A combination of nanosystems for the delivery of cancer chemoimmunotherapeutic combinations: 1-Methyltryptophan nanocrystals and paclitaxel nanoparticles.

IDO is an enzyme that tumors use to create a state of immunosupression. 1-d-methyltryptophan (1-MT) is an IDO pathway inhibitor. After being successfully evaluated in preclinical studies, current clinical trials are actually analyzing its efficacy as monotherapy or in combination with multiple chemotherapeutic agents such as paclitaxel. 1-MT very poor solubility in water and many other solvents precludes its ease parenteral administration. It is currently administered by oral route because high daily doses were well-tolerated and effectively inhibited the IDO activity although only 25% of dose was recovered in plasma. The present work describes the preparation and characterization of 1-MT nanocrystals in order to enhance its solubility, dissolution rate, biodisponibility as well as facilitate its administration by parenteral route. A bottom-down approach of nanoprecipitation with an antisolvent was used for the fabrication of the nanocrystals and the choice of stabilizers was critical for reducing the size. Thermal analysis and x-ray diffraction indicated modifications in the drug crystalline state by the process. Through the reduction size and crystalline state modifications the dissolution characteristics of raw material were significantly increased. In a Lewis Lung cancer mice model, the nanocrystals strategy facilitated the sc administration and its antitumoral activity was similar to that of i.v. paclitaxel. The best efficacy was achieved when sc 1-MT nanocrystals were administered in combination with oral paclitaxel loaded in poly(anhydride) nanoparticles. Take together, 1-MT nanocrystals delivery performs a nanotechnological strategy suitable to modify the current route and schedule for its administration.

Towards a non-living vaccine against Shigella flexneri: from the inactivation procedure to protection studies.

Shigellosis is one of the leading causes of diarrhea worldwide with more than 165 million cases annually. Hence, a vaccine against this disease is a priority, but no licensed vaccine is still available. Considering target population as well as intrinsic risks of live attenuated vaccines, non-living strategies appear as the most promising candidates. Remarkably, the preservation of antigenic properties is a major concern since inactivation methods of bacteria affect these qualities. We previously reported the use of a subcellular antigen complex for vaccination against shigellosis, based on outer membrane vesicles (OMVs) released from Shigella flexneri. Now, we describe in more detail the employment of binary ethylenimine (BEI) for inactivation of Shigella and its subsequent effect on the antigenic conservation of the vaccinal product. Results demonstrate the effectiveness of BEI treatment to completely inactivate Shigella cells without disturbing the antigenicity and immunogenicity of the OMVs. Thus, OMVs harvested after BEI inactivation were able to protect mice against an experimental infection with S. flexneri.

[Mucopenetrating nanoparticles: vehicles for the oral administration of paclitaxel]. / Nanoparticules mucopénétrantes: véhicules pour l'administration orale du paclitaxel.

Paclitaxel is an anticancer drug used as solution for perfusion for the treatment of certain types of cancers. In the last years, a number of strategies have been proposed for the development of an oral formulation of this drug. However, this task is quite complicated due to the poor aqueous solubility of paclitaxel as well as the fact that this compound is substrate of the intestinal P-glycoprotein and the cytochrome P450 enzymatic complex. In this work, we have developed pegylated nanoparticles with mucopenetrating properties in order to conduct paclitaxel onto the surface of the enterocyte. These nanoparticles displayed a size of about 180 nm and a drug loading close to 15% by weight. The pharmacokinetic study in mice has shown that these nanoparticles were capable to offer therapeutic plasma levels of paclitaxel up to 72 hours. In addition, the oral relative bioavailability of paclitaxel when loaded in nanoparticles pegylated with poly(ethylene glycol) 2000 (PEG) was found to be 85%. In a subcutaneous model of tumour in mice, these pegylated nanoparticles administered orally every 3 days have demonstrated a similar efficacy than Taxol® administered intravenously every day during 9 days. All of these results suggested that these pegylated nanoparticles were capable to cross the mucus layer of the gut and, then, reach the surface of the enterocytes. The PEG molecules would facilitate the adhesion of nanoparticles to this epithelial surface, minimise the pre-systemic metabolism of paclitaxel and, thus, promote its absorption.

Gantrez AN nanoparticles for ocular delivery of memantine: in vitro release evaluation in albino rabbits.

