Estudos de síntese de dendrons dirigidos de NFOH com potencial antichagásico e modelagem molecular do receptor de manose de macrófagos / Studying the synthesis of NFOH targeted dendrons, potentially active in Chagas disease, and the molecular modeling of manose receptor from macrophages

A doença de Chagas, considerada doença extremamente negligenciada, acomete mais de 6 milhões de pessoas ao redor do mundo e mais de 75 milhões de pessoas vivem sob risco da doença. Considerada endêmica em 21 países da América Latina. No Brasil, grassa, sobretudo, na região Norte, especialmente, na região amazônica. Apesar de se constituir em risco global, a doença de Chagas conta com apenas com dois fármacos, o benznidazol e o nifurtimox, que, além de tóxicos, não apresentam eficácia significativa na fase crônica da parasitose. Assim sendo, torna-se imperativa a busca por quimioterápicos mais eficazes, mormente na fase crônica da doença. A introdução de novos fármacos da terapêutica várias fases, consumindo tempo e recursos. No entanto, há processos que permitem a otimização de fármacos já existentes e de compostos bioativos, com vistas à busca de candidatos a fármacos, que, uma vez bem-sucedidos nos ensaios clínicos, são aprovados para uso terapêutico. Entre esses processos, destaca-se a latenciação, forma de aprimoramento de propriedades farmacêuticas, farmacocinéticas e, indiretamente, farmacodinâmicas, que utiliza, em geral, transportadores para a resolução de problemas dessas naturezas. Os transportadores variam de acordo com o problema a ser resolvido e, entre eles, os dendrons e dendrímeros podem ser ressaltados pela sua natureza química, que permite a ligação de várias moléculas de fármacos/compostos bioativos e, também, de grupos diretores para certos compartimentos ou células. Dessa forma, podem-se obter fármacos dirigidos, que se constituem em formas latentes de alta seletividade. Face ao exposto e, estimulados pela busca de novas alternativas terapêuticas para a doença de Chagas, o objetivo deste trabalho foi a obtenção de dendrons dirigidos, por meio de manose, derivados de hidroximetilnitrofural (NFOH). Esse composto foi mostrou-se altamente ativo contra T. cruzi, também na fase crônica NFOH e menos tóxico que o protótipo e o benznidazol. Efetuaram-se estudos para a síntese desses compostos derivados de dendron triazólico, sintetizado através de click chemistry, tendo a manose como grupo diretor para os macrófagos, onde, também, são encontrados os amastigotas de Trypanosoma cruzi. Obtiveram-se alguns intermediários, que foram caracterizados por RMN 1H e 13C. A rota sintética proposta não pôde ser finalizada. Por outro lado, efetuaram-se estudos de modelagem molecular, utilizando-se dinâmica molecular, com o intuito de conhecer como se dá a interação da manose e de polimanosídeos com seu respectivo receptor e como se realiza a liberação do composto bioativo da ligação com o dendron. Anteriormente, procedeu-se à caracterização da biologia estrutural do receptor de manose e de suas estruturas primárias, secundárias e terciárias, com ênfase para o domínio CRD4 o papel do cálcio principal na interação com o monossacarídeo. A movimentação do domínio foi muito pouco diferente nos meios simulados (neutro, ácido, contendo ligantes e contendo o cálcio auxiliar), evidenciado pelo RMSF e estudo de PCA desses sistemas. Foi possível concluir que este domínio não apresenta nenhuma alteração conformacional responsável pela liberação de ligantes em meio lisossômico, e que o cálcio auxiliar e os ligantes não causam impactos na estabilidade conformacional do CRD4. Há necessidade de mais estudos para o conhecimento dos requisitos estruturais envolvidos na da formação do complexo receptor-composto bioativo

