Lectin-mediated Drug Delivery Research Articles

A Novel Preparation Technique for Human Nasal Respiratory Mucosa to Disclose Its Glycosylation Pattern for Bioadhesive Drug Delivery

To shed some light on glycotargeting as a potential strategy for nasal drug delivery, a reliable preparation method for human nasal mucosa samples and a tool to investigate the carbohydrate building blocks of the glycocalyx of the respiratory epithelium are required. Applying a simple experimental setup in a 96-well plate format together with a panel of six fluorescein-labeled lectins with different carbohydrate specificities allowed for the detection and quantification of accessible carbohydrates in the mucosa. As confirmed by binding experiments at 4 °C, both quantitatively by fluorimetry and qualitatively by microscopy, the binding of wheat germ agglutinin exceeded that of the others by 150% on average, indicating a high content of N-acetyl-D-glucosamine and sialic acid. Providing energy by raising the temperature to 37 °C revealed uptake of the carbohydrate-bound lectin into the cell. Moreover, repeated washing steps during the assay gave a slight hint as to the influence of mucus renewal on bioadhesive drug delivery. All in all, the experimental setup reported here for the first time is not only a suitable approach to estimating the basics and potential of nasal lectin-mediated drug delivery but also meets the needs for answering a broad variety of scientific questions involving the use of ex vivo tissue samples.

Alginate Modification and Lectin-Conjugation Approach to Synthesize the Mucoadhesive Matrix

Alginates are natural anionic polyelectrolytes investigated in various biomedical applications, such as drug delivery, tissue engineering, and 3D bioprinting. Functionalization of alginates is one possible way to provide a broad range of requirements for those applications. A range of techniques, including esterification, amidation, acetylation, phosphorylation, sulfation, graft copolymerization, and oxidation and reduction, have been implemented for this purpose. The rationale behind these investigations is often the combination of such modified alginates with different molecules. Particularly promising are lectin conjugate macromolecules for lectin-mediated drug delivery, which enhance the bioavailability of active ingredients on a specific site. Most interesting for such application are alginate derivatives, because these macromolecules are more resistant to acidic and enzymatic degradation. This review will report recent progress in alginate modification and conjugation, focusing on alginate-lectin conjugation, which is proposed as a matrix for mucoadhesive drug delivery and provides a new perspective for future studies with these conjugation methods.

A doxorubicin loaded colloidal delivery system for the intravesical therapy of non-muscle invasive bladder cancer using wheat germ agglutinin as targeter

Non-muscle invasive bladder cancer is one of the most frequent forms of cancer. Commonly, the superficial tumor is removed by a minimal invasive surgery, mostly followed by the intravesical instillation of cytostatic agents. Although the initial treatment is usually successful, the recurrence rate is extraordinary high, which might be grounded in limiting factors during instillation, such as the high resistance of the bladder wall and the constant dilution with the permanently produced urine reducing the probability of interaction between the drug and the target cell. To overcome these limitations, we introduce a lectin-mediated drug delivery system consisting of wheat germ agglutinin as targeter, poly-l-glutamic acid as backbone and the active pharmaceutical ingredient (API) doxorubicin that should prolong the residence time in the bladder cavity. The optimized WGA-PGA-Doxo conjugate revealed an API load of 81 molecules per mole backbone and convinced in urothelial cell monolayer studies with an up to 56-fold higher cell-binding potential than pure doxorubicin. Additionally, the highly substituted drug delivery system not only internalized into urothelial cells, but also inhibited cell viability up to 99%.The combination of the high drug load with the promising cell-associative properties, which also revealed a higher affinity to malignant than to healthy urothelial cells, and the proven cytotoxic potential might lead to an improved efficacy of intravesical chemotherapy.

