Effect of polyhydroxy-terminated PAMAM dendrimer on dentin matrix metalloproteinases within the hybrid layers.

BACKGROUND: Intrafibrillar remineralization within the hybrid layers (HLs) has recently attracted extensive attention in achieving durable resin-dentin bonds. The polyhydroxy-terminated poly(amidoamine) dendrimer (PAMAM-OH) at fourth generation becomes a desirable candidate to induce intrafibrillar remineralization to protect exposed collagen fibrils within HLs based on the size exclusion effect of fibrillar collagen. However, the remineralization process in vivo is time-consuming, during which the exposed collagen fibrils are vulnerable to enzymatic degradation, resulting in unsatisfactory remineralization. Thereby, if PAMAM-OH itself possesses concomitant anti-proteolytic activity during the induction of remineralization, it would be very beneficial to obtain satisfactory remineralization. METHODS: Binding capacity tests using adsorption isotherm and confocal laser scanning microscopy (CLSM) were performed to assess if the PAMAM-OH had adsorption capacity on dentin. Anti-proteolytic testings were detected by MMPs assay kit, in-situ zymography and ICTP assay. Adhesive infiltration of resin-dentin interface and tensile bond strength before and after thermomechanical cycling were implemented to assess if the PAMAM-OH adversely affected resin-dentin bonds. RESULTS: Anti-proteolytic testings performed using MMPs assay kit, in-situ zymography and ICTP assay indicated that PAMAM-OH inhibited exogenous soluble MMP-9 as well as had inhibitory effect on the endogenous proteases. Adhesive infiltration of resin-dentin interface and tensile bond strength before and after thermomechanical cycling were implemented to indicate that the PAMAM-OH pretreatment had no adverse effects on immediate dentin bonding and prolonged the durability of resin-dentin bonds. CONCLUSIONS: PAMAM-OH possesses anti-proteolytic activity and prevents exposed collagen fibrils within HLs from degradation, which lays the foundation for the satisfactory intrafibrillar remineralization induced by PAMAM-OH within HLs to achieve durable resin-dentin bonds in the next work.

Intermolecular interactions determined by NOE build-up in macromolecules from hyperpolarized small molecules.

The nuclear Overhauser effect (NOE) is a primary means to characterize intermolecular interactions using modern NMR spectroscopy. Multiple experiments measured using different mixing time can be used for quantifying NOE buildup and measuring cross-relaxation rates. However, this approach using conventional multi-dimensional NMR is time consuming. Hyperpolarization by dissolution dynamic nuclear polarization (D-DNP) can generate deviations from equilibrium spin polarization by orders of magnitude, thereby enhancing signals and allowing to characterize NOE build up in real-time. Since most small molecules can be hyperpolarized using D-DNP, this method is applicable to the study of intermolecular interactions between small molecules and macromolecules. This application is demonstrated using a model system for host-guest interactions including the third generation polyamidoamine dendrimer (G3 PAMAM) and the pharmaceutical phenylbutazone (PBZ). After mixing 1H hyperpolarized PBZ with PAMAM, the NOE build up is directly observed at different sites of the dendrimer in series of one-dimensional NMR spectra. Cross-relaxation rates specific to individual source and target spins are determined from the build up curves. Further, the polarization enhancement is shown to be sufficiently large to allow identification of cross-peaks not observed in a conventional 2D-NOESY spectrum. The improved signal-to-noise ratio provided by hyperpolarization allows for characterizing the intermolecular interaction in an almost instantaneous measurement, opening an application to macromolecular and biomacromolecular NMR.

A mechanistic investigation exploring the differential transfection efficiencies between the easy-to-transfect SK-BR3 and difficult-to-transfect CT26 cell lines.