AIM: To prepare and evaluate the in vitro release of memantine-loaded poly(anhydride) (Gantrez®) nanoparticles (NPs). The clinical safety and retinal toxicity caused by unloaded NPs after sub-Tenon and intravitreal ocular injections were also evaluated. METHODS: Preparation and characterization of this type of NP as well as the in vitro release study are described. Twenty-three healthy New Zealand rabbits were used for clinical and histological assessment after sub-Tenon and intravitreal ocular injections of unloaded NPs. RESULTS: The amount of drug associated with NPs was 55 µg of memantine/mg of NP. The release profile of memantine from this type of NPs was characterized by an initial burst effect, followed by continuous release of the drug for at least 15 days. No relevant complications were found during the clinical follow-up. The histological evaluation suggested that Gantrez NPs are well tolerated after sub-Tenon ocular injection and that signs of inflammation during the first days after intravitreal ocular injections can be considered a normal reaction of the eye's defence mechanism.

Preparation and evaluation of PEG-coated zein nanoparticles for oral drug delivery purposes.

The aim was to produce PEG-coated nanoparticles (NP-PEG), with mucus-permeating properties, for oral drug delivery purposes by using simple procedures and regulatory-approved compounds in order to facilitate a potential clinical development. For this purpose, zein nanoparticles were prepared by desolvation and, then, coated by incubation with PEG 35,000. The resulting nanocarriers displayed a mean size of about 200 nm and a negative zeta potential. The presence of PEG on the surface of nanoparticles was evidenced by electron microscopy and confirmed by FTIR analysis. Likely, the hydrophobic surface of zein nanoparticles (NP) was significantly reduce by their coating with PEG. This increase of the hydrophilicity of PEG-coated nanoparticles was associated with an important increase of their mobility in pig intestinal mucus. In laboratory animals, NP-PEG (fluorescently labelled with Lumogen® Red 305) displayed a different behavior when compared with bare nanoparticles. After oral administration, NP appeared to be trapped in the mucus mesh, whereas NP-PEG were capable of crossing the protective mucus layer and reach the epithelium. Finally, PEG-coated zein nanoparticles, prepared by a simple and reproducible method without employing reactive reagents, may be adequate carriers for promoting the oral bioavailability of biomacromolecules and other biologically active compounds with low permeability properties.

Different approaches for determination of the attachment degree of polyethylene glycols to poly(anhydride) nanoparticles.

PURPOSE: The aim of the study was to find an appropriate method for determination of the attachment degree of polyethylene glycol (PEG) to poly(anhydride) nanoparticles. METHODS: The nanoparticles were modified with hydroxy-functionalized or with amino-functionalized PEGs. Three methods for their determination were applied and examined: in particular colorimetry, nuclear magnetic resonance ( (1) H-NMR), and elemental analysis (EA). RESULTS: The attachment degrees determined by (1) H-NMR and colorimetry were similar. The associated amounts of PEGs estimated on the basis of EA differed significantly than those determined by colorimetry and (1) H-NMR. CONCLUSION: The colorimetric determination was considered fast and simple technique, but (1) H-NMR contributed to more precise results.

Preparation, radiolabeling with 99mTc and 67Ga and biodistribution studies of albumin nanoparticles covered with polymers. / Preparación, radiomarcaje con 99mTc y 67Ga y estudios de biodistribución de nanopartículas de albúmina con recubrimientos poliméricos.