Lectin Mediated Biorecognition as a Novel Strategy for Targeted Delivery to Bladder Cancer

Inadequate urothelial delivery of drugs is considered a primary cause of current shortcomings in adjuvant intravesical chemotherapy for bladder cancer. We report what is to our knowledge a novel biorecognitive approach to achieve more regionally selective targeting of malignant tissue and improve urothelial uptake based on specific interaction between lectins and bladder cell glycocalyces. We assessed the cytoadhesive and cytoinvasive potential of selected plant lectins in 3 human urothelial cell lines, corresponding to healthy tissue, and low and high grade carcinoma, respectively. Flow cytometry and fluorimetry were used to determine binding capacity and specificity in single cells and confluent monolayers. Monensin quenching experiments, microscopic analysis and enzyme treatment allowed further characterization of internalization, the uptake pathway and the potential cause of tumor selectivity. Wheat germ agglutinin had the highest bioadhesive potential while peanut agglutinin was the most potent discriminator between healthy and cancerous tissue (p <0.01). In each case cell interaction was highly specific (greater than 80%) and proved decisive for efficient uptake. Within 60 minutes after exposure greater than 50% of membrane bound lectins were internalized in acidic compartments. Cancer associated aberrant glycosylation likely represents the determining cause of peanut agglutinin selectivity. Given careful choice of the targeting ligand, the development of carbohydrate based delivery strategies for bladder cancer therapy seems feasible. Lectin bioadhesion may not only mediate preferential accumulation in malignant tissue but also promote cellular internalization via increased recruitment of membrane bound material to physiological uptake routes.

Alteration of the glycosylation pattern of monocytic THP-1 cells upon differentiation and its impact on lectin-mediated drug delivery

In the present study, human monocytic THP-1 cells were treated with phorbol-12-myristate-13-acetate (PMA) in order to obtain macrophage-like cells. Before and after treatment, plant lectins with distinct sugar specificities were applied in order to elucidate the glycosylation patterns of both monocytic and macrophage-like cell types and to follow changes during differentiation. As a result of flow-cytometric analyses, for untreated as well as for PMA-differentiated cells WGA yielded the highest binding rate without significant changes in the binding capacity. For the other lectins, divergent results were obtained which point to reorganization of sugar residues on the cell surface during differentiation. Additionally, cytoinvasion being beneficial for enhanced drug absorption was studied with WGA which had displayed a high binding capacity together with a high specificity. For both untreated and PMA-differentiated cells decreased fluorescence intensity at 37 °C as compared to 4 °C was observable pointing to internalization and accumulation within acidic compartments. Moreover, WGA-functionalized PLGA nanoparticles were prepared, and their uptake evaluated. Uptake rates of 55% in case of PMA-differentiated cells suggested that WGA-grafted drug delivery systems might be an interesting approach for treatment of infectious diseases provoked by parasites, facultative intracellular bacteria, or viruses such as HIV.

Development of a sensitive and reliable ELISA for quantification of wheat germ agglutinin

Among others, lectin-mediated drug delivery is currently discussed as a promising strategy towards improved bioavailability of biotech drugs. For quantitative determination of the lectin from wheat germ (WGA), a sandwich ELISA relying on capture of the lectin by pig gastric mucin coated wells and detection of bound WGA by a lectin specific first antibody followed by peroxidase-labelled second antibody was elaborated. The stepwise optimised protocol allows quantification over the range from 10 to 1000 ng/ml WGA with a coefficient of determination of 0.9991. The day to day variation was ± 0.09 OD at 500 ng/ml WGA. Additionally, the presented ELISA-protocol allows determination of WGA in serum with the same sensitivity and reliability as in buffer. Lectins with different carbohydrate specificity such as those from jack beans and peanuts exhibited no cross-reactivity. Among the lectins with the same carbohydrate specificity that from potatoes interfered with the assay, whereas that from tomatoes was not recognised by the first antibody. Since the potato lectin is fully degraded in the intestine, no cross-reactivity with WGA is expected in serum samples. Following on from these results, the absorption rate of WGA in biologically active form might be determined as a basis for further steps towards improved drug delivery systems.

Chemical biology of the sugar code.