BACKGROUND: Gold-polyamidoamine (AuPAMAM) has previously been shown to successfully transfect cells with high efficiency. However, we have observed that certain cell types are more amenable to Au-PAMAM transfection than others. Here we utilized two representative cell lines-a "difficult to transfect" CT26 cell line and an "easy to transfect" SK-BR3 cell line-and attempted to determine the underlying mechanism for differential transfection in both cell types. Using a commonly established poly-cationic polymer similar to PAMAM (polyethyleneimine, or PEI), we additionally sought to quantify the relative transfection efficiencies of each vector in CT26 and SK-BR3 cells, in the hopes of elucidating any mechanistic differences that may exist between the two transfection vectors. RESULTS: A comparative time course analysis of green fluorescent protein reporter-gene expression and DNA uptake was conducted to quantitatively compare PEI- and AuPAMAM-mediated transfection in CT26 and SK-BR3, while flow cytometry and confocal microscopy were used to determine the contribution of cellular uptake, endosomal escape, and cytoplasmic transport to the overall gene delivery process. Results from the time course analysis and flow cytometry studies revealed that initial complex uptake and cytoplasmic trafficking to the nucleus are likely the two main factors limiting CT26 transfectability. CONCLUSIONS: The cell type-dependent uptake and intracellular transport mechanisms impacting gene therapy remain largely unexplored and present a major hurdle in the application-specific design and efficiency of gene delivery vectors. This systematic investigation offers insights into the intracellular mechanistic processes that may account for cell-to-cell differences, as well as vector-to-vector differences, in gene transfectability.

Fluorophore:dendrimer ratio impacts cellular uptake and intracellular fluorescence lifetime.

G5-NH2-TAMRAn (n = 1-4, 5+, and 1.5(avg)) were prepared with n = 1-4 as a precise dye:dendrimer ratio, 5+ as a mixture of dendrimers with 5 or more dye per dendrimer, and 1.5(avg) as a Poisson distribution of dye:dendrimer ratios with a mean of 1.5 dye per dendrimer. The absorption intensity increased sublinearly with n whereas the fluorescence emission and lifetime decreased with an increasing number of dyes per dendrimer. Flow cytometry was employed to quantify uptake into HEK293A cells. Dendrimers with 2-4 dyes were found to have greater uptake than dendrimer with a single dye. Fluorescence lifetime imaging microscopy (FLIM) showed that the different dye:dendrimer ratio alone was sufficient to change the fluorescence lifetime of the material observed inside cells. We also observed that the lifetime of G5-NH2-TAMRA5+ increased when present in the cell as compared to solution. However, cells treated with G5-NH2-TAMRA1.5(avg) did not exhibit the high lifetime components present in G5-NH2-TAMRA1 and G5-NH2-TAMRA5+. In general, the effects of the dye:dendrimer ratio on fluorescence lifetime were of similar magnitude to environmentally induced lifetime shifts.

Difference in microchip electrophoretic mobility between partially and fully PEGylated poly(amidoamine) dendrimers.

The objective of this study was to investigate the difference in electrophoretic mobility between partially and fully poly(ethylene glycol)-conjugated poly(amidoamine) dendrimers (part-PEG-PAMAM and full-PEG-PAMAM, respectively) using a microchip capillary gel electrophoresis (MCGE). While MCGE allowed size-based separation of PEG-PAMAMs prepared with monomethoxy PEG-nitrophenyl carbonate, full-PEG-PAMAMs migrated slower than part-PEG-PAMAMs that were similar in size or larger. When the measured molecular weights obtained from MCGE analysis and the calculated molecular weights were plotted, each part-PEG-PAMAM and full-PEG-PAMAM showed correlation coefficients greater than 0.98. This study indicates that MCGE would be useful for characterizing PEG-PAMAMs with different PEGylation degrees.

Letter: The potential of amine-containing dendrimer mass standards for internal calibration of peptides.

In order to ensure accurate mass determinations, MALDI-TOF mass spectrometers must be calibrated regularly. While peptides and proteins represent the most widely used calibration standards due to their monodispersity, known masses and availability, their limited shelf-life complicates their use. Recently, polyester dendrimer calibrants have been introduced as an alternative because, in addition to monodispersity and relative molecular masses as high as 30,000, they exhibit vastly improved stability and broad compatibility with both matrices and solvents. However, the use of these initially reported polyester dendrimers as internal calibrants for the analysis of peptides or proteins presents a unique problem because these dendrimers typically require ionization with metal cations, while amino acid-based compounds preferentially ionize via protonation of an amine. To address this complication, dendrimers bearing a single amine were prepared which demonstrate the ability to easily ionize via protonation with either acidic matrices or dilute solutions of trifluoroacetic acid. This class of amine-containing dendrimers shows promise as a calibrant system specifically designed for the internal calibration of peptides.

An amphiphilic perylene imido diester for selective cellular imaging.

