Cryopreservation of spermatozoa of black marlin, Makaira indica (Teleostei : Istiophoredae)

As a first step towards the development of a method for the cryopreservation of black marlin spermatozoa, this study investigated the effect of dimethylsulfoxide (DMSO) concentration and pellet size on post-thaw spermatozoal motility. Spermatozoa were recovered from the spermatic duct of testes retrieved post-mortem from four adult black marlin caught in the Coral Sea spawning grounds of Australia. Undiluted spermatozoa. were stored on ice for 4 to 10 hours during transport to shore, then evaluated for motility after activation in seawater (1:10 v:v). Spermatozoa were prepared for cryopreservation in pellets by extension (1:3 v:v) in a defined fish Ringer's solution to give two final DMSO concentrations of 2.5% or 5.0%. Diluted spermatozoa were frozen directly on a dry ice block in pellet sizes of either 0.25 ml or 0.50 ml. Frozen pellets were thawed in a water bath at 40 degrees C for 60 seconds and assessed for post-thaw motility following activation in seawater. Spermatozoa recovered within 50 minutes of death and chilled on ice for 4 to 10 hours showed a mean (+/- SEM) motility immediately following activation of 91.6 +/- 7.9%. 50% of the spermatozoa remained motile for approximately 4 to 5 minutes. Following cryopreservation; mean motility declined significantly across all cryoprotectant and pellet size combinations (P < 0.001) but spermatozoa frozen in 2.5% DMSO showed higher motility than those frozen in 5.0% DMSO (P = 0.014). Pellet size had no effect on post-thaw motility (P = 0.179).

DOSY NMR studies of chemical exchange behavior of poly(2-hydroxyethyl methacrylate)

PFG-NMR was used to study the chemical exchange of linear PHEMA having a range of molecular weights with water in DMSO containing varying quantities of water. The aim was to investigate the use of PFG-NMR to study chemical exchange between a polymer with exchangeable protons and a small fast diffusing molecule to provide insight into the conformation adopted by a polymer in solution. The experimental data were simulated closely for the two-site exchange case using the Bloch equations modified for chemical exchange and diffusion. The exchange rate could be used to detect changes in polymer conformation resulting from changes in the solvent. PHEMA of MW 10 000 showed significant time-dependent changes in exchange rate, resulting from preferential solvation of the OH sites by water, and subsequent conformational changes which altered accessibility of the OH sites to water. This behavior was not observed for larger MW PHEMA, which adopted a stable conformation immediately. Large changes in the exchange rate were not reflected in changes to the hydrodynamic radius, suggesting that a minimal overall change in the chain dimensions occurred. DMSO was found to be a poor solvent for PHEMA, which adopts a compact conformation in DMSO. This work has demonstrated that PFG-NMR is a sensitive method for detecting subtle changes in polymer conformation in polymers with exchangeable protons.

Immunochemical detection of glyoxal DNA damage

The relevance of reactive oxygen species (ROS) in the pathogenesis of inflammatory diseases is widely documented. Immunochemical detection of ROS DNA adducts has been developed, however, recognition of glyoxal-DNA adducts has not previously been described. We have generated a polyclonal antibody that has shown increased antibody binding to ROS-modified DNA in comparison to native DNA. In addition, dose-dependent antibody binding to DNA modified with ascorbate alone was shown, with significant inhibition by desferrioxamine, catalase, and ethanol. Minimal inhibition was observed with uric acid, 1,10-phenanthroline and DMSO. However, antibody binding in the presence of EDTA increased 3500-fold. The involvement of hydrogen peroxide and hydroxyl radical in ascorbate-mediated DNA damage is consistent with ascorbate acting as a reducing agent for DNA-bound metal ions. Glyoxal is known to be formed during oxidation of ascorbate. Glyoxylated DNA, that previously had been proposed as a marker of oxidative damage, was recognised in a dose dependent manner using the antibody. We describe the potential use of our anti-ROS DNA antibody, that detects predominantly Fenton-type mediated damage to DNA and report on its specificity for the recognition of glyoxal-DNA adducts.

Skin delivery potency and antitumor activities of temozolomide ester prodrugs

Clinical trials have shown temozolomide to be an effective agent for treatment of malignant melanoma. In order to investigate its suitability for delivery via the skin, a series of temozolomide esters was synthesized as prodrugs. In vitro assays demonstrated temozolomide, temozolomide acid and the hexyl ester equi-effective against selected cancer cell lines. The susceptibility of the esters to enzyme hydrolysis and their effectiveness for application to the skin were investigated. The esters effectively diffuse through rat skin and the hexyl ester demonstrated profound potency for penetrating through skin. Topical application of 5% (w/v) hexyl ester in DMSO solution on a mouse model demonstrated a significant inhibition of tumor growth. These results suggest that temozolomide esters could be an effective alternative to temozolomide in the treatment of skin cancer. © 2005 Elsevier Ireland Ltd. All rights reserved.

