iodine’ into protein, that comprise the over-all iodination of proteins have been studied separately and together. (2) With IgG, pH, 7·0 and 2° were optimal and reproducible for both reactions (a) and (b), and for the over-all reaction. Among the parameters which were found to influence the individual and over-all reactions and which were studied to establish optimal conditions were the concentrations of oxidant, iodide, and protein as well as pH, time, temperature, ionic strength, and particular batch of protein. (3) Different proteins and separate lots of the same protein show variable amounts of residues which are oxidized prior to the incorporation of iodine. (4) No differences in the immunochemical precipitation characteristics were detected between untreated IgG and IgG iodinated by the present method. (5) Calculations of the distribution of IgG species, iodinated at a level of 1 atom iodine per 4000–10,000 moles IgG, shows the population of iodinated molecules to be essentially homogeneous and in all likelihood, monosubstituted. (6) Typical conditions for labeling IgG at a level of approximately 1 atom iodine/5000 molecules IgG are described in detail.
The effect of enterocyte-like differentiation on the transferrin receptor (TfR) polarity in filter-grown Caco-2 cells was studied. The ratio of apical to basolateral TfRs which was found to be approximately 1:1 on the first day after the cells had reached confluence, changed to 1:40 eight days after reaching confluence. The transepithelial electrical resistance (TEER), transport of horseradish peroxidase (HRP) across the monolayer, and total cellular TfR number remained constant over this period. However, the activity of brush border membrane-associated alkaline phosphatase, an established marker for enterocyte differentiation, increased over this 8-day period concurrent with a decrease in apical TfR number. These results suggest that enterocyte-like differentiation rather than tight junction formation is most likely responsible for the polarized distribution of TfRs in Caco-2 cells. The effects of the fungal metabolite brefeldin A (BFA) on TfR distribution and TfR-mediated transcytosis in Caco-2 cells were also studied. BFA caused a marked decrease in the number of basolateral TfRs along with a slight increase in the number of apical TfR. BFA enhanced the TfR-mediated transcytosis of both 125I-Tf and the horseradish peroxidase-Tf conjugate across Caco-2 cells in both apical-to-basolateral and basolateral-to-apical directions. These findings imply a potential application of BFA as an enhancer for TfR-mediated delivery of protein drugs across the intestinal epithelium.
In cancer treatment, one of the approaches is targeting of the drug to tumor cells via receptor specific ligands. Transferrin (molecular weight 80,000) has been used as a ligand for delivering anticancer drugs or drug containing liposomes mostly due to the increased number of transferrin (trf) receptors found on tumor cells as compared to normal cells. Transferrin was linked to methotrexate (MTX) containing small unilamellar liposomes and its activity was compared to antitransferrin receptor antibody (7D-3) linked to MTX liposomes. In each of these conjugates, the method of coupling was the same and a disulphide linkage was formed between the ligand and MTX liposomes. No significant differences in the potency of 7D-3 conjugate or trf conjugate with MTX liposomes were observed in studies performed in vitro against various human tumor cell lines (Hela, KB and Colon). Trf was also linked to adriamycin via a schiff base which was formed by using glutaraldehyde. This conjugate was found to be effective in vitro against various human tumors (Lovo, HL-60, SW 403 and Hep2) and also in vivo against H-mesothelioma tumors. Transferrin receptor has also been used for gene delivery. Gene delivery to K562 haematopoietic leukaemic cells was achieved by using a transferrin-polycation (poly-L-lysine or protamine) conjugate. This review will cover the various important applications of transferrin based drug delivery formulations in the chemotherapy of cancer and the related work performed in our and other laboratories.
Alkylphosphocholines (APC) constitute a new group of antineoplastic agents without haematological toxicity. Their first clinically available derivative hexadecylphosphocholine (miltefosine) is locally used to control skin metastases of breast cancer. Since intravesical chemotherapy represents a form of topical treatment we investigated whether a new APC with a long alkyl chain would be active against 5637 and EJ bladder cancer cell lines. Their antineoplastic activity was inversely related to the alkyl chain length of the respective APC. Erucylphosphocholine and its congener with modified phosphocholine head erucylphospho-N,N,N-trimethylpropanolamine were the most effective derivatives. APC with alkyl chains over 16 carbons in length induced programmed cell death in both cell lines, as determined by oligonucleosomal DNA fragmentation and morphology. The distinct antineoplastic effects lead us to predict that urinary bladder instillation of APC will be of therapeutic benefit for patients with urinary bladder neoplasia.