A new amphiphilic membrane marker based on a water-soluble dendritic polyglycerol perylene imido dialkylester has been designed, synthesized, and its optical properties characterized. In water it forms fluorescently quenched micellar self-aggregates, but when incorporated into a lipophilic environment, it monomerizes, and the highly fluorescent properties of the perylene core are recovered. These properties make it an ideal candidate for the imaging of artificial and cellular membranes as demonstrated by biophysical studies.

Identification and quantification of poly(amidoamine) PAMAM dendrimers of generations 0 to 3 by liquid chromatography/hybrid quadrupole time-of-flight mass spectrometry in aqueous medium.

RATIONALE: Poly(amidoamine) PAMAM dendrimers are highly water soluble and are used as flexible scaffolding or nanocontainers to conjugate, complex or encapsulate therapeutic drugs to overcome intrinsically weak characteristics such as solubilization in aqueous medium. To provide a reliable method for the quantitation of PAMAM dendrimers in aqueous medium, we report here a validation study which was developed in a complex wastewater matrix to evaluate the matrix effect in the electrospray ionization (ESI) source. METHODS: PAMAM dendrimers (generations G0 to G3) were identified and quantitated in aqueous medium using liquid chromatography interfaced to a hybrid quadrupole/time-of-flight mass analyzer. This approach used the high resolving power of isotopic clusters and mass accuracy of the instrument, with especial attention to the tandem mass spectrometric (MS/MS) capabilities. The formation of multiply charged ions of PAMAM dendrimers in the ESI source and their later fragmentation allowed fragmentation paths to be determined and structural assignments to be made. RESULTS: The analytical strategy allowed dendrimer identification with a high degree of confidence obtained by accurate mass and high resolution with mass errors below 5 ppm and 10 ppm in MS and MS/MS modes. The parameters of validation in spiked matrix were: limits of quantification in the range of 0.12 to 1.25 µM depending on the generation, linearity (R >0.996), repeatability (R.S.D. <6.7%) and reproducibility (R.S.D. <10.8%). CONCLUSIONS: Accurate mass measurement, elemental composition, and charge state assignment through the resolution of isotopic clusters of product and precursor ions, confers enhanced confidence on PAMAM dendrimer characterization. This selectivity provided high discriminating capacity of PAMAM dendrimers against matrix interferences. Because of the reliable and reproducible quantitation by LC/ESI-QTOF-MS, analysis of PAMAM dendrimers in an aqueous matrix is feasible.

Qualitative and quantitative analysis of poly(amidoamine) dendrimers in an aqueous matrix by liquid chromatography-electrospray ionization-hybrid quadrupole/time-of-flight mass spectrometry (LC-ESI-QTOF-MS).

This work introduces a liquid chromatography-electrospray ionization-hybrid quadrupole/time-of-flight mass spectrometry (LC-ESI-QTOF-MS)-based method for qualitative and quantitative analysis of poly(amidoamine) (PAMAM) dendrimers of generations 0 to 3 in an aqueous matrix. The multiple charging of PAMAM dendrimers generated by means of ESI has provided key advantages in dendrimer identification by assignation of charge state through high resolution of isotopic clusters. Isotopic distribution in function of abundance of isotopes (12)C and (13)C yielded valuable and complementarity data for confident characterization. A mass accuracy below 3.8 ppm for the most abundant isotopes (diagnostic ions) provided unambiguous identification of PAMAM dendrimers. Validation of the LC-ESI-QTOF-MS method and matrix effect evaluation enabled reliable and reproducible quantification. The validation parameters, limits of quantification in the range of 0.012 to 1.73 µM, depending on the generation, good linear range (R > 0.996), repeatability (RSD < 13.4%), and reproducibility (RSD < 10.9%) demonstrated the suitability of the method for the quantification of dendrimers in aqueous matrices (water and wastewater). The added selectivity, achieved by multicharge phenomena, represents a clear advantage in screening aqueous mixtures due to the fact that the matrix had no significant effect on ionization, with what is evidenced by an absence of sensitivity loss in most generations of PAMAM dendrimers. Fig Liquid chromatography-electrospray ionization-hybrid quadrupole/time of flight mass spectrometry (LC-ESI-QTOF-MS) based method for qualitative and quantitative analysis of PAMAM dendrimers in aqueous matrix.

