Removal of Erythromycin from Water by Ibuprofen-Driven Pre-Organized Divinyl Sulfone Cross-Linked Dextrin.

Water recycling and reuse are cornerstones of water management, which can be compromised by the presence of pollutants. Among these, pharmaceuticals can overcome standard water treatments and require sophisticated approaches to remove them. Sorption is an economically viable alternative limited by the need for sorbents with a sorption coefficient (Kd) higher than 500 L/kg. The cross-linking of dextrin (Dx) with divinyl sulfone (DVS) in the presence of 1 mmol or 5 mmol of ibuprofen (IBU) yields the insoluble polymers pDx1 and pDx5 with improved affinity for IBU and high selectivity towards erythromycin (ERY) and ERY Kd higher than 4 × 103 L/kg, when tested against a cocktail of six drugs. Characterization of the polymers shows that both pDx1 and pDx5 have similar properties, fast sorption kinetics, and ERY Kd of 13.3 × 103 for pDx1 and 6.4 × 103 for pDx5, representing 26.6 and 12.0 times the 500 L/kg threshold. The fact that new affinities and improvements in Kd can be achieved by cross-linking Dx in the presence of other molecules that promote pre-organization expands the applications of DVS cross-linked polysaccharides as sustainable, scalable, and environmentally friendly sorbents with a potential application in wastewater treatment plants (WTPs).

Removal of the Water Pollutant Ciprofloxacin Using Biodegradable Sorbent Polymers Obtained from Polysaccharides.

Water use has been increasing globally by 1% per year, and recycling and re-use are critical issues compromised by the presence of pollutants. In this context, the design of novel materials and/or procedures for the large scale-removal of pollutants must be economically and environmentally feasible in order to be considered as part of the solution by emerging economies. We demonstrate that the cross-linking of biodegradable polysaccharides such as starch, dextrin, or dextrin and ß-cyclodextrin with divinyl sulfone is an innovative strategy for synthesizing insoluble and eco-friendly sorbent polymers, including pSt, pDx and pCD-Dx. The evaluation of these polymers' ability to remove ciprofloxacin (CIP), a prime example of antibiotic pollution, revealed that pSt, with a Kd of 1469 L/kg and a removal rate higher than 92%, is a favorable material. Its sorption is pH-dependent and enhanced at a mildly alkaline pH, allowing for the desorption (i.e., cleaning) and reuse of pSt through an environmentally friendly treatment with 20 mM AcONa pH 4.6. The facts that pSt (i) shows a high affinity for CIP even at high NaCl concentrations, (ii) can be obtained from affordable starting materials, and (iii) is synthesized and regenerated through organic, solvent-free procedures make pSt a novel sustainable material for inland water and seawater remediation, especially in less developed countries, due to its simplicity and low cost.

Molecular Recognition of Surface Trans-Sialidases in Extracellular Vesicles of the Parasite Trypanosoma cruzi Using Atomic Force Microscopy (AFM).

Trans-sialidases (TS) are important constitutive macromolecules of the secretome present on the surface of Trypanosoma cruzi (T. cruzi) that play a central role as a virulence factor in Chagas disease. These enzymes have been related to infectivity, escape from immune surveillance and pathogenesis exhibited by this protozoan parasite. In this work, atomic force microscopy (AFM)-based single molecule-force spectroscopy is implemented as a suitable technique for the detection and location of functional TS on the surface of extracellular vesicles (EVs) released by tissue-culture cell-derived trypomastigotes (Ex-TcT). For that purpose, AFM cantilevers with functionalized tips bearing the anti-TS monoclonal antibody mAb 39 as a sense biomolecule are engineered using a covalent chemical ligation based on vinyl sulfonate click chemistry; a reliable, simple and efficient methodology for the molecular recognition of TS using the antibody-antigen interaction. Measurements of the breakdown forces between anti-TS mAb 39 antibodies and EVs performed to elucidate adhesion and forces involved in the recognition events demonstrate that EVs isolated from tissue-culture cell-derived trypomastigotes of T. cruzi are enriched in TS. Additionally, a mapping of the TS binding sites with submicrometer-scale resolution is provided. This work represents the first AFM-based molecular recognition study of Ex-TcT using an antibody-tethered AFM probe.

Polyethylenimine-Bisphosphonate-Cyclodextrin Ternary Conjugates: Supramolecular Systems for the Delivery of Antineoplastic Drugs.