Chagas disease, considered an extremely neglected one, affects more than 6 million people all over de world, with more than 75 million people living under its risk, while endemics in 21 countries in Latin America. In Brazil, it propagates, mainly in North region, especially in Amazon region. Although being a global risk, only two drugs, benznidazole and nifurtimox, are currently available for Chagas disease. These drugs are toxic and not significantly efficient against the chronic phase of the disease. Therefore, the search for more active chemotherapeutic agents, mainly against the chronic phase of the parasitosis, is imperative. The introduction of new drugs in the therapeutics involves many phases, consuming time, and money. Notwithstanding, there are processes that allow either drugs or bioactive compounds to be optimized, towards drug candidates. These derivatives, once well-succeeded in the clinical trials, can be approved for therapeutic uses. Among those processes, prodrug design stands out. It is a way to improve the pharmaceutics, pharmacokinetics and, indirectly, pharmacodynamics, properties of drugs/bioactive compounds, which requires adequate carriers, in general, for these problems´ solution. The carriers vary according to the problem to be solved, and, among them, dendrons and dendrimers can be emphasized due to their chemical nature, which allows the link of many molecules/bioactive compounds and of directing groups to specific compartments or cells. Thus, targeted drugs, which are latent forms of drugs/bioactive compounds with high selectivity. In this connection and stimulated by the search for new therapeutic alternatives for Chagas disease, the objective of this work was obtaining hydroxymethylnitrofurazone (NFOH) targeted dendrons, by means of mannose, as directing groups. NFOH is highly active against T. cruzi, even in chronic phase of the disease, and less toxic than the prototype and benznidazole. Studies have been developed to synthesize these compounds with a triazole dendron, planned to be obtained by click chemistry. Mannose was designed to be the directing groups to macrophages, where the T. cruzi amastigotes can also be found. Some intermediaries have been obtained and structurally characterized by 1H and 13C NMR, but the proposed synthetic route could not be finished. On the other hand, molecular modeling studies have been developed, using molecular dynamics, with the aim to know how the interaction of mannose, and also of polymannoside, occur with the specific receptor, and how NFOH is released from its linkage to the dendron. The structural biology characterization, as well as of primary, secondary and tertiary structures of the mannose receptor was previously performed, with emphasis onCRD4 and main calcium role in the interaction of the mannoside. All systems simulated (neutral medium, acid medium, complexes with ligands and auxiliary calcium) showed little movement differences when analyzed by RMSF and PCA calculations. It was possible to conclude that this domain shows no conformational changes involved in ligand releasing in lysosomal environment and its conformation is not altered when in presence of ligands or the auxiliary calcium. Much more studies are needed to the knowledge of the structural requirements to the complex receptor-drug-compound bioactive to the receptor

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.

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.

Estudo de síntese e estabilidades química e enzimática de fármaco dirigido dendrimérico potencialmente ativo em doença de Chagas / Synthesis, chemical and enzymatic stability studies of targeted dendrimer potentially active in Chagas disease

A doença de Chagas representa um problema de saúde pública em muitos países e regiões. O tratamento consiste em fármacos tóxicos, com eficácia discutível, principalmente, na fase crônica da doença. Assim, faz-se necessário o planejamento de novos quimioterápicos, mais seguros e eficazes. Os dendrímeros são novas arquiteturas moleculares formadas por um foco central e ramificações partindo desse foco. Apresentam diversas aplicações biológicas como, por exemplo, atuar como transportadores de fármacos. Face ao exposto, o objetivo deste trabalho foi o estudo de condições para ligar o ácido anacárdico (AA) em derivado dendrimérico com potencial ação na doença de Chagas, o qual tem como foco central o ácido succínico (AS) e ramificações compostas por arginina (Arg) e lisina (Lys). Sabe-se que a cruzaína, uma cisteíno-protease do T. cruzi, catalisa a hidrólise de ligação peptídica entre lisina e arginina. A síntese dos compostos em fase sólida forneceu os derivados brutos: (1) pró-fármaco AA-K-R-NH2 e (2) G.05 AA-K(AS)-R-NH2, que foram purificados e caracterizados por Cromatografia Líquida de Alta Eficiência e espectrometria de massas. Os compostos purificados AA-K-R-NH2 e AA-K(AS)-R-NH2 apresentaram rendimentos de 34% e 47%, com pureza de 88% e 98%, respectivamente. Os resultados dos experimentos enzimáticos utilizando o AA-K-R-NH2 não foram conclusivos. Acredita-se que a baixa solubilidade e/ou baixa concentração podem ter contribuído para tal. Já na estabilidade química em pH 7,4 (que simula pH sanguíneo), pH 1,2 (que simula pH estomacal) e pH 8,5 (que simula pH intestinal), observou-se que o AA-K(AS)-R-NH2 foi estável durante as 24 h de ensaio. Estes últimos resultados são interessantes, pois espera-se que o pró-fármaco dendrimérico alcance o T. cruzi estruturalmente integro, sofrendo hidrólise e liberação do composto ativo no interior do parasita