A polymer network prepared by the thiol-yne photocrosslinking of a liquid crystalline dendrimer.

A polymer network is prepared by the thiol-yne photopolymerization of multifunctional dendrimer with a tetrathiol crosslinker. The network obtained shows a liquid crystalline phase at room temperature, which has been characterized by optical microscopy, differential scanning calorimetry, and X-ray diffraction. Photoinduced deformation of uniaxially aligned free-standing films of the photocrosslinked material has been demonstrated.

A unique aliphatic tertiary amine chromophore: fluorescence, polymer structure, and application in cell imaging.

Although photoluminescence of tertiary aliphatic amines has been extensively studied, the usage of this fundamental chromophore as a fluorescent probe for various applications has unfortunately not been realized because their uncommon fluorescence is easily quenched, and strong fluorescence has been observed only in vapor phase. The objective of this study is how to retain the strong fluorescence of tertiary amines in polymers. Tertiary amines as branching units of the hyperbranched poly(amine-ester) (HypET) display relatively strong fluorescence (Φ = 0.11-0.43). The linear polymers with tertiary amines in the backbone or as the side group are only very weakly fluorescent. The tertiary amine of HypET is easily oxidized under ambient conditions, and red-shifting of fluorescence for the oxidized products has been observed. The galactopyranose-modified HypET exhibits low cytotoxicity and bright cell imaging. Thus, this study opens a new route of synthesizing fluorescent materials for cell imaging, biosensing, and drug delivery.

Peripheral functionalization of dendrimers regulates internalization and intracellular trafficking in living cells.

GATG (gallic acid-triethylene glycol) dendrimers represent appealing nanostructures for biomedical applications. The incorporation of specific ligands and targeting and imaging agents on their surface has resulted in promising tools in diagnosis and drug delivery. With the aim to further explore the versatility of GATG dendrimers in the biomedical field, in this work we study the effect of peripheral substitution on their uptake and intracellular trafficking in living cells. To this end, peripheral groups with different physicochemical properties and biological relevance have been installed on the surface of GATG dendrimers, and their interactions, uptake efficacy, and specificity for certain cell populations studied by confocal microscopy. Finally, this information was used to design a pH-sensitive drug delivery system for the selective release of cargo molecules inside cells after lysosomal localization. These results along with the easy functionalization and modular architecture of GATG dendrimers reveal these systems as promising nanotools in biomedicine.

Heterogeneous ligand-nanoparticle distributions: a major obstacle to scientific understanding and commercial translation.

Nanoparticles conjugated with functional ligands are expected to have a major impact in medicine, photonics, sensing, and nanoarchitecture design. One major obstacle to realizing the promise of these materials, however, is the difficulty in controlling the ligand/nanoparticle ratio. This obstacle can be segmented into three key areas: First, many designs of these systems have failed to account for the true heterogeneity of ligand/nanoparticle ratios that compose each material. Second, studies in the field often use the mean ligand/nanoparticle ratio as the accepted level of characterization of these materials. This measure is insufficient because it does not provide information about the distribution of ligand/nanoparticle species within a sample or the number and relative amount of the different species that compose a material. Without these data, researchers do not have an accurate definition of material composition necessary both to understand the material-property relationships and to monitor the consistency of the material. Third, some synthetic approaches now in use may not produce consistent materials because of their sensitivity to reaction kinetics and to the synthetic history of the nanoparticle. In this Account, we describe recent advances that we have made in under standing the material composition of ligand-nanoparticle systems. Our work has been enabled by a model system using poly(amidoamine) dendrimers and two small molecule ligands. Using reverse phase high-pressure liquid chromatography (HPLC), we have successfully resolved and quantified the relative amounts and ratios of each ligand/dendrimer combination. This type of information is rare within the field of ligand-nanoparticle materials because most analytical techniques have been unable to identify the components in the distribution. Our experimental data indicate that the actual distribution of ligand-nanoparticle components is much more heterogeneous than is commonly assumed. The mean ligand/nanoparticle ratio that is typically the only information known about a material is insufficient because the mean does not provide information on the diversity of components in the material and often does not describe the most common component (the mode). Additionally, our experimental data has provided examples of material batches with the same mean ligand/nanoparticle ratio and very different distributions. This discrepancy indicates that the mean cannot be used as the sole metric to assess the reproducibility of a system. We further found that distribution profiles can be highly sensitive to the synthetic history of the starting material as well as slight changes in reaction conditions. We have incorporated the lessons from our experimental data into the design of new ligand-nanoparticle systems to provide improved control over these ratios.