Bisphosphonates (BPs) are bone-binding molecules that provide targeting capabilities to bone cancer cells when conjugated with drug-carrying polymers. This work reports the design, synthesis, and biological evaluation of polyethyleneimine-BP-cyclodextrin (PEI-BP-CD) ternary conjugates with supramolecular capabilities for the loading of antineoplastic drugs. A straightforward, modular, and versatile strategy based on the click aza-Michael addition reaction of vinyl sulfones (VSs) allows the grafting of BPs targeting ligands and ßCD carrier appendages to the PEI polymeric scaffold. The in vitro evaluation (cytotoxicity, cellular uptake, internalization routes, and subcellular distribution) for the ternary conjugates and their doxorubicin inclusion complexes in different bone-related cancer cell lines (MC3T3-E1 osteoblasts, MG-63 sarcoma cells, and MDA-MB-231 breast cancer cells) confirmed specificity, mitochondrial targeting, and overall capability to mediate a targeted drug transport to those cells. The in vivo evaluation using xenografts of MG-63 and MDA-MB-231 cells on mice also confirmed the targeting of the conjugates.

Single chain variable fragment fused to maltose binding protein: a modular nanocarrier platform for the targeted delivery of antitumorals.

The use of the specific binding properties of monoclonal antibody fragments such as single-chain variable fragments (ScFv) for the selective delivery of antitumor therapeutics for cancer cells is attractive due to their smaller size, low immunogenicity, and low-cost production. Although covalent strategies for the preparation of such ScFv-based therapeutic conjugates are prevalent, this approach is not straightforward, as it requires prior chemical activation and/or modification of both the ScFv and the therapeutics for the application of robust chemistries. A non-covalent alternative based on ScFv fused to maltose-binding protein (MBP) acting as a binding adapter is proposed for active targeted delivery. MBP-ScFv proves to be a valuable modular platform to synergistically bind maltose-derivatized therapeutic cargos through the MBP, while preserving the targeting competences provided by the ScFv. The methodology has been tested by using a mutated maltose-binding protein (MBP I334W) with an enhanced affinity toward maltose and an ScFv coding sequence toward the human epidermal growth factor receptor 2 (HER2). Non-covalent binding complexes of the resulting MBP-ScFv fusion protein with diverse maltosylated therapeutic cargos (a near-infrared dye, a maltosylated supramolecular ß-cyclodextrin container for doxorubicin, and non-viral polyplex gene vector) were easily prepared and characterized. In vitro and in vivo assays using cell lines that express or not the HER2 epitope, and mice xenografts of HER2 expressing cells demonstrated the capability and versatility of MBP-ScFv for diagnosis, imaging, and drug and plasmid active targeted tumor delivery. Remarkably, the modularity of the MBP-ScFv platform allows the flexible interchange of both the cargos and the coding sequence for the ScFv, allowing ad hoc solutions in targeting delivery without any further optimization since the MBP acts as a pivotal element.

A vinyl sulfone clicked carbon dot-engineered microfluidic paper-based analytical device for fluorometric determination of biothiols.

A microfluidic paper-based analytical device integrating carbon dot (CDs) is fabricated and used for a fluorometric off-on assay of biothiols. Vinyl sulfone (VS) click immobilization of carbon dots (CDs) on paper was accomplished by a one-pot simplified protocol that uses divinyl sulfone (DVS) as a homobifunctional reagent. This reagent mediated both the click oxa-Michael addition to the hydroxyl groups of cellulose and ulterior covalent grafting of the resulting VS paper to NH2-functionalized CDs by means of click aza-Michael addition. The resulting cellulose nanocomposite was used to engineer an inexpensive and robust microfluidic paper-based analytical device (µPAD) that is used for a reaction-based off-on fluorometric assay of biothiols (GSH, Cys, and Hcy). The intrinsic blue fluorescence of CDs (with excitation/emission maxima at 365/450 nm) is turned off via the heavy atom effect of an introduced iodo group. Fluorescence is turned on again due to the displacement of iodine by reaction with a biothiol. The increase in fluorescence is related to the concentration over a wide range (1 to 200 µM for GSH and 5-200 µM for Cys and Hcy, respectively), and the assay exhibits a low detection limit (0.3 µM for GSH and Cys and 0.4 µM for Hcy). The method allows for rapid screening and can also be used in combination with a digital camera readout. Graphical abstract Schematic representation of a µPAD based on click immobilized carbon dots and used for a reaction-based fluorometric off-on assay of biothiols. The intrinsic blue fluorescence of carbon dots is turned off via the heavy atom effect of an introduced iodo group and turned on by the displacement of this atom by reaction with a biothiol.