A high-density coding system is essential to allow cells to communicate efficiently and swiftly through complex surface interactions. All the structural requirements for forming a wide array of signals with a system of minimal size are met by oligomers of carbohydrates. These molecules surpass amino acids and nucleotides by far in information-storing capacity and serve as ligands in biorecognition processes for the transfer of information. The results of work aiming to reveal the intricate ways in which oligosaccharide determinants of cellular glycoconjugates interact with tissue lectins and thereby trigger multifarious cellular responses (e.g. in adhesion or growth regulation) are teaching amazing lessons about the range of finely tuned activities involved. The ability of enzymes to generate an enormous diversity of biochemical signals is matched by receptor proteins (lectins), which are equally elaborate. The multiformity of lectins ensures accurate signal decoding and transmission. The exquisite refinement of both sides of the protein-carbohydrate recognition system turns the structural complexity of glycans--a demanding but essentially mastered problem for analytical chemistry--into a biochemical virtue. The emerging medical importance of protein-carbohydrate recognition, for example in combating infection and the spread of tumors or in targeting drugs, also explains why this interaction system is no longer below industrial radarscopes. Our review sketches the concept of the sugar code, with a solid description of the historical background. We also place emphasis on a distinctive feature of the code, that is, the potential of a carbohydrate ligand to adopt various defined shapes, each with its own particular ligand properties (differential conformer selection). Proper consideration of the structure and shape of the ligand enables us to envision the chemical design of potent binding partners for a target (in lectin-mediated drug delivery) or ways to block lectins of medical importance (in infection, tumor spread, or inflammation).

Lectin-mediated drug delivery in the oral cavity

The delivery of therapeutic agents to, or via, the oral cavity is limited by the efficient removal mechanisms that exist in this area. Lectins are proteins or glycoproteins that bind to specific sugar residues, and can, therefore, interact with the glycoconjugates present on cell surfaces or salivary mucins. Endogenous lectins could also be used as points of attachment for carbohydrate-containing delivery systems. This review considers the possibility of using lectins as targeting agents within the oral cavity and reports on some of the limited number of studies completed to date. As lectins are multifunctional molecules, the possibility of using them as both targeting and therapeutic agents is considered. Lectin-containing delivery systems are a potential innovation for targeted and prolonged therapy within the oral cavity, but considerations such as toxicity and cost will need to be addressed before their routine use becomes a reality.

The lectin–cell interaction and its implications to intestinal lectin-mediated drug delivery

Based on the fact that oligosaccharides encode biological information, the biorecognition between lectinised drug delivery systems and glycosylated structures in the intestine can be exploited for improved peroral therapy. Basic research revealed that some lectins can mediate mucoadhesion, cytoadhesion, and cytoinvasion of drugs. Entering the vesicular pathway by receptor mediated endocytosis, part of the conjugated drug is accumulated within the lysosomes. Additionally, part of the drug is supposed to be transported across the epithelium. Moreover, factors probably adversely influencing feasibility of the concept such as toxicity, immunogenicity, and intestinal stability of plant lectins are discussed. As exemplified by lectin-grafted prodrug and carrier systems, this strategy is expected to improve absorption and probably bioavailability of poorly absorbable drugs, peptides and proteins as well as therapeutic DNA.

Lectin-mediated drug delivery: binding and uptake of BSA-WGA conjugates using the Caco-2 model

To examine whether the dietary lectin wheat germ agglutinin (WGA) can facilitate binding and uptake of protein drugs due to its cytoadhesive and cytoinvasive properties, conjugates were prepared by covalent coupling of fluorescein-labeled bovine serum albumin (F-BSA) to WGA using divinylsulfone for crosslinking. Increasing the molar ratio of F-BSA/WGA resulted in 2.6–8.7 times higher Caco-2 binding as compared with glycyl-F-BSA. About 75% of F-BSA-WGA were bound specifically to Caco-2 cells according to inhibition studies in presence of the complementary carbohydrate. The Caco-2 association of F-BSA-WGA was temperature-dependent indicating active uptake of membrane bound conjugate, which was confirmed by confocal microscopy. The conjugate accumulated within lysosomal compartments followed by proteolytic degradation of F-BSA-WGA 1–4 h after conjugate loading as observed by equilibrating the intracellular pH with monensin. Finally low molecular weight degradation products of the proteinaceous prodrug appear in the extracellular medium. Contrary to Caco-2 single cells, a minor part of the conjugate is degraded by brush border proteases already 30 min after exposure to Caco-2 monolayers. But most of the conjugate is taken up into differentiated cells and processed as in single cells. Though the enzymic barrier remains to be surmounted, WGA-mediated drug delivery is a promising strategy for peroral delivery of even high molecular weight drugs to overcome the mucosal barrier.