Chlorhexidine-loaded poly (amido amine) dendrimer and a dental adhesive containing amorphous calcium phosphate nanofillers for enhancing bonding durability.

OBJECTIVE: A novel method of combining chlorhexidine (CHX) loaded poly (amido amine) (PAMAM) dendrimers with a dental adhesive containing amorphous calcium phosphate (ACP) nanofillers are proposed for etch-and-rinse bonding system to enhance resin-dentin bonding durability. METHODS: The CHX-loaded PAMAM and ACP nanofillers were synthesized and characterized. Their effects on the cytotoxicity were tested by MTT assay. Micro-tensile bond strength (µTBS) before and after thermomechanical challenges were used to evaluate the bonding durability. Anti-matrix metalloproteinase (MMPs) property was examined using in-situ zymography. A double-fluorescence technique was used to examine interfacial permeability after bonding. Dentin remineralization in Ca/P lacking solution was observed under scanning electron microscopy. RESULTS: Compared with a 0.2 wt% CHX solution, the PAMAM loaded CHX had less cytotoxicity, while the in situ zymography showed it could still inhibit MMPs activity within the hybrid layer after released from PAMAM. The application of the novel method maintained the µTBS better than the control group after thermomechanical challenges, and it did not negatively affect water permeability of the bonding interfaces. CHX-loaded PAMAM regulated the calcium (Ca) and phosphate (P) ions provided by the ACP-containing adhesives to remineralize the demineralized dentin surfaces without initial Ca/P in the environment. SIGNIFICANCE: The novel method can reduce the cytotoxicity of CHX, inhibit MMPs activities, maintain µTBS, and induce dentin remineralization, which are crucial factors for enhancing bonding durability.

[Polyamidoamine dendrimer-functionalized silica nanocomposite with polydopamine coating for dispersive micro solid-phase extraction of benzoylurea insecticides in water samples].

Pesticides are used in the agricultural production process to ensure the yield and quality of agricultural products. However, in recent years, environmental pollution issues caused by pesticide residues have sparked widespread concern in society. It is important to develop convenient and efficient approaches to detect and monitor pesticide residues. In this study, targeting benzoylurea insecticides (BUs), polyamidoamine dendrimer-functionalized silica nanocomposite with polydopamine coating (SiO2-PAMAM-PDA) was designed and successfully synthesized. First, monodisperse silica nanoparticles were prepared by the hydrolysis of tetraethyl orthosilicate (TEOS) in mixed solution of ethanol, water and ammonia. The silane coupling agent (3-aminopropyl)triethoxysilane was then employed to introduce amino groups into the silica. Silica with the zeroth generation of polyamidoamine (PAMAM) modification (SiO2-PAMAM-G0) was obtained through Michael addition reaction of methyl acrylate. Ethylenediamine was added to polymerize with methyl acrylate using an amidation reaction to form the first-generation PAMAM (SiO2-PAMAM-G1). Finally, by polymerizing dopamine under alkaline conditions (pH=8.5), the SiO2-PAMAM-G1 was coated with PDA. Thus, the final product named SiO2-PAMAM-PDA was obtained. The composite was characterized using a transmission electron microscope (TEM) and an increase in surface roughness indicated the successful grafting of PDA coating. Dopamine structure contains abundant benzene rings and amino and hydroxyl groups. It could bind with BUs through multiple secondary interactions, such as hydrogen bond and π-π stacking interaction. Therefore, the introduction of PDA could effectively enhance the affinity of the material toward benzoylurea insecticides. The prepared nanocomposites were used as sorbents in a dispersive micro solid-phase extraction approach (D-µ-SPE). The established approach was employed to extract and enrich the BUs in water samples before high-performance liquid chromatography (HPLC) analysis. Diflubenzuron, triflumuron, hexaflumuron, and teflubenzuron were chosen as target analytes. The following was a typical D-µ-SPE procedure. The prepared adsorbents measuring 40 mg were first dispersed in an 8-mL sample solution containing 150 g/L NaCl. The dispersion was assisted by 120-s vortexing to ensure full contact between the SiO2-PAMAM-PDA and the targets. Next, the adsorbents were separated from the liquid phase by 4-min centrifugation (5000 r/min). Thereafter, the adsorbed benzoylureas were eluted using 1 mL acetonitrile as desorption solvent by 120-s vortexing. Separated by centrifugation, the eluate was dried under a mild nitrogen stream. The solid remains were redissolved in 0.1 mL of acetonitrile, filtered by filter membrane (0.22 µm), and then analyzed by HPLC. The experimental conditions in the D-µ-SPE process could have a great impact on the extraction efficiency. Experimental conditions were optimized using a single factor optimization approach to further enhance the extraction recoveries. The optimized conditions included adsorbent amount, extraction time, desorption solvent type, desorption solvent volume, desorption time, and NaCl addition amount. Under the optimal conditions, a linearity range of 10-500 µg/L and limits of detection (LODs, S/N=3) of 1.1-2.1 µg/L were obtained. The extraction recoveries and relative standard deviations (RSDs) of the four BUs were 82.8%-94.1% and 2.1%-8.0%, respectively. The established approach was compared with reported approaches targeting benzoylurea insecticides. It was discovered that this approach consumed less sample, material, organic solvent, and pretreatment time. It provided a more rapid and green choice for the determination of benzoylurea pesticides. To determine the applicability, the proposed approach was applied to analyze the four benzoylurea insecticides in three river water samples. The real water samples were pretreated using the developed approach ahead of instrumental analysis, and no benzoylurea pesticides residue was detected. Next, standard addition experiments were performed under three spiking levels, including 15, 50, and 200 µg/L. The established approach had good accuracy and feasibility with satisfactory recoveries (69.5%-99.4%) and RSDs (0.2%-9.5%).