Chagas disease is a public health problem in many countries and regions. The treatment consists of toxic drugs, with debatable efficacy, mainly, in the chronic phase of the disease. Thus, it is necessary to plan new chemotherapeutics, safer and more effective than those drugs. Dendrimers are new molecular architectures composed by a central focus and branching from that focus. They present several biological applications, such as acting as drug carriers. Thereby, the goal of this work was the study of conditions to bind anacardic acid (AA) in a dendrimeric derivative with potential action in Chagas disease, which was composed by a central focus of succinic acid (AS) and branches of arginine (Arg) and lysine (Lys). Cruzain, a T. cruzi cysteine protease, is known to catalyze the peptide-binding hydrolysis between lysine and arginine. Synthesis of the solid phase compounds provided the crude derivatives: (1) prodrug AA-KR-NH2 and (2) G.05 AA-K(AS)-R-NH2, which were purified and characterized by High Performance Liquid Chromatography (HPLC) and mass spectrometry. The purified AA-K-R-NH2 and AA-K(AS)-R-NH2 compounds showed yields of 34% and 47%, with purity of 88% and 98% respectively. The results of the enzymatic experiments using AA-K-R-NH2 were not conclusive. It is believed that the low solubility and/or low concentration may have contributed for this. On the chemical stability at pH 7.4 (which simulates blood pH), pH 1.2 (which simulates stomach pH) and pH 8.5 (which simulates intestinal pH), it was observed that AA-K(AS)R-NH2 was stable for 24 hours. These latter results are interesting because the dendrimeric prodrug is expected to reach structurally integral T. cruzi, undergoing hydrolysis and release of the active compound within the parasite

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.

DNA delivery with hyperbranched polylysine: a comparative study with linear and dendritic polylysine.

PEI and polylysine are among the most investigated synthetic polymeric carriers for DNA delivery. Apart from their practical use, these 2 classes of polymers are also of interest from a fundamental point of view as they both can be prepared in different architectures (linear and branched/dendritic) and in a wide range of molecular weights, which is attractive to establish basic structure-activity relationships. This manuscript reports the results of an extensive study on the influence of molecular weight and architecture of a library of polylysine variants that includes linear, dendritic and hyperbranched polylysine. Hyperbranched polylysine is a new polylysine-based carrier that is structurally related to dendritic polylysine but possesses a randomly branched structure. Hyperbranched polylysine is attractive as it can be prepared in a one-step process on a large scale. The performance of these 3 classes of polylysine analogs was evaluated by assessing eGFP and IgG production in transient gene expression experiments with CHO DG44 cells, which revealed that protein production generally increased with increasing molecular weight and that at comparable molecular weight, the hyperbranched analogs were superior as compared to the dendritic and linear polylysines. To understand the differences between the gene delivery properties of the hyperbranched polylysine analogs on the one hand and the dendritic and linear polylysines on the other hand, the uptake and trafficking of the corresponding polyplexes were investigated. These experiments allowed us to identify (i) polyplex-external cell membrane binding, (ii) free, unbound polylysine coexisting with polyplexes as well as (iii) polymer buffer capacity as three possible factors that may contribute to the superior transfection properties of the hyperbranched polylysines as compared to their linear and dendritic analogs. Altogether, the results of this study indicate that hyperbranched polylysine is an interesting, alternative synthetic gene carrier. Hyperbranched polylysine can be produced at low costs and in large quantities, is partially biodegradable, which may help to prevent cumulative cytotoxicity, and possesses transfection properties that can approach those of PEI.