The co-ordination chemistry of tellurium and related selenium ligands

2-(2-pyridyl)phenyl(p-ethoxyphenyl)tellurium(II), (RR1Te) reacts with HgC12 at room temperature to give white HgCl2.RR1Te. On setting aside, or on warming the reaction mixture a yellow material, [R1HgCl.(RTeCl)2] is formed. Multinuclear NMR(125Te, 199Hg, 1H) and mass spectroscopy confirm the formulation, and confirm the ease of transfer of the p-ethoxyphenyl group (R1) between the metal centres. The crystal structure of the yellow material consists of two discrete RTeCl molecules together with a R1HgCl molecule. There is no dative bond formation between these species, hence the preferred description of the formation of an inclusion complex. The reaction of RR1Te with Copper(I) chloride in the cold gives an air sensitive yellow product Cu3Cl3(RR1Te)2(0.5CH3CN); under reflux in air changes to the green Cu2Cl(RR1Te)(0.5 EtOH). By contrast, the reaction of RR1Te with acetonitrile solution of Copper(II) salts under mild conditions affords the white materials CuCl(RR1Te) and CuBr(RR1Te)H2O. RR1Te reacts with PdCl2 and PtCl2 to give materials albeit not well defined, can be seen as intermediates to the synthesis of inorganic phase of the type M3XTe2XCl2X. Paramagnetism is associated with some of the palladium and platinum products. The 195Pt NMR measurement in DMSO establishes the presence of six platinum species, which are assigned to Pt(IV), Pt(III) or Pt(II). The reactions show that in the presence of PdCl2 or PtCl2 both R and R1 are very labile. The reaction of RHgCl(R= 2-(2-pyridyl)phenyl) with SeX4(X= Cl, Br) gives compounds which suggest that both Trans-metallation and redox processes are involved. By varying reaction conditions materials which appear to be intermediates in the trans-metallation process are isolated. Potentially bidentate tellurium ligands having molecular formula RTe(CH2)nTeR,Ln, (R= Ph,(t-Bu). C6H4, n = 5,10) are prepared. Palladium and Platinum complexes containing these ligands are prepared. Also complex Ph3SnC1L(L = p-EtO.C6H4) is prepared.

Functional polymers for biomedical application : synthesis and applications

Aromatic and aliphatic diacid chlorides were used to condense naturally occurring diamino acids and their esterified derivatives. It was anticipated the resulting functional polyamides would biodegrade to physiologically acceptable compounds and show pH dependant solubility could be used for biomedical applications ranging from enteric coatings to hydrosoluble drug delivery vehicles capable of targeting areas of low physiological pH. With these applications in mind the polymers were characterised by infra red spectroscopy, gel permeation chromatography and in the case of aqueous soluble polymers by potentiometric titration. Thin films of poly (lysine ethyl ester isophthalamide) plasticised with poly (caprolactone) were cast from DMSO/chloroform solutions and their mechanical properties measured on a Hounsfield Hti tensiometer. Interfacial synthesis was investigated as a synthetic route for the production of linear functional polyamides. High molecular weight polymer was obtained only when esterified diamino acids were condensed with aromatic diacid chlorides. The method was unsuitable for the production of copolymers of free and esterified amino acids with a diacid chloride. A novel miscible mixed solvent single phase reaction was investigated for production of copolymers of esterified and non-esterified amino acids with diacid chlorides. Aliphatic diacid chlorides were unsuitable for condensing diamino acids using this technique because of high rates of hydrolysis. The technique gave high molecular weight homopolymers from esterified diamino acids and aromatic diacid chlorides.

Novel imprinted polymers as artificial enzymes

Derivatives of L-histidine were investigated as suitable models for the Asp-His couple found in the catalytic triad of serine proteases. A combination of molecular dynamics and IH NMR spectroscopy suggested that the most populous conformations of N-acetyl-L-histidine and the N-acetyl-L-histidine anion were predominated by those in which the carboxylate group was gauche to the imidazole ring overcoming steric and electrostatic repulsion, suggesting there is an interaction between the carboxylate group and the imidazole ring. Kinetic studies, using imidazole, N-acetyl-L-histidine and the N-acetyl-L-histidine anion showed that in a DMSO/H20 9: 1 v/v solution, the N-acetyl-L-histidine anion catalysed the hydrolysis of p-nitrophenyl acetate at a greater rate than using either imidazole or N-acetyl-L-histidine as catalyst. This indicates that the carboxylate group affects the nucleophilicity of the unprotonated imidazole ring. 31P MAS NMR spectroscopy was investigated as a new technique for the study of the template molecule environment within the polymer networks. It was found that it was possible to distinguish between template associated with the polymer and that which was precipitated onto the surface, though it was not possible to distinguish between polymer within imprinted cavities and that which was not. Attempts to study the effect of the carboxylate group/imidazole ring interaction in the imprinted cavity of a molecularly imprinted polymer network were hindered by the method used to follow the reaction. It was found though that in a pH 8.0 buffered solution the presence of imprinted cavities increased the rate of reaction for those polymers derived from L-histidine. Some preliminary investigations into the design and synthesis of an MIP which would catalyse the oxy-Cope rearrangement were carried out but the results were inconclusive.