OBJECTIVE: To optimize radiolabeling with 99mTc and 67Ga of albumin nanoparticles coated with 4 differents synthetic polymers and to evaluate their stability in vivo and in vitro, as well as their biodistribution in vivo after intravenous administration. MATERIAL AND METHODS: The nanoparticles were prepared using albumin and NOTA-modified albumin by the desolvation method and coated with 4 different polymers; HPMC, GMN2, GPM2 and GTM2. They were purified, lyophilized and characterized. Radiolabelling with 99mTc was perfomed with 74 MBq of 99mTc sodium pertechnetate, previously reduced with and acid solution of tin chloride at different concentrations (0.003, 0.005, 0.007, 0.01, 0.05 and 0.1mg/ml) and at different times (5, 10, 15, 30 and 60minutes) and temperatures (room temperature, 40°C and 60°C). Radiolabelling with 67Ga was perfomed by incubation of the nanoparticles with 37 MBq of 67Gallium chloride (obtained from commercial gallium-67 citrate) at different times (10 and 30minutes) and temperatures (room temperature, 30°C and 60°C), and posterior purification with microconcentrators. The radiochemical purity was evaluated by TLC. Stability studies of radiolabeled nanoparticles in physiological serum and blood plasma were perfomed. Biodistribution studies of nanoparticles coated with GPM2 polymer were carried out in Wistar rats after intravenous administration of the nanoparticles. Control animals were carried out with 99mTc sodium pertechnetate and 67Ga chloride. To do so, the animals were killed and activity in organs was measured in a gamma counter. RESULTS: 99mTc labeling was carried out optimally with a tin concentration of 0.007mg/ ml for the GPM2 nanoparticles and 0.005mg / ml for the rest of the formulations, with a radiolabelling time of 10minutes at room temperature. In the case of 67Ga the label was optimized at 30° C temperature and 30minutes of incubation. In both cases the radiochemical purity obtained was greater than 97%. The nanoparticles showed high stability in vitro after 48hours of labeling (70% nanoparticles labeled with 99mTc and 90% those labeled with 67Ga). Biodistribution studies of nanoparticles 99mTc -GPM2 and 67Ga -NOTA-GPM2 showed a high accumulation of activity in the liver at 2 and 24hours after intravenous administration. CONCLUSION: The labeling procedure with 99mTc and 67Ga of albumin and albumin modified with NOTA nanoparticles allows obtaining nanoparticles with high labeling yields and adequate in vitro stability, allowing their use for in vivo studies.

Soybean protein-based microparticles for oral delivery of probiotics with improved stability during storage and gut resistance.

The present work describes the encapsulation of probiotics using a by-product as wall material and a process feasible to be scaled-up: coacervation of soybean protein concentrate (SPC) by using calcium salts and spray-drying. SPC was extracted from soybean flour, produced during the processing of soybean milk, by alkaline extraction following isoelectric precipitation. Two probiotic strains were selected for encapsulation (Lactobacillus plantarum CECT 220 and Lactobacillus casei CECT 475) in order to evaluate the ability of SPC to encapsulate and protect bacteria from stress conditions. The viability of these encapsulated strains under in vitro gastrointestinal conditions and shelf-life during storage were compared with the most common forms commercialized nowadays. Results show that SPC is a feasible material for the development of probiotic microparticles with adequate physicochemical properties and enhanced significantly both probiotic viability and tolerance against simulated gastrointestinal fluids when compared to current available commercial forms.

Evaluation of bioadhesive potential and intestinal transport of pegylated poly(anhydride) nanoparticles.

Nanoparticles based on the poly(methyl vinyl ether-co-maleic anhydride) were pegylated with different types of PEGs, namely, two hydroxyl-functionalized PEGs (PEG and mPEG) and two amino-PEGs (DAE-PEG and DAP-PEG). The resulted nanoparticles demonstrated reduction of the negative surface charge compared to the non-modified particles. Further, in vivo experiments showed that all types of pegylated particles possessed higher affinity to adhere to intestinal rather than to the stomach mucosa. Higher bioadhesive potential was observed in the case of PEG-NP and DAE-PEG-NP which was attributed to the flexibility and specific properties of the surface "brush" layer of these particles. The lower bioadhesive potential of mPEG-NP was due to the low presence of coating "brush" layer, whereas for DAP-PEG-NP to the fact that the double end coupled chains ("loop"-conformation) were not available for intensive interactions with the mucosa. The observations made by optic microscopy illustrated an intracellular transport of PEG-NP in vivo with preferable location in the apical area of enterocytes.

In vitro evaluation of the genotoxicity of poly(anhydride) nanoparticles designed for oral drug delivery.