Autoinductive exponential signal amplification: a diagnostic probe for direct detection of fluoride.

A new example of an exponential signal amplification strategy for the direct detection of fluoride is demonstrated. The amplification occurred through reaction of fluoride with a responsive chromogenic probe. The probe activity is based on a unique dendritic chain reaction that generates a fluoride anion, which is the analyte of interest, during the disassembly pathway of the dendritic probe. This autoinductive amplification mechanism may be applied for detection of other analytes by coupling activity of a modified probe with that of the fluoride amplifier.

Analysis of poly(amidoamine) dendrimer structure by UV-vis spectroscopy.

We report a UV-vis spectroscopic study of four different types of poly(amidoamine) dendrimers. The results indicate that the degree of protonation of the interior tertiary amines of these dendrimers correlates directly to an absorption band with λ(max) in the range of 280-285 nm. Specifically, at low pH, the tertiary amines are protonated and the 280-285 nm band is absent. However, at elevated pH, when these groups are deprotonated, this band appears. Similar results were obtained for a simple model compound. The dependence of the 280-285 nm band on the chemical state of the tertiary amines of the dendrimers was confirmed by complexing them with Pd(2+) and Pt(2+). In this case the band disappears, and it only reappears when the metal ions are decomplexed following reduction with BH(4)(-). Finally, filtration experiments showed that the absorption band between 280-285 nm arises exclusively from intact, or nearly intact, dendrimers rather than low-molecular-weight fragments.

Chemical characterization (LC-MS-ESI), cytotoxic activity and intracellular localization of PAMAM G4 in leukemia cells.

Generation 4 of polyamidoamine dendrimer (G4-PAMAM) has several biological effects due to its tridimensional globular structure, repetitive branched amides, tertiary amines, and amino-terminal subunit groups liked to a common core. G4-PAMAM is cytotoxic due to its positive charges. However, its cytotoxicity could increase in cancer cells due to the excessive intracellular negative charges in these cells. Furthermore, this work reports G4-PAMAM chemical structural characterization using UHPLC-QTOF-MS/MS (LC-MS) by electrospray ionization to measure its population according to its positive charges. Additionally, the antiproliferative effects and intracellular localization were explored in the HMC-1 and K-562 cell lines by confocal microscopy. The LC-MS results show that G4-PAMAM generated multivalent mass spectrum values, and its protonated terminal amino groups produced numerous positive charges, which allowed us to determine its exact mass despite having a high molecular weight. Additionally, G4-PAMAM showed antiproliferative activity in the HMC-1 tumor cell line after 24 h (IC50 = 16.97 µM), 48 h (IC50 = 7.02 µM) and 72 h (IC50 = 5.98 µM) and in the K-562 cell line after 24 h (IC50 = 15.14 µM), 48 h (IC50 = 14.18 µM) and 72 h (IC50 = 9.91 µM). Finally, our results showed that the G4-PAMAM dendrimers were located in the cytoplasm and nucleus in both tumor cell lines studied.