Chemical and physical methods of enhancing the percutaneous absorption of antimicrobial agents

Contrary to previously held beliefs, it is now known that bacteria exist not only on the surface of the skin but they are also distributed at varying depths beneath the skin surface. Hence, in order to sterilise the skin, antimicrobial agents are required to penetrate across the skin and eliminate the bacteria residing at all depths. Chlorhexidine is an antimicrobial agent with the widest use for skin sterilisation. However, due to its poor permeation rate across the skin, sterilisation of the skin cannot be achieved and, therefore, the remaining bacteria can act as a source of infection during an operation or insertion of catheters. The underlying theme of this study is to enhance the permeation of this antimicrobial agent in the skin by employing chemical (enhancers and supersaturated systems) or physical (iontophoresis) techniques. The hydrochloride salt of chlorhexidine (CHX), a poorly soluble salt, was used throughout this study. The effect of ionisation on in vitro permeation rate across the excised human epidennis was investigated using Franz-type diffusion cells. Saturated solutions of CHX were used as donor and the variable studied was vehicle pH. Permeation rate was increased with increasing vehicle pH. The pH effect was not related to the level of ionisation of the drug. The effect of donor vehicle was also studied using saturated solutions of CHX in 10% and 20% ethanol as the donor solutions. Permeation of CHX was enhanced by increasing the concentration of ethanol which could be due to the higher concentration of CHX in the donor phase and the effect of ethanol itself on the membrane. The interplay between drug diffusion and enhancer pretreatment of the epidennis was studied. Pretreatment of the membrane with 10% Azone/PG demonstrated the highest diffusion rate followed by 10% olcic acid/PG pretreatment compared to other pretreatment regimens (ethanol, dimethyl sulfoxide (DMSO), propylene glycol (PG), sodium dodecyl sulphate (SDS) and dodecyl trimethyl ammonium bromide (DT AB). Differential Scanning Calorimetry (DSC) was also employed to study the mode of action of these enhancers. The potential of supersaturated solutions in enhancing percutaneous absorption of CHX was investigated. Various anti-nucleating polymers were screened in order to establish the most effective agent. Polyvinylpyrrolidone (PVP, K30) was found to be a better candidate than its lower molecular weight counterpart (K25) and hydroxypropyl methyleellulose (HPMC). The permeation studies showed an increase in diffusion rate by increasing the degree of saturation. Iontophoresis is a physical means of transdemal drug delivery enhancement that causes an increased penetration of molecules into or through the skin by the application of an electric field. This technique was employed in conjunction with chemical enhancers to assess the effect on CHX permeation across the human epidermis. An improved transport of CHX, which was pH dependant was observed upon application of the current. Combined use of iontophoresis and chemical enhancers further increased the CHX transport indicating a synergistic effect. Pretreatment of the membrane with 10% Azone/PG demonstrated the greatest effect.