In the last years, the development of nanomaterials has significantly increased due to the immense variety of potential applications in technological sectors, such as medicine, pharmacy and food safety. Focusing on the nanodevices for oral drug delivery, poly(anhydride) nanoparticles have received extensive attention due to their unique properties, such as their capability to develop intense adhesive interactions within the gut mucosa, their modifiable surface and their biodegradable and easy-to-produce profile. However, current knowledge of the possible adverse health effects as well as, toxicological information, is still exceedingly limited. Thus, we investigated the capacity of two poly(anhydride) nanoparticles, Gantrez® AN 119-NP (GN-NP) and Gantrez® AN 119 covered with mannosamine (GN-MA-NP), and their main bulk material (Gantrez® AN 119-Polymer), to induce DNA damage and thymidine kinase (TK+/-) mutations in L5178Y TK+/- mouse lymphoma cells after 24h of exposure. The results showed that GN-NP, GN-MA-NP and their polymer did not induce DNA strand breaks or oxidative damage at concentrations ranging from 7.4 to 600µg/mL. Besides, the mutagenic potential of these nanoparticles and their polymer revealed no significant or biologically relevant gene mutation induction at concentrations up to 600µg/mL under our experimental settings. Considering the non-genotoxic effects of GN-NP and GN-MA-NP, as well as their exceptional properties, these nanoparticles are promising nanocarriers for oral medical administrations.

Evaluation of the cytotoxicity, genotoxicity and mucus permeation capacity of several surface modified poly(anhydride) nanoparticles designed for oral drug delivery.

The main concerns with drugs designed for oral administration are their inactivation or degradation in the harsh conditions of the gastrointestinal tract, their poor solubility through the gastrointestinal mucus gel layer, the poor intestinal epithelium permeability that limits their absorption, and their toxicity. In this context, poly(anhydride) nanoparticles are capable of protecting the drug from the harsh environment, reduce the drug's toxicity and, by virtue of surface modification, to enhance or reduce their mucus permeability and the bioadhesion to specific target cells. The copolymer between methyl vinyl ether and maleic anhydride (commercialized as Gantrez® AN 119) are part of the poly(anhydride) nanoparticles. These biocompatible and biodegradable nanoparticles (NPs) can be modified by using different ligands. Their usefulness as drug carriers and their bioadhesion with components of the intestinal mucosa have been described. However, their toxicity, genotoxicity and mucus permeation capacity has not been thoroughly studied. The aim of this work was to evaluate and compare the in vitro toxicity, cell viability and in vitro genotoxicity of the bioadhesive empty Gantrez® AN 119 NPs modified with dextran, aminodextran, 2-hydroxypropyl-ß-cyclodextrin, mannosamine and poly-ethylene glycol of different molecular weights. Results showed that, in general, coated NPs exhibit better mucus permeability than the bare ones, those coated with mannosamine being the most permeable ones. The NPs studied did not affect cell metabolism, membrane integrity or viability of Caco-2 cells at the different conditions tested. Moreover, they did not induce a relevant level of DNA strand breaks and FPG-sensitive sites (as detected with the comet assay).

Genotoxic evaluation of poly(anhydride) nanoparticles in the gastrointestinal tract of mice.

Gantrez® AN 119-based NPs have been developed as oral drug carriers due to their strong bioadhesive interaction with components of the gastrointestinal mucosa and to their adaptable surface. The use of mannosamine to coat Gantrez® AN 119-based NPs results in a high mucus-permeable carrier, able to reach the gastrointestinal epithelium. Although their efficacy to transport a therapeutic agent has been demonstrated, their safety has not yet been thoroughly studied. They have proved to be non-cytotoxic, non-genotoxic and non-mutagenic in vitro; however, the in vivo toxicity profile has not yet been determined. In this study, the in vivo genotoxic potential of Gantrez® AN 119 NPs coated with mannosamine (GN-MA-NP) has been assessed using the in vivo comet assay in combination with the enzyme formamidopyrimidine DNA glycosylase in mice, following the OECD test guideline 489. To determine the relevant organs to analyse and the sampling times, an in vivo biodistribution study was also carried out. Results showed a statistically significant induction of DNA strand breaks and oxidized bases in the duodenum of animals exposed to 2000 mg/kg bw. However, this effect was not observed at lower doses (i.e. 500 and 1000 mg/kg which are closer to the potential therapeutic doses) or in other organs. In conclusion, GN-MA-NP are promising nanocarriers as oral drug delivery systems.

Albumin nanoparticles for the intravitreal delivery of anticytomegaloviral drugs.

Albumin nanoparticles (NP) were proved to be effective and safe carriers for delivering anticytomegaloviral compounds in the vitreous. NP improved the antiviral activity of both ganciclovir and the phosphodiester oligonucleotide analog to formivirsen. NP appeared to be fusogenic carriers able to target the nucleus of cells. In addition, these drug carriers were well tolerated when administered by the intravitreal route and did not induce autoimmune reactions.