Bioanalytical method development for a generation 5 polyamidoamine folic acid methotrexate conjugated nanoparticle.

Generation 5 poly(amidoamine) dendrimer nanoparticles conjugated with folic acid and methotrexate (G5-MTX-FA) for targeted treatment of cancer are of recent interest. The increased efficacy of these nanodevices over the free methotrexate has been shown in vitro and in vivo. The heterogeneous nature of this nanoparticle together with possible release of active compounds complicated the method development. This work presents a bioanalytical assay for the detection of nanoparticle-conjugated methotrexate, released methotrexate, and its main plasma metabolite 7-hydroxymethotrexate in rat plasma. Determination of G5-MTX-FA-associated methotrexate occurred by a reductive cleavage of the C9-N10 bond in methotrexate, resulting in a highly fluorescent 2,4-diamino-6-methylpteridine reporter molecule that could be measured by reversed-phase chromatography and fluorescence detection. It was found that reduction should occur directly in the plasma matrix to avoid irreversible adsorption of the nanodevice during sample preparation. The method was linear over a range from 50 to 10,000 nM G5-MTX-FA utilizing 100 microL of plasma. Nanoparticle-released methotrexate and its metabolite 7-hydroxymethotrexate were determined by reversed-phase chromatography followed by online post-column photochemical derivatization and fluorescence detection. The method was specific for these analytes irrespective of nanoparticle concentration. Sample preparation consisted of perchloric acid protein precipitation followed by a strong anion exchange solid-phase extraction. Limits of quantification were about 50 nM for methotrexate and 10 nM for 7-hydroxymethotrexate. Preliminary pharmacokinetic profiles of intravenous and subcutaneous administered G5-MTX-FA in rats were obtained. These data indicated that less than 0.1% of the methotrexate mass is released from the nanoparticle in plasma.

MALDI-MS Imaging Analysis of Noninflammatory Type III Rotaxane Dendrimers.

Rotaxane dendrimers with hyperbranched macromolecular interlocked structures and size modulation capacity demonstrate drug binding and release ability upon external stimuli. Mass spectrometry imaging (MSI) can offer the high-throughput screening of endogenous/exogenous compounds. Herein, we reported a novel method to display the in situ spatial distribution of label-free monodispersed type III rotaxane dendrimers (RDs) G1 (first generation, size ∼1.5 nm) and G2 (second generation, size ∼5 nm) that were explored as potential drug vehicles in spleen tissue by using matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-MSI). Experimental results indicated that the trans-2-[3-(4-tert-butylphenyl)-2-methyl-2-propenylidene]malononitrile (DCTB) matrix exhibited the best performance for monodispersed type III RDs G1 and G2. The optimized method was successfully applied to map the in vivo spatial distribution of type III RDs G1 and G2 in the spleen from intraperitoneally injected mice. The MALDI-MSI images revealed that RDs G1 and G2 were relatively stable in the spleen within 24 h after administration. It was found that the identified type III RDs G1 and G2 penetrated through the tunica serosa and were predominantly localized in red pulp regions of spleens. They were also mapped in a marginal zone of spleens simultaneously. There was almost no toxicity of type III RDs G1 and G2 to mice spleens from the H&E results. Furthermore, the type III RDs did not induce the expression of inflammatory cytokines from peripheral blood mononuclear cells (PBMCs) or THP-1 monocytes. The MSI analysis not only demonstrated its ability to image select rotaxane dendrimers in a rapid and efficient manner but also provided tremendous assistance on the applications of the further treatment of cancerous tissue as safe drug carriers. Furthermore, the new strategy demonstrated in this study could be applied on other label-free mechanically interlocked molecules, molecular machines, and macromolecules, which opened a new path to evaluate the toxicological and pharmacokinetic characteristics of these novel materials at the suborgan level.