The role of metabolism in the toxicity of N-alkylformamides

Using ionspray tandem mass spectrometry the glutathione conjugate SMG was identified as a biliary metabolite of DMF in rats (0.003% of a dose of 5OOmg/kg DMF i.p.). Formation of this metabolite was increased five fold after induction of CYP2E1 by acetone, and was inhibited to 20% of control values following pretreatment with disulfrram. Generation of SMG from DMF in vivo was shown to exhibit a large kinetic deuterium isotope effect (KWKD=10.1 ± 1.3), which most likely represents the product of 2 discrete isotope effects on N-demethylation and formyl oxidation reactions.The industrial solvent N,N-dimethylformamide (DMF) and the investigational anti-tumour agent N-methylformamide (NMF) cause liver damage in rodents and humans. The hepatotoxicity of N-alkylformamides is linked to their metabolism to N-alkylcarbamic acid thioesters. The enzymatic details of this pathway were investigated. Hepatocytes isolated from BALB/c mice which had been pretreated with acetone, an inducer of the cytochrome P-450 isozyme CYP2E1, were incubated with NMF (10mM). NMF caused extensive toxicity (> 90% ) as determined by lactate dehydrogenase (LDH) release, compared to cells from untreated animals. Incubation of liver cells with NMF for 6 hrs caused 60±17% LDH release whilst in the presence of DMSO (10mM), an alternative substrate for CYP2E1, LDH release was reduced to 20±10% . The metabolism of NMF to S-(N-methylcarbamoyl)glutathione (SMG) was measured in incubates with liver microsomes from mice, rats or humans. Metabolism of NMF was elevated in microsomes isolated from rats and mice pretreated with acetone, by 339% and 183% respectively. Pretreatment of animals with 4-methylpyrazole induced the metabolism of NMF to 280% by rat microsomes, but was without effect on NMF metabolism by mouse microsomes. The CYP2E1 inhibitors or alternative substrates diethyl dithiocarbamate (DEDTC), p-nitrophenol (PNP) and dimethyl sulphoxide (DMSO) strongly inhibited the metabolism of NMF in suspensions of rat liver microsomes, at concentrations which did not effect aminopyrine N-demethylation. The rate of metabolism of NMF to SMG in human microsomes correlated (r> 0.8) with the rate of metabolism of chlorzoxazone, a CYP2E1 probe. A polyclonal antibody against rat CYP2E1 (10mg/nmol P-450) inhibited NMF metabolism in microsomes from rats and humans by 75% and 80% , respectively. The amount of immunoblottable enzyme in human microsomes, determined using an anti-rat CYP2E1 antibody, correlated with the rate of NMF metabolism (r> 0.8). Purified rat CYP2E1 catalysed the generation of SMG from NMF. Formation of the DMF metabolite N-hydroxymethyl-N-methylformamide (HMMF) in incubations with rat liver microsomes was elevated by 200% following pretreatment of animals with acetone. Co-incubation with DEDTC (100μM) inhibited HMMF generation from DMF by 88% . Co-incubation of DMF (10mM) with NMF (1mM) inhibited the formation of SMG by 95% . A polyclonal antibody against rat CYP2E1 (10mg/nmol P-450) inhibited generation of HMMF in incubates with rat and human liver microsomes by 68.4% and 67.5% , respectively. Purified rat CYP2E1 catalysed the generation of HMMF from DMF. Using ionspray tandem mass spectrometry the glutathione conjugate SMG was identified as a biliary metabolite of DMF in rats (0.003% of a dose of 5OOmg/kg DMF i.p.). Formation of this metabolite was increased five fold after induction of CYP2E1 by acetone, and was inhibited to 20% of control values following pretreatment with disulfrram. Generation of SMG from DMF in vivo was shown to exhibit a large kinetic deuterium isotope effect (KHKD=10.1 ± 1.3), which most likely represents the product of 2 discrete isotope effects on N-demethylation and formyl oxidation reactions.

Preliminary in vitro toxicological evaluation of a series of 2-pyridylcarboxamidrazone candidate anti-tuberculosis compounds III

We have evaluated the cytotoxicity of a series of novel anti-tubercular 2-pyridyl carboxamidrazones through incubation with human mononuclear leucocytes (MNL), with and without a rat microsomal metabolising system. Isoniazid (INH), the closest structurally related agent, was used as a positive control. Incubation of the 3-benzyloxy-benzylidene, dimethylpropyl-benzylidene and 4-phenyl-benzylidene with MNL showed no significant toxicity in comparison with either INH or DMSO vehicle control. However, the 4-N,N-dimethylamino-1-naphthylidene derivative exerted more than sevenfold greater toxicity compared with INH, while the 4-N,N-dimethylamino-1-naphthylidene, 2-benzyloxy-3-methoxy-benzylidene, 2-t-butylthio-benzylidene and 4-i-propyl-benzylidene derivatives showed toxicity which ranged from five to fourfold that of INH. In the presence of either rat microsomes with or without NADPH, the 3-benzyloxy-benzylidene, dimethylpropyl-benzylidene and 4-phenyl-benzylidene derivatives showed no metabolically-mediated cytotoxicity. The latter two derivatives showed a combination of low toxicity and considerable efficacy against Mycobacteria tuberculosis in vitro and show promise for future development. © 2001 Elsevier Science B.V.

Reactions of copper macrocycles with antioxidants and HOCl:potential for biological redox sensing

A series of simple copper N(2)S(2) macrocycles were examined for their potential as biological redox sensors, following previous characterization of their redox potentials and crystal structures. The divalent species were reduced by glutathione or ascorbate at a biologically relevant pH in aqueous buffer. A less efficient reduction was also achieved by vitamin E in DMSO. Oxidation of the corresponding univalent copper species by sodium hypochlorite resulted in only partial (~65 %) recovery of the divalent form. This was concluded to be due to competition between metal oxidation and ligand oxidation, which is believed to contribute to macrocycle demetallation. Electrospray mass spectrometry confirmed that ligand oxidation had occurred. Moreover, the macrocyclic complexes could be demetallated by incubation with EDTA and bovine serum albumin, demonstrating that they would be inappropriate for use in biological systems. The susceptibility to oxidation and demetallation was hypothesized to be due to