Toxicity evaluation of nanocarriers for the oral delivery of macromolecular drugs.

Oral administration is the most commonly used and accepted route for drug administration. However, two of the main concerns are the poor intestinal epithelium permeability and rapid degradation, which limit absorption of drugs. In this context, nanocarriers have shown great potential for oral drug delivery. Nevertheless, special importance should be given to the possible toxic effect of these nanocarriers, such as their bioaccumulation in different tissues of the body, as well as, the different physicochemical parameters influencing their properties and so their potential toxic effect. This review describes first some aspects related to the behavior of nanosystems within the gastrointestinal tract and then some aspects of nanotoxicology and its evaluation, including the most popular techniques and approaches used for in vitro and in vivo toxicity studies. It also reviews the physicochemical characteristics of polymeric nanoparticles that may influence the development of toxicological effects, and finally it summarizes the toxicity results that have been published regarding polymeric nanocarriers.

Mimicking microbial strategies for the design of mucus-permeating nanoparticles for oral immunization.

Dealing with mucosal delivery systems means dealing with mucus. The name mucosa comes from mucus, a dense fluid enriched in glycoproteins, such as mucin, which main function is to protect the delicate mucosal epithelium. Mucus provides a barrier against physiological chemical and physical aggressors (i.e., host secreted digestive products such as bile acids and enzymes, food particles) but also against the potentially noxious microbiota and their products. Intestinal mucosa covers 400m(2) in the human host, and, as a consequence, is the major portal of entry of the majority of known pathogens. But, in turn, some microorganisms have evolved many different approaches to circumvent this barrier, a direct consequence of natural co-evolution. The understanding of these mechanisms (known as virulence factors) used to interact and/or disrupt mucosal barriers should instruct us to a rational design of nanoparticulate delivery systems intended for oral vaccination and immunotherapy. This review deals with this mimetic approach to obtain nanocarriers capable to reach the epithelial cells after oral delivery and, in parallel, induce strong and long-lasting immune and protective responses.

Interactions of poly (anhydride) nanoparticles with macrophages in light of their vaccine adjuvant properties.

Understanding how nanoparticles are formed and how those processes ultimately determine the nanoparticles' properties and their impact on their capture by immune cells is key in vaccination studies. Accordingly, we wanted to evaluate how the previously described poly (anhydride)-based nanoparticles of the copolymer of methyl vinyl ether and maleic anhydride (NP) interact with macrophages, and how this process depends on the physicochemical properties derived from the method of preparation. First, we studied the influence of the desolvation and drying processes used to obtain the nanoparticles. NP prepared by the desolvation of the polymers in acetone with a mixture of ethanol and water yielded higher mean diameters than those obtained in the presence of water (250nm vs. 180nm). In addition, nanoparticles dried by lyophilization presented higher negative zeta potentials than those dried by spray-drying (-47mV vs. -35mV). Second, the influence of the NP formulation on the phagocytosis by J774 murine macrophage-like cell line was investigated. The data indicated that NPs prepared in the presence of water were at least three-times more efficiently internalized by cells than NPs prepared with the mixture of ethanol and water. Besides, lyophilized nanoparticles appeared to be more efficiently taken up by J744 cells than those dried by spray-drying. To further understand the specific mechanisms involved in the cellular internalization of NPs, different pharmacological inhibitors were used to interfere with specific uptake pathways. Results suggest that the NP formulations, particularly, nanoparticles prepared by the addition of ethanol:water, are internalized by the clathrin-mediated endocytosis, rather than caveolae-mediated mechanisms, supporting their previously described vaccine adjuvant properties.

Preparation and characterization of lectin-latex conjugates for specific bioadhesion.

This paper reports on the preparation and characterization of certain bioadhesive model drug deliver systems formed by a carrier (e.g. modified nanoparticles of polystyrene) and a ligand (e.g. tomato lectin, asparagus pea lectin, Mycoplasma gallisepticum lectin or albumin). Three different manufacturing methods (carbodiimide and glutaraldehyde coupling and physical adsorption) were studied. The activity of the lectin-latex conjugates and albumin-latex conjugate (control) were tested with gastric pig mucin. The manufacturing method had an insignificant effect on the activity, but all lectin-latex conjugates interacted two or three times more with mucin than with the control.