Transferrin (Tf) receptor-mediated transcytosis of insulin-transferrin conjugate (In-Tf) has been demonstrated in cultured human enterocyte-like Caco-2 cells. In the present report, oral delivery of insulin as a Tf conjugate in streptozotocin (STZ)-induced diabetic rats was investigated. Human insulin was conjugated at a 1:1 molar ratio to iron-loaded human Tf by a disulfide linkage. The stability of In-Tf and the free insulin released from In-Tf was studied in the presence of rat liver slices by using radioimmunoassay. The release of free insulin involved a disulfide reduction reaction that was inhibited by the pretreatment of the liver slice with a sulfhydryl-reactive reagent N-ethylmaleimide. A protease inhibitor cocktail also showed a partial inhibition of insulin degradation. The biological activity of the conjugate was tested in STZ-induced diabetic rats with s.c. administration, and the conjugate exhibited a slow but prolonged hypoglycemic effect compared with that of the native human insulin. In-Tf also displayed a slow but prolonged hypoglycemic effect after oral administration in fasted STZ-induced diabetic rats in a dose-dependent manner. Furthermore, In-Tf was detected in the serum of rats at 4 h after oral administration of the conjugate, indicating that In-Tf can overcome the barriers in the gastrointestinal tract and be absorbed as an intact conjugate. These results demonstrate that transepithelial transport via TfR-mediated transcytosis is a feasible approach for developing the oral delivery of insulin, as well as other peptide drugs.
To investigate the effect of tyrphostin 8 (T-8), a GTPase inhibitor, on transferrin receptor (TfR)-mediated transcytosis of insulin-transferrin (In-Tf) conjugate in cultured enterocyte-like Caco-2 cells and on gastrointestinal (GI) absorption of In-Tf in streptozotocin (STZ)-induced diabetic rats.
Caco-2 cells and diabetic rats were used as in vitro and in vivo models, respectively. TfR-mediated transcytosis was measured using 125I-In-Tf. The absorption of insulin in diabetic rats was demonstrated by the hypoglycemic effect. Rat blood glucose level was determined using a ONE TOUCH blood glucose monitoring system.
T-8 increased apical-to-basolateral transport of In-Tf conjugate by enhancing TfR-mediated transcytosis in filter-grown Caco-2 cell monolayer, and this enhancement was higher and faster than the previously reported brefeldin A (BFA)-induced effect. The measurement of transepithelial electrical resistance (TEER) during the transport study showed that T-8 was less destructive on the cell tight junction than BFA. The GI absorption of In-Tf was evaluated by its hypoglycemic effect after oral administration in STZ-induced diabetic rats. The glucose-lowering effect of orally administered In-Tf in STZ-induced diabetic rats was improved by either T-8 or BFA. However, the effect of T-8 was more potent than that of BFA, especially at 7 h after administration. Either non-conjugated insulin or insulin-human serum albumin (In-HSA) conjugate by itself or in combination with T-8 did not show any hypoglycemic effect after oral administration, indicating that T-8-enhanced hypoglycemic activity of In-Tf was due to a selective enhancement of TfR-mediated transcytosis.
Our data indicated that T-8 could be used to increase the GI absorption of insulin as a transferrin conjugate. T-8, as an enhancer of TfR-mediated transcytosis, is better than the previously reported BFA. T-8 produces a higher increase on the transport of In-Tf and a lower toxicity on epithelial cells. Our findings provide an alternative approach to promote the GI absorption of insulin, as well as other peptide or protein drugs.
In most cells the endocytic system is organized following a common concept that allows for the integrative handling of a variety of housekeeping functions. In addition, variations on the general scheme provide for specialized endosome-based pathways that occur only in specific cell types. The diversity of endosomal functions is not only reflected by characteristic molecular compositions, but also mirrored in their morphological organization. In this review we will first describe the general outline of the endocytic system by combining kinetic, morphological, molecular, and functional definitions. In the second part, adaptations of endosomes that allow their functioning in specialized processes, such as antigen presentation, synaptic vesicle formation, and glucose transport, will be addressed.
The purpose of this study was to use primary cultured rat alveolar epithelial cell monolayers to examine the potential of using transferrin receptor (TfR)-mediated transcytosis for noninvasive systemic protein drug delivery via the pulmonary route.