Carbon dots-inspired fluorescent cyclodextrins: competitive supramolecular "off-on" (bio)sensors.

Chromophore-appended cyclodextrins combine the supramolecular loading capabilities of cyclodextrins (CDs) with the optical properties of the affixed chromophores. Among fluorescent materials, carbon dots (CNDs) are attractive and the feasibility of CND-appended CDs as sensors has been demonstrated by different authors. However, CNDs are intrinsically heterogeneous materials and their ulterior functionalization yields hybrid composites that are not well defined in terms of structure and composition. Inspired by the fluorescence properties of 5-oxo-1,2,3,5-tetrahydroimidazo[1,2-a]pyridine-7-carboxylic acid (IPCA), the most paradigmatic of the molecular fluorophores detected in CNDs, herein we report two highly efficient synthetic chemical strategies for the preparation of IPCA-appended CDs that behave as CND-based CD "turn off-on" biosensors suitable for the analysis of cholesterol and ß-galactosidase activity. We have deconstructed the CND-CD systems to demonstrate that (i) the role of CNDs is limited to acting as a support for the molecular fluorophores produced during their synthesis and (ii) the molecular fluorophores suffice for the determination of the enzymatic activity based on the quenching by p-nitrophenol as a sacrificial quencher.

Acid anhydride coated carbon nanodots: activated platforms for engineering clicked (bio)nanoconstructs.

Activated carbon nanodots functionalized with acid anhydride groups (AA-CNDs) are prepared by one-pot water-free green thermolysis of citric acid. As a proof of concept of their capabilities as appealing and versatile platforms for accessing engineering nanoconstructs, the as-prepared AA-CNDs have been reacted to yield clickable CNDs. Their click bioconjugation with relevant recognizable complementary clickable sugars has led to multivalent CND-based glyconanoparticles that are non-toxic and biorecognizable. The accessibility and intrinsic reactivity of AA-CNDs expand the current toolbox of covalent surface grafting methodologies and provide a wide range of potential applications for engineering (bio)nanoconstructs.

Biological Evaluation and Docking Studies of Synthetic Oleanane-type Triterpenoids.

Saponins are potential wide-spectrum antitumor drugs, and copper(I) catalyzed azide-alkyne 1,3-dipolar cycloaddition is a suitable approach to synthesizing saponin-like compounds by regioselective glycosylation of the C2/C3 hydroxyl and C28 carboxylic groups of triterpene aglycones maslinic acid (MA) and oleanolic acid (OA). Biological studies on the T-84 human colon carcinoma cell line support the role of the hydroxyl groups at C2/C3, the influence of the aglycone, and the bulky nature of the substituents in C28. OA bearing a α-d-mannose moiety at C28 (compound 18) focused our interest because the estimated inhibitory concentration 50 was similar to that reported for ginsenoside Rh2 against colon cancer cells and it inhibits the G1-S phase transition affecting the cell viability and apoptosis. Considering that triterpenoids from natural sources have been identified as inhibitors of nuclear factor kappa-light-chain-enhancer of activated B cell (NF-κB) signaling, docking studies were conducted to evaluate whether NF-κB may be a potential target. Results are consistent with the biological study and predict a similar binding mode of MA and compound 18 to the p52 subunit from NF-κB but not for OA. The fact that the binding site is shared by the NF-κB inhibitor 6,6-dimethyl-2-(phenylimino)-6,7-dihydrobenzo[d][1,3]oxathiol-4(5H)-one supports the result and points to NF-κB as a potential target of both MA and compound 18.

PEI-NIR Heptamethine Cyanine Nanotheranostics for Tumor Targeted Gene Delivery.