Loading of plasmid DNA into PLGA microparticles using TROMS (Total Recirculation One-Machine System): evaluation of its integrity and controlled release properties.

Loading plasmid DNA into poly(ester) microparticles usually involves the formation of a multiple emulsion, using homogenisation techniques such as sonication or Ultra-Turrax. These procedures may negatively affect the integrity of the macromolecule and consequently its activity. The aim of this study was to prepare and evaluate DNA-loaded microparticles by TROMS (Total Recirculation One-Machine System), a new procedure that is based on the formation of a multiple emulsion by the injection of the phases under a turbulent regime. Microparticles were prepared with either Resomer) RG 502 (MP 502) or RG 756 (MP 756) and DNA loading was quantified fluorimetrically. DNA loading in MP 756 was almost twice as high as in MP 502 (510 vs. 285 ng/mg, respectively). Under both formulations, the loaded plasmid was released while maintaining its integrity for at least 24 days (MP 502) and 40 days (MP 756). Finally, the transfection efficiency was studied after injection of the microparticles (MP 502) into rat skeletal muscle and compared with naked DNA injection. Injection of naked DNA (150 microg DNA per muscle) achieved higher but variable expression levels that decreased after 3 weeks. In contrast, the muscles injected with microparticles (6.8 microg DNA per muscle) showed lower but homogeneous expression values, which were maintained for at least 3 weeks.

Albumin nanoparticles improved the stability, nuclear accumulation and anticytomegaloviral activity of a phosphodiester oligonucleotide.

The goal of this study was to evaluate the potential of albumin nanoparticles as a delivery system for antisense oligonucleotides. Nanoparticles were prepared by a coacervation process and cross-linkage with glutaraldehyde. Phosphodiester (PO) and phosphorotioate (PS) oligonucleotides were either adsorbed on the surface of nanoparticles (PO-NPA and PS-NPA) or incorporated in the nanoparticle matrix (PO-NPB and PS-NPB). When PO-loaded nanoparticles were incubated with phosphodiesterase, only NPB was able to keep the oligonucleotide hybridization capability for at least 60 min. The antiviral activity was evaluated in MRC-5 fibroblasts infected with human cytomegalovirus at a MOI of 0.0035. Both PO nanoparticle formulations significantly increased the antiviral activity of free PO (P<0.001) and NPB showed slightly higher efficacies than NPA (P<0.05). On the other hand, PS exhibited significant higher activity than free PO (P<0.001), however, no significant differences were found between PS-nanoparticle and PO-nanoparticle formulations. These findings were well correlated with the intracellular distribution observed for fluorescent oligonucleotide-loaded albumin nanoparticles. Even these carriers delayed and decreased the uptake of PO by MRC-5 cells, they finally induced a diffused cytoplasmic distribution and major nuclear accumulation. In summary, albumin nanoparticles partially protected a PO against enzymatic degradation and improved their presence in the nucleus and thus, increased its efficiency.

Gantrez AN as a new polymer for the preparation of ligand-nanoparticle conjugates.

The aim of this study was to evaluate the feasibility and in vitro activity of ligand-conjugates based on the use of poly(methyl vinyl ether-co-maleic anhydride) (PVM/MA or Gantrez AN). Fluorescently labelled PVM/MA nanoparticles were prepared by desolvation and cross-linkage with 1,3-diaminopropane (DP). Conjugates were obtained by incubation between the carriers and Sambucus nigra agglutinin (SNA) for 1 h in an aqueous medium. The lectin binding to the surface of nanoparticles was increased by both increasing the bulk ligand concentration and decreasing the amount of cross-linker. However, a concentration of about 0.3-0.4 mg DP per mg polymer was necessary to obtain maximum agglutination activity. Under optimal conditions, the amount of fixed ligand was 46 microg/mg nanoparticle (binding efficiency of 86%); although the activity of SNA conjugates was 13.3 microg/mg particle. The activity of nanoparticles, measured by the association to Caco-2 monolayers, was higher when SNA was covalently bound onto their surface. The lectin-conjugate interaction was 6-fold higher than conventional nanoparticles. Moreover, energy-dependent mechanisms were only observed in SNA-PVM/MA particles. Finally, the decrease in association in the presence of lactose demonstrates that both SNA- and SNA-conjugate-binding was due to a true lectin-sugar interaction.