Freshly isolated rat type II pneumocytes were plated onto tissue culture-treated polycarbonate 12-mm Transwells. AEC monolayers (> or = 2500 omega(cm2)) were treated with keratinocyte growth factor (10 ng/mL) for maintenance of type II cell-like characteristics. Filgrastim (GCSF)-Tf conjugates were prepared using the linkers SPDP and DPDPB. TfR-specific binding and uptake were determined using 125I-Tf and 59Fe-Tf treatment, respectively. Apical-to-basolateral (A-to-B) transferrin receptor (TfR)-mediated transcytosis was determined by dosing the apical compartment with 1.5 microg/mL of 125I-Tf or 125I-GCSF-Tf. Nonspecific TfR-independent transport of 125I-Tf and 125I-GCSF-Tf was determined in parallel by including 150 microg/mL of nonradiolabeled Tf. Basolateral samples (500 microL) were taken at 2, 4, and 6 h post-dosing, subjected to 15% trichloroacetic acid precipitation, and assayed in a Packard gamma counter. TfR-specific transport was determined as the difference between total and nonspecifc transport. The effects of brefeldin-A (BFA) on TfR distribution and (A-to-B) transport of 125I-Tf, 125I-GCSF and 125I-GCSF-Tf was studied by including the agent in the apical fluid at 1 microg/mL.
BFA treatment resulted in a small significant reduction in TfR at the basolateral surface of type II cell-like monolayers, while it had no effect on TfR distribution in type I cell-like monolayers. In contrast, BFA treatment significantly altered the endocytosis of TfR, reducing the basolateral uptake of 59Fe-Tf while greatly increasing the apical uptake of 59Fe-Tf. BFA treatment, however, did not affect the TfR-specific uptake of 59Fe-Tf in type I cell-like monolayers. TfR-specific apical-to-basolateral transcytosis of 125I-Tf and 126I-GCSF-Tf conjugates was significantly enhanced in the presence of BFA in type II cell-like monolayers, whereas it had no effect on apical-to-basolateral transport of 125I-GCSF. BFA-enhanced transport of 125I-GCSF-Tf was approximately 3-fold higher than that of 125I-GCSF in the presence or absence of BFA. Moreover, 125I-GCSF transport in the presence of BFA was not significantly different from non-specific 125I-GCSF-Tf transport. Chromatographic analyses and bio-assays revealed that GCSF-Tf was not degraded during transport via TfR-specific processes, and that GCSF retained biologic activity when liberated from the conjugate via dithiothreitol reduction.
This study suggests the possibility of using TfR-mediated transcytosis for systemic delivery of therapeutic proteins via the alveolar epithelium.
Transferrin receptor has been an important protein for many of the advances made in understanding the intricacies of the intramolecular sorting pathways of endocytosed molecules. The unique internalization and recycling functions of transferrin receptor have also made it an attractive choice for drug targeting and delivery of large protein-based therapeutics and toxins. Recent advances in elucidating the role of the intracellular controllers of transferrin recycling and sorting, such as Rab proteins and their effectors, have led to enhancement of transferrin receptor as a drug delivery vehicle. This review focuses on the use of transferrin receptor as an agent for facilitating drug delivery and targeting, and the role that mechanisms of transferrin receptor sorting and transcytosis play in these events.
The authors review the preclinical and clinical results of the ligand-targeted toxin conjugate Transferrin-CRM107 (Tf-CRM107), for the treatment of malignant gliomas. Tf-CRM107 is a conjugate protein of diphtheria toxin with a point mutation (CRM107) linked by a thioester bond to human transferrin (Tf). This conjugate exhibits potent cytotoxicity in vitro against mammalian cells expressing the transferrin receptor with activity at picomolar concentrations. Phase I clinical trial results demonstrated that Tf-CRM107, delivered via a high-flow convection method utilizing stereotactically placed catheters, produced tumor response in patients with malignant brain tumors refractory to conventional therapy without severe neurologic or systemic toxicity. The results of a Phase II study are also summarized. Tf-CRM107 treatment results in complete and partial tumor response without severe toxicity in 35% of the evaluable patients. These data warrant a Phase III study as well as continued research in the field of targeted toxin therapy. Future directions of research include optimizing Tf-CRM107 delivery to targeted brain regions, and improving the treatment efficacy by combining with other toxin conjugates targeted to different receptors.
Purpose:
The purpose of this study was to investigate the transferrin-receptor (TfR)-mediated transepithelial transport of G-CSF-transferrin (Tf) conjugate in cultured enterocyte-like Caco-2 monolayers and the myelopoietic effect of subcutaneously and orally administered G-CSF-Tf in BDF1 mice.