Nitric oxide releasing-dendrimers: an overview

Platforms able to storage, release or scavenge NO in a controlled and specific manner is interesting for biological applications. Among the possible matrices for these purposes, dendrimers are excellent candidates for that. These molecules have been used as drug delivery systems and exhibit interesting properties, like the possibility to perform chemical modifications on dendrimers surface, the capacity of storage high concentrations of compounds of interest in the same molecule and the ability to improve the solubility and the biocompatibility of the compounds bonded to it. This review emphasizes the recent progress in the development and in the biological applications of different NO-releasing dendrimers and the nitric oxide release pathways in these compounds.

Plataformas capazes de armazenar, liberar ou capturar NO de forma controlada e específica são de grande interesse tendo-se em vista aplicações biológicas. Dentre as possíveis matrizes que podem ser utilizadas para esse fim, os dendrímeros são excelentes candidatos. Essas moléculas têm sido empregadas em sistemas para o transporte de fármacos e apresentam propriedades interessantes tais como a possibilidade de modificações químicas em sua superfície, a capacidade de estocar altas concentrações de compostos de interesse em uma só molécula e a possibilidade de aumentar a solubilidade e a biocompatibilidade dos compostos a eles ligados. Esta revisão enfatiza os recentes avanços no desenvolvimento e nas aplicações biológicas de diferentes dendrímeros liberadores de NO e a forma em que o óxido nítrico é liberado nesses compostos.

Dendrimers as anti-inflammatory agents

Dendrimers constitute an intriguing class of macromolecules which find applications in a variety of areas including biology. These hyperbranched macromolecules with tailored backbone and surface groups have been extensively investigated as nanocarriers for gene and drug delivery, by molecular encapsulation or covalent conjugation. Dendrimers have provided an excellent platform to develop multivalent and multifunctional nanoconjugates incorporating a variety of functional groups including drugs which are known to be anti-inflammatory agents. Recently, dendrimers have been shown to possess anti-inflammatory properties themselves. This unexpected and intriguing discovery has provided an additional impetus in designing novel active pharmaceutical agents. In this review, we highlight some of the recent developments in the field of dendrimers as nanoscale anti-inflammatory agents.

Dendrímeros constituem uma classe intrigante de macromoléculas que apresentam aplicações em diversas áreas incluindo biologia. Essas macromoléculas extremamente ramificadas com esqueleto planejado e grupos de superfície foram extensivamente investigadas como nanotransportadores de genes e de fármacos, por encapsulamento molecular ou conjugação covalente. Dendrímeros têm proporcionado uma plataforma excelente de desenvolvimento nanoconjugados multivalentes e multifuncionais incorporando uma variedade de grupos funcionais, incluindo fármacos que são conhecidos por atuarem agentes antiinflamatórios. Recentemente, os dendrímeros mostraram propriedades antiinflamatórias. Esta inesperada e intrigante descoberta tem proporcionado um impulso adicional no planejamento de novos agente farmacêuticos ativos. Nesta revisão, nós destacamos alguns dos desenvolvimentos recentes no campo dos dendrímeros como agentes antiinflamatórios em nanoescala.