Polymer-based nanotheranostics are appealing tools for cancer treatment and diagnosis in the fast-growing field of nanomedicine. A straightforward preparation of novel engineered PEI-based nanotheranostics incorporating NIR fluorescence heptamethine cyanine dyes (NIRF-HC) to enable them with tumor targeted gene delivery capabilities is reported. Branched PEI-2 kDa (b2kPEI) is conjugated with IR-780 and IR-783 dyes by both covalent and noncovalent simple preparative methodologies varying their stoichiometry ratio. The as-prepared set of PEI-NIR-HC nanocarriers are assayed in vitro and in vivo to evaluate their gene transfection efficiency, cellular uptake, cytotoxicity, internalization and trafficking mechanisms, subcellular distribution, and tumor specific gene delivery. The results show the validity of the approach particularly for one of the covalent IR783-b2kPEI conjugates that exhibit an enhanced tumor uptake, probably mediated by organic anion transporting peptides, and favorable intracellular transport to the nucleus. The compound behaves as an efficient nanotheranostic transfection agent in NSG mice bearing melanoma G361 xenographs with concomitant imaging signal and gene concentration in the targeted tumor. By this way, advanced nanotheranostics with multifunctional capabilities (gene delivery, tumor-specific targeting, and NIR fluorescence imaging) are generated in which the NIRF-HC dye component accounts for simultaneous targeting and diagnostics, avoiding additional incorporation of additional tumor-specific targeting bioligands.

Polyethyleneimine-Coated Gold Nanoparticles: Straightforward Preparation of Efficient DNA Delivery Nanocarriers.

A novel one-pot method for the synthesis of polyethyleneimine (PEI)-coated gold nanoparticles (AuPEI-NPs) that combines the reductant-stabilizer properties of PEI with microwave irradiation starting from hydrogen tetrachloroaurate acid (HAuCl4 ) and branched PEI 25 kDa (b25kPEI) was explored. The method was straightforward, green, and low costing, for which the Au/PEI ratio (1:1 to 1:128 w/w) was a key parameter to modulate their capabilities as DNA delivery nanocarriers. Transfection assays in CHO-k1 cells demonstrated that AuPEI-NPs with 1:16 and 1:32 w/w ratios behaved as effective DNA gene vectors with improved transfection efficiencies (twofold) and significantly lower toxicity than unmodified b25kPEI and Lipofectamine 2000. The transfection mediated by these AuPEI-NP-DNA polyplexes preferentially used the caveolae-mediated route for intracellular internalization, as shown by studies performed by using specific internalization inhibitors as well as colocalization with markers of clathrin- and caveolae-dependent pathways. The AuPEI-NP polyplexes preferentially used the more efficient caveolae internalization pathway to promote transfection, a fact that supports their higher transfection efficiency relative to that of Lipofectamine 2000. In addition, intracellular trafficking of the AuPEI-NPs was studied by transmission electron microscopy.

Polyelectrolyte Complexes of Low Molecular Weight PEI and Citric Acid as Efficient and Nontoxic Vectors for in Vitro and in Vivo Gene Delivery.

Gene transfection mediated by the cationic polymer polyethylenimine (PEI) is considered a standard methodology. However, while highly branched PEIs form smaller polyplexes with DNA that exhibit high transfection efficiencies, they have significant cell toxicity. Conversely, low molecular weight PEIs (LMW-PEIs) with favorable cytotoxicity profiles display minimum transfection activities as a result of inadequate DNA complexation and protection. To solve this paradox, a novel polyelectrolyte complex was prepared by the ionic cross-linking of branched 1.8 kDa PEI with citric acid (CA). This system synergistically exploits the good cytotoxicity profile exhibited by LMW-PEI with the high transfection efficiencies shown by highly branched and high molecular weight PEIs. The polyectrolyte complex (1.8 kDa-PEI@CA) was obtained by a simple synthetic protocol based on the microwave irradiation of a solution of 1.8 kDa PEI and CA. Upon complexation with DNA, intrinsic properties of the resulting particles (size and surface charge) were measured and their ability to form stable polyplexes was determined. Compared with unmodified PEIs the new complexes behave as efficient gene vectors and showed enhanced DNA binding capability associated with facilitated intracellular DNA release and enhanced DNA protection from endonuclease degradation. In addition, while transfection values for LMW-PEIs are almost null, transfection efficiencies of the new reagent range from 2.5- to 3.8-fold to those of Lipofectamine 2000 and 25 kDa PEI in several cell lines in culture such as CHO-k1, FTO2B hepatomas, L6 myoblasts, or NRK cells, simultaneously showing a negligible toxicity. Furthermore, the 1.8 kDa-PEI@CA polyelectrolyte complexes retained the capability to transfect eukaryotic cells in the presence of serum and exhibited the capability to promote in vivo transfection in mouse (as an animal model) with an enhanced efficiency compared to 25 kDa PEI. Results support the polyelectrolyte complex of LMW-PEI and CA as promising generic nonviral gene carriers.

In vitro and in vivo evaluation of novel cross-linked saccharide based polymers as bile acid sequestrants.