Methods:
Caco-2 monolayers exhibiting a minimum transepithelial electrical resistance of 500 ohms-cm2 and BDF1 mice were used as in vitro and in vivo models, respectively. TfR-mediated transcytosis wa measured by using 125I-G-CSF-Tf and analyzing the downstream compartment by gamma counter. The efficacy of subcutaneously and orally administered G-CSF-Tf was determined by performing daily absolute neutrophil counts.
Results:
Transport experiments in Caco-2 cells revealed that the monolayers that received 125I-G-CSF-Tf exhibited significantly higher apical-to-basolateral transport rates compared to the monolayers that received 125I-G-CSF. Inclusion of 100-fold excess unlabeled Tf reduced the extent of 125I-G-CSF-Tf transport by 80%. Chromatographic and bioactivity assays revealed that the protein recovered from the basolateral compartment was the intact conjugate, and it retained full ability to stimulate the proliferation of the granulocyte-colony stimulating factor (G-CSF) dependent cell line, NFS-60, upon reduction. Subcutaneous administration of G-CSF-Tf in BDF1 mice demonstrated that the conjugate is able to elicit a statistically significant enhancement in therapeutic effect relative to filgrastim, which includes a longer duration of action with higher absolute neutrophil counts. Oral administration of G-CSF-Tf in BDF1 mice demonstrated that G-CSF-Tf is able to elicit a significant, and apparently dose-dependent, increase in absolute neutrophil counts whereas filgrastim had no effect.
Conclusions:
Our data indicate that G-CSF-Tf is transported across Caco-2 monolayers by TfR-specific processes at a rate that is significantly higher than the nonspecific flux of G-CSF. G-CSF-Tf is also able to elicit a prolonged myelopoietic effect relative to filgrastim when administered subcutaneously or orally in BDF1 mice. The development of an orally bioavailable G-CSF has the potential to provide great benefit to patients under sustained G-CSF dosing regimes.
The number of newly identified genes participating in the regulation of iron homeostasis has continued to expand at a remarkable pace. The roles for many have begun to be elucidated and there is an increasing indication that hepatocytes play a central role in determining the level of intestinal iron absorption. Total body iron homeostasis is dependent upon carefully regulated absorption of dietary iron, thus these genes are of fundamental importance in understanding of pathophysiology of such common disorders as hereditary hemochromatosis (HH) and the anaemia of chronic diseases.
The hepatic peptide hepcidin plays a key role as a circulating hormone that regulates the absorption of dietary iron from the duodenum. Hepcidin expression is inappropriately decreased in hereditary hemochromatosis and is abnormally increased in the anaemia of chronic diseases. Other hepatic proteins essential for normal iron homeostasis, including HFE, transferrin receptor 2 (TfR2), and hemojuvelin, function at least in part, by modulating the expression of hepcidin.
New insights into the pathophysiology of hereditary hemochromatosis and the anaemia of chronic diseases have been achieved with the recognition of the central role for hepcidin as an iron regulatory hormone. Investigations into the biologic control of this hormone and its mechanism of action offer the possibility of new therapeutic approaches to disorders of iron metabolism.
To improve the oral efficacy of the recombinant fusion protein containing granulocyte colony-stimulating factor (G-CSF) and transferrin (Tf) by inserting a linker between the two protein domains.
Oligonucleotides encoding flexible and helix-forming peptides were inserted to the recombinant plasmids. The fusion protein without linker insertion was used for comparison. The G-CSF cell-proliferation and Tf receptor-binding activities of the fusion proteins were tested in NFS-60 cells and Caco-2 cells, respectively, and in vivo myelopoietic assay with both subcutaneous and oral administration was performed in BDF1 mice.
All fusion proteins produced from transfected HEK293 cells were positive in Western-blotting assay with anti-G-CSF and anti-Tf antibodies. Among them, the fusion protein with a long helical (H4-2) linker showed the highest activity in NFS-60 cell proliferation assay, with an EC50 about ten-fold lower than that of the non-linker fusion protein. The fusion protein with H4-2 linker also showed a significantly higher myelopoietic effect when administered either subcutaneously or orally in BDF1 mice.
The insertion of a linker peptide, such as the helix linker H4-2, between G-CSF and Tf domains in the recombinant fusion protein can improve significantly both in vitro and in vivo myelopoietic activity over the non-linker fusion protein.