Lectin-mediated Drug Delivery: Discrimination Between Cytoadhesion and Cytoinvasion and Evidence for Lysosomal Accumulation of Wheat Germ Agglutinin in the Caco-2 Model

Lectin-mediated drug delivery may become a promising strategy to improve the efficacy of poorly permeable drugs by utilising active high-capacity transport pathways of epithelial tissues. This requires the elucidation of the basic mechanisms of lectin uptake prior to their practical use. We studied the interaction between the dietary lectin wheat germ agglutinin (WGA) and Caco-2 cells (single cells and monolayers) by a newly established assay design that is able to discriminate between cellular binding and uptake as well as by confocal microscopy: (i) All binding sites available for WGA at the cell membrane were occupied within 10 min of incubation. (ii) Cytoadhesion was followed by immediate uptake. After 20 min, 60% (single cells) or 30% (monolayers) of the membrane bound lectin were internalised. However, regardless of cell arrangement, 80% of the surface bound lectin was taken up into the cells during the course of the experiment. (iii) About 50% of the internalised lectin accumulated within the lysosomes after 1 h. This was confirmed by assays in the presence of monensin, an inhibitor of endosomal acidification, and by colocalisation with lysosomal cathepsin followed by semiquantitative image analysis. Further analysis by immunocytochemistry suggested that the trans-Golgi complex and the caveoli were not involved. Due to cytoadhesion, cytoinvasion and partial lysosomal accumulation, WGA-mediated drug delivery may provide for improved intracellular availability of conjugated drugs or colloidal carrier systems.

Lectin-mediated drug delivery: influence of mucin on cytoadhesion of plant lectins in vitro

As the mucous layer represents the first barrier to peroral lectin-mediated drug delivery, the influence of mucin on the cytoadhesive properties of lectins was studied in vitro by establishing a rapid and simple microplate format assay using pig gastric mucin (PGM) for coating the wells. The lectin-binding capacity of mucin followed the order WGA≫UEA-I≫LCA=STL>PNA>DBA. The PGM-binding of wheat germ agglutinin (WGA) was strongly dependent on pH being highest at pH 5.0. In comparison, PGM-binding of WGA was about 15% at gastric pH and 60–70% at intestinal pH. This points to unimpeded gastric transit of WGA-grafted formulations and favorable conditions within the intestine for binding to mucus coated enterocytes. Moreover the WGA–PGM interaction was concentration-dependent, specific and fully reversible. According to a competitive assay in the presence of Caco-2 monolayers, the PGM-binding of WGA was saturated and influenced by the lectin-concentration yielding 28% Caco-2 bound WGA (125 ng WGA/0.29 cm 2 monolayer) and 68% Caco-2 bound WGA (4 μg WGA/0.29 cm 2 monolayer), respectively. Following on from these results, lectins are expected to suffer at least partially from premature inactivation by shed off mucus like bioadhesives of the first generation, however initial but reversible mucus-binding of lectins offers partititioning to the cell membrane followed by uptake into the enterocyte.

Lectin-mediated drug delivery:: The second generation of bioadhesives

This paper reviews some recent developments in the area of bioadhesive drug delivery systems. The area of bioadhesion in drug delivery had started some 20 years ago by using so-called mucoadhesive polymers. Many of these polymers were already used as excipients in pharmaceutical formulations. This has facilitated the development of the first bioadhesive drug products, which are now commercially available. A major disadvantage of the hitherto known mucoadhesives, however, is their non-specificity with respect to the substrate. In particular for gastro-intestinal applications, this may cause some premature inactivation and moreover limits the duration of mucoadhesive bonds to the relatively fast mucus turnover. Nevertheless, for some mucoadhesive polymers other interesting functionalities were discovered, such as their ability to modulate epithelial permeability and to inhibit proteolytic enzymes. In contrast to the mucoadhesive polymers, lectins and some other adhesion molecules specifically recognize receptor-like structures of the cell membrane and therefore bind directly to the epithelial cells themselves (“cytoadhesion”) rather than to the mucus gel layer. Furthermore, when bioadhesion is receptor-mediated, it is not only restricted to mere binding, but may subsequently trigger the active transport of large molecules or nanoscalic drug carrier systems by vesicular transport processes (endo-/transcytosis). Rather than only acting as a platform for controlled release systems, the concept of lectin-mediated bioadhesion therefore bears the potential for the controlled delivery of macromolecular biopharmaceuticals at relevant biological barriers, such as the epithelia of the intestinal or respiratory tract.

