Methods for Intracellular Delivery of Quantum Dots.

Quantum dots (QDs) have attracted considerable attention as fluorescent probes for life sciences. The advantages of using QDs in fluorescence-based studies include high brilliance, a narrow emission band allowing multicolor labeling, a chemically active surface for conjugation, and especially, high photostability. Despite these advantageous features, the size of the QDs prevents their free transport across the plasma membrane, limiting their use for specific labeling of intracellular structures. Over the years, various methods have been evaluated to overcome this issue to explore the full potential of the QDs. Thus, in this review, we focused our attention on physical and biochemical QD delivery methods-electroporation, microinjection, cell-penetrating peptides, molecular coatings, and liposomes-discussing the benefits and drawbacks of each strategy, as well as presenting recent studies in the field. We hope that this review can be a useful reference source for researches that already work or intend to work in this area. Strategies for the intracellular delivery of quantum dots discussed in this review (electroporation, microinjection, cell-penetrating peptides, molecular coatings, and liposomes).

Evaluating the glycophenotype on breast cancer tissues with quantum dots-Cramoll lectin conjugates.

During carcinogenesis, changes in the glycosylation can modulate many biological processes. Thus, the interest in exploring and understanding the roles of carbohydrates as cancer biomarkers has been increasing. Lectins have been applied as useful tools in glycobiology, especially when associated with fluorescent reporters. Therefore, to take advantage of the physicochemical properties of quantum dots (QDs), herein, we conjugated Cramoll, a lectin that recognizes glucose/mannose residues, with those nanoparticles. We applied the conjugates to investigate the glycocode of normal, fibroadenoma (FB), and invasive ductal carcinoma (IDC) human breast tissues. Additionally, we proposed a method to quantitatively evaluate the tissue labeling intensity by a fluorescence microplate assay (FMA). Conjugates showed intense fluorescence and specificity. The lectin activity and secondary structure were also preserved after the conjugation with QDs. Moreover, fluorescence images showed that ductal cells of normal and FB tissues were preferentially labeled by conjugates, whereas both cells and stroma were strongly labeled in IDC. FMA showed in a quantitative, practical, and sensitive way that the level of exposed glucose/mannose residues increased accordingly to the sample malignancy degree. In conclusion, QDs-Cramoll conjugates can be considered effective, specific, and versatile probes to evaluate glycan profiles in normal and transformed tissues, by fluorescence microscopy as well as FMA quantification. Furthermore, FMA showed to be a potential method that can be applied with other fluorescent conjugates.

Quantum dot-Cramoll lectin as novel conjugates to glycobiology.

The optical properties of quantum dots (QDs) make them useful tools for biology, especially when combined with biomolecules such as lectins. QDs conjugated to lectins can be used as nanoprobes for carbohydrate expression analysis, which can provide valuable information about glycosylation changes related to cancer and pathogenicity of microorganisms, for example. In this study, we evaluated the best strategy to conjugate Cramoll lectin to QDs and used the fluorescent labeling of Candida albicans cells as a proof-of-concept. Cramoll is a mannose/glucose-binding lectin with unique biological properties such as immunomodulatory, antiparasitic, and antitumor activities. We probed covalent coupling and adsorption as conjugation strategies at different pH values. QDs conjugated to Cramoll at pH7.0 showed the best labeling efficiency in the fluorescence microscopy analysis. Moreover, QD-Cramoll conjugates remained brightly fluorescent and preserved identical biological activity according to hemagglutination assays. Flow cytometry revealed that approximately 17% of C. albicans cells were labeled after incubation with covalent conjugates, while approximately 92% of cells were labeled by adsorption conjugates (both at pH7.0). Inhibition assays confirmed QD-Cramoll specificity, which reduced the labeling to at most 3%. Therefore, the conjugates obtained by adsorption (pH7.0) proved to be promising and versatile fluorescent tools for glycobiology.

Encapsulation into Stealth Liposomes Enhances the Antitumor Action of Recombinant Cratylia mollis Lectin Expressed in Escherichia coli.

This study evaluated the in vivo antitumor potential of the recombinant lectin from seeds of Cratylia mollis (rCramoll) expressed in Escherichia coli, free or encapsulated in stealth liposomes, using mice transplanted with sarcoma 180. rCramoll-loaded stealth liposomes (rCramoll-lipo) were formulated by hydration of the lipid film followed by cycles of freezing and thawing, and about 60% of rCramoll was encapsulated. This novel preparation showed particle size, polydispersity index, and pH suitable for the evaluation of antitumor activity in vivo. Tumor growth inhibition rates were 59% for rCramoll and 75% for rCramoll-lipo. Histopathological analysis of the experimental groups showed that both free and encapsulated lectin caused no changes in the kidneys of animals. Hematological analysis revealed that treatment with rCramoll-lipo significantly increased leukocyte concentration when compared with the untreated and rCramoll group. In conclusion, the encapsulation of rCramoll in stealth liposomes improves its antitumor activity without substantial toxicity; this approach was more successful than the previous results reported for pCramoll loaded into conventional liposomes. At this point, a crucial difference between the antitumor action of free and encapsulated rCramoll was found along with their effects on immune cells. Further investigations are required to elucidate the mechanism(s) of the antitumor effect induced by rCramoll.