Bile acid sequestrants (BAS) represent a therapeutic approach for the management of hypercholesterolemia that relies on the cationic polymeric nature of BAS to selectively bind negatively charged bile acids. We hypothesized that the cross-linking of ß-cyclodextrin (ß-CD) and saccharides such as starch or dextrin with divinyl sulfone (DVS) yields homo- and hetero-polymeric materials with the ability to trap sterols. Our hypothesis was put to test by synthesizing a library of 22 polymers that were screened to evaluate their capability to sequester both cholesterol (CHOL) and cholic and deoxycholic acids (CA and DCA). Three polymers synthesized in high yield were identified as promising. Two were neutral hetero-polymers of ß-CD and starch or dextrin and the third was a weakly cationic homo-polymer of starch, highlighting the importance of the cavity effect. They were tested in hypercholesterolemic male Wistar rats and their ability to regulate hypercholesterolemia was similar to that for the reference BAS cholestyramine, but with two additional advantages: (i) they normalized the TG level and (ii) they did not increase the creatinine level. Neither hepatotoxicity nor kidney injury was detected, further supporting them as therapeutical candidates to manage hypercholesterolemia.

Divinyl sulfone cross-linked cyclodextrin-based polymeric materials: synthesis and applications as sorbents and encapsulating agents.

The aim of this study was to evaluate the crosslinking abilities of divinyl sulfone (DVS) for the preparation of novel water-insoluble cyclodextrin-based polymers (CDPs) capable of forming inclusion complexes with different guest molecules. Reaction of DVS with native α-cyclodextrin (α-CD), ß-cyclodextrin (ß-CD) and/or starch generates a variety of homo- and hetero-CDPs with different degrees of crosslinking as a function of the reactants' stoichiometric ratio. The novel materials were characterized by powder X-ray diffraction, electron microscopy and for their sorption of phenol and 4-nitrophenol. They were further evaluated as sorbents with phenolic pollutants (bisphenol A and ß-naphthol) and bioactive compounds (the hormone progesterone and curcumin). Data obtained from the inclusion experiments show that the degree of cross-linking has a minor influence on the yield of inclusion complex formation and highlight the important role of the CDs, supporting a sorption process based on the formation of inclusion complexes. In general, the inclusion processes are better described by a Freundlich isotherm although an important number of them can also be fitted to the Langmuir isotherm with R2 ≥ 0.9, suggesting a sorption onto a monolayer of homogeneous sites.

Engineered glycated amino dendritic polymers as specific nonviral gene delivery vectors targeting the receptor for advanced glycation end products.

The receptor for advanced glycation end products (RAGE) is involved in diabetes or angiogenesis in tumors. Under pathological conditions, RAGE is overexpressed and upon ligand binding and internalization stimulates signaling pathways that promote cell proliferation. In this work, amino dendritic polymers PEI 25 kDa and alkylated derivatives of PAMAM-G2 were engineered by the nonenzymatic Maillard glycation reaction to generate novel AGE-containing gene delivery vectors targeting the RAGE. The glycated dendritic polymers were easily prepared and retained the capability to bind and protect DNA from endonucleases. Furthermore, while glycation decreased the transfection efficiency of the dendriplexes in CHO-k1 cells which do not express RAGE, glycated dendriplexes acted as efficient transfection reagents in CHO-k1 cells which stably express recombinant RAGE. In addition, preincubation with BSA-AGEs, a natural ligand of the RAGE, or dansyl cadaverine, an inhibitor of the RAGE internalization, blocked transfection, confirming their specificity toward RAGE. The results were confirmed in NRK and RAW264.7 cell lines, which naturally express the receptor. The glycated compounds retain their transfection efficiency in the presence of serum and promote in vivo transfection in a mouse model. Accordingly, RAGE is a suitable molecular target for the development of site-directed engineered glycated nonviral gene vectors.

Masked thiol sugars: chemical behavior and synthetic applications of S-glycopyranosyl-N-monoalkyl dithiocarbamates.