Lectin-Mediated Drug Targeting: Quantification of Binding and Internalization of Wheat germ Agglutinin and Solatium tuberosum Lectin Using Caco-2 and HT-29 Cells

For the potential use of Wheat germ agglutinin (WGA) and Solarium tuberosum lectin (STL) as auxiliary excipients for targeting drugs to colonocytes, the number of Caco-2 and HT-29-bound lectins was determined by fluorimetry using fluorescein-labelled derivatives of the N-acetylglucosamine-specific lectins. After 1 h of incubation, the WGA-binding capacity of 5 × 104 Caco-2 cells was 26.9 ± 0.5 pmol at 4°C and 27.2 ± 1.0 pmol at 37°C respectively. In comparison, 19.5 ±2.9 pmol (37°C) and 16.7 ±0.9 pmol (4°C) WGA were bound within 1 h to 5 × 104 HT-29 cells referring to about 80% of the total amount of WGA bound within 4 h of incubation. In contrast, binding of STL to the colon carcinoma cell lines was independent of incubation times and temperatures tested exhibiting a binding rate of 8.4 ± 0.6 pmol (HT-29) and 9.9 ± 0.8 pmol (Caco-2) STL/ 5 × 104 cells. As determined by flow cytometry, non-specific binding is lower than 1.0% (WGA) and 3.4% (STL). Uptake and intracellular accumulation of the lectins were investigated by confocal laser scanning microscopy at 4°C and 37°C respectively. A decrease of initially membrane-bound lectins concurrent with increasing cytoplasmic enrichment by time was observed by digital cell image analysis. Due to specific and numerically sufficient adhesion as well as internalization, WGA and STL are anticipated as targeting tools in lectin-mediated drug delivery systems.

Lectin-mediated bioadhesion: Proteolytic stability and binding-characteristics of wheat germ agglutinin and Solanum tuberosum lectin on Caco-2, HT-29 and human colonocytes

For the development of lectin-mediated drug delivery systems, the proteolytic stability of the non-toxic lectins from Arachis hypogea, Lens culinaris, Dolichus biflorus, Solanum tuberosum (STL) and Triticum vulgare was investigated by in vitro exposure to gastrointestine-located enzymes. No degradation products were observed within 24 h of incubation on sodium dodecyl sulfate polyacrylamide gels. Binding to human colon carcinoma cell lines was investigated by flow cytometry. The fluorescein-labelled derivatives of N-acetylglucosamine-specific wheat germ agglutinin (WGA) and STL exhibited the highest cell-associated fluorescence intensity. As determined by dilution experiments, the number of WGA-binding sites on Caco-2, HT-29 and human colonocytes exceeded those for STL by 5-, 1.7- and 1.4-fold, respectively. By a competitive flow cytometric assay using N,N′, N″-triacetylchitotriose for inhibition, WGA-affinity exceeded STL-affinity by ten-fold. The affinity of each lectin to Caco-2, HT-29 and human colonocytes was about the same, indicating that similar lectin receptors were involved. Preventing N-glycosylation of the carcinoma cells by pretreatment with 0.001% tunicamycin for 40 h resulted in 30% inhibition of WGA- and STL-binding. When WGA was covalently attached to Sepharose beads (250–350 μm), the interaction with HT-29 and Caco-2 cells showed stable and tight binding. Therefore, especially considering the comparable affinity of human colonocytes and monolayer-forming Caco-2 and HT-29 cells, this system is proposed as a model for the development of lectin-mediated particulate pharmaceutical devices.