From dendrimers to fractal polymers and beyond

The advent of dendritic chemistry has facilitated materials research by allowing precise control of functional component placement in macromolecular architecture. The iterative synthetic protocols used for dendrimer construction were developed based on the desire to craft highly branched, high molecular weight, molecules with exact mass and tailored functionality. Arborols, inspired by trees and precursors of the utilitarian macromolecules known as dendrimers today, were the first examples to employ predesigned, 1 → 3 C-branched, building blocks; physical characteristics of the arborols, including their globular shapes, excellent solubilities, and demonstrated aggregation, combined to reveal the inherent supramolecular potential (e.g., the unimolecular micelle) of these unique species. The architecture that is a characteristic of dendritic materials also exhibits fractal qualities based on self-similar, repetitive, branched frameworks. Thus, the fractal design and supramolecular aspects of these constructs are suggestive of a larger field of fractal materials that incorporates repeating geometries and are derived by complementary building block recognition and assembly. Use of terpyridine-M2+-terpyridine (where, M = Ru, Zn, Fe, etc) connectivity in concert with mathematical algorithms, such as forms the basis for the Seirpinski gasket, has allowed the beginning exploration of fractal materials construction. The propensity of the fractal molecules to self-assemble into higher order architectures adds another dimension to this new arena of materials and composite construction.

O advento da química dendrítica tem facilitado a pesquisa de materiais por permitir o controle preciso do posicionamento do componente funcional na arquitetura macromolecular. Os protocolos sintéticos iterativos usados para construção dos dendrímeros foram desenvolvidos baseados no desejo de elaborar moléculas extremamente ramificadas, com alta massa molecular, massa exata e funcionalidade planejada. Arborols, inspirados em árvores e precursores de macromoléculas utilitárias, conhecidas hoje como dendrímeros, foram os primeiros exemplos a empregar blocos de construção de ramificação-C 1→3; Características físicas dos Arborols, incluindo a sua forma globular, excelente solubilidade, e agregação, combinam-se para revelar o potencial supramolecular inerente (isto é, a micela unimolecular) destas espécies únicas. A arquitetura que é característica dos materiais dendríticos também exibe qualidades fractais com base em estruturas repetitivas, ramificadas e auto-similares. Assim, o design fractal e os aspectos supramoleculares destas construções são sugestivas de um campo maior de materiais fractais que incorporam geometrias repetidas. O uso de terpiridina-M2+-terpiridina (onde, M = Ru, Zn, Fe, etc) em conjunto com algoritmos matemáticos tais como as formas da base do Triângulo de Seirpinski, tem permitido o início da exploração da construção de materiais fractais. A propensão da auto-criação de moléculas fractais para arquiteturas de ordem superior adiciona outra dimensão para essa nova arena de materiais e construção de compostos.

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.

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.

Development and validation of a reversed-phase high-performance liquid chromatographic method for quantification of peptide dendrimers in human skin permeation experiments.

The aim of the present work was to develop and validate a simple RP-HPLC method with UV detection to quantify peptide dendrimers in skin permeation experiments. Six dendrimers of varying positive charges (4(+), 8(+) and 16(+)) containing either histidine or arginine as terminal aminoacids were prepared by solid phase peptide synthesis. Mobile phase containing 0.02% (v/v) heptafluorobutyric acid in 90% acetonitrile-water was capable of separating all dendrimers from interfering peaks of receptor fluid. For the calibration of each dendrimer, a different dendrimer from the same class was selected as the internal standard. The results of preliminary human skin permeation studies showed that the developed analytical method can be successfully used for the quantification of cationic poly(aminoacid)-based dendrimers in skin permeation experiments.

Amphiphilic nanoassemblies for the detection of peptides and proteins using fluorescence and mass spectrometry.

Amphiphilic nanostructures provide unique environments for molecules that are incompatible with the solvent to be sequestered within their interior. These internal environments provide opportunities for concentrating an analyte or transducer molecule for detection, and the functional groups within the amphiphiles provide an opportunity for incorporating specificity or selectivity toward analytes. In this review, we discuss ways in which amphiphilic assemblies can be used to detect peptides and proteins with a particular emphasis on facially amphiphilic polymers and dendrimers.