The chemical behavior of S-glycopyranosyl-N-monoalkyl dithiocarbamates (DTCs) as masked 1-glycosyl thiols, easily prepared by the nucleophilic displacement of 1-halo sugars with dithiocarbamate salts of primary amines, has been studied and synthetically exploited. This behavior relies on the abstraction of the proton of the carbamate functionality that allows controlled access to thiolate sugar intermediates. The basic character of the DTC salts used as reagents leads to thiolates that evolve in situ to symmetrical diglycosyldisulfides (DGDSs) when long reaction times are allowed. Alternatively, controlled unmasking of the thiolate function can be efficiently attained by treatment with an external base of isolated anomeric glycosyl DTCs, the formation of which is prevalent when using short reaction times. In this manner, a second methodology for the preparation of symmetrical DGDSs and a chemical protocol for the S-glycosylation of any electrophilic substrate are established. The applications of this last strategy for the preparation of thioglycosyl vinyl sulfones, thiodisaccharides, and S-linked homo- and heterodivalent neoglycoconjugates are described as a proof-of-concept of the great potential of the sugar DTCs in any chemical scenario in which the covalent attachment of a thiol sugar is required. The evaluation of the biological functionality of some divalent sulfurated sugar systems is also described.

Vinyl sulfone-based ferrocenylation reagents: applications in conjugation and bioconjugation.

The easy vinyl sulfone derivatization of ferrocene allows the preparation of some effective, versatile and valuable ferrocenylation reagents. The applicability of such compounds in conjugation and bioconjugation of amine and/or thiol containing molecules and biomolecules through Michael-type addition under mild conditions that preserve the biological function of the latter is described. The feasibility of the methodology is demonstrated by the preparation of a variety of conjugates and bioconjugates (ferrocenyl terminated dendrimers and ferrocene-sugar, ferrocene-cyclodextrin, ferrocene-peptide and ferrocene-protein conjugates).

Vinyl sulfone functionalization: a feasible approach for the study of the lectin-carbohydrate interactions.

Carbohydrate-mediated molecular recognition is involved in many biological aspects such as cellular adhesion, immune response, blood coagulation, inflammation, and infection. Considering the crucial importance of such biological events in which proteins are normally involved, synthetic saccharide-based systems have emerged as powerful tools for the understanding of protein-carbohydrate interactions. As a new approach to create saccharide-based systems, a set of representative monosaccharides (D-mannose, D-glucose, N-acetyl-D-glucosamine, and L-fucose) and disaccharides (lactose, maltose, and melibiose) were derivatized at their anomeric carbon with a vinyl sulfone group spanned by an ethylthio linker. This vinyl sulfone functionalization is demonstrated to be a general strategy for the covalent linkage of a saccharide in mild conditions via Michael-type additions with the amine and thiol groups from functionalized supports and those naturally present in biomolecules. The introduction of the ethylthio linker between the biorecognizable element (i.e., saccharide) and the reactive group (i.e., vinyl sulfone) was found to preserve the functionality of the former. The capability of the vinyl sulfone saccharides for the study of lectin-carbohydrate interactions was demonstrated by (i) immobilizing them on both amine-functionalized supports (glass slides and microwell plates) and polylysine-coated glass slides to create sugar arrays that selectively bind lectins (ii) coupling to model proteins to yield neoglycoproteins that are recognized by lectins and (iii) using vinyl sulfone saccharides as tags to allow the detection of the labeled biomolecule by HRP-lectins. The above results were further put tothe test with a real case: detection of carbohydrate binding proteins present in rice ( Oryza sativa ).

Alkyl sulfonyl derivatized PAMAM-G2 dendrimers as nonviral gene delivery vectors with improved transfection efficiencies.

Amphiphilic dendrimer-based gene delivery vectors bearing peripheral alkyl sulfonyl hydrophobic tails were constructed using low-generation PAMAM-G2 as the core and functionalized by means of the aza-Michael type addition of its primary amino groups to vinylsulfone derivatives as an efficient tool for surface engineering. While the unmodified PAMAM-G2 was unable to efficiently transfect eukaryotic cells, functionalized PAMAM-G2 dendrimers were able to bind DNA at low N/P ratios, protect DNA from digestion with DNase I and showed high transfection efficiencies and low cytotoxicity. Dendrimers with a C18 alkyl chain produced transfection efficiencies up to 3.1 fold higher than LipofectAMINE™ 2000 in CHO-k1 cells. The dendriplexes based in functionalized PAMAM-G2 also showed the ability to retain their transfection properties in the presence of serum and the ability to transfect different eukaryotic cell lines such as Neuro-2A and RAW 264.7. Taking advantage of the vinylsulfone chemistry, fluorescent PAMAM-G2 derivatives of these vectors were prepared as molecular probes to determine cellular uptake and internalization through a clathrin-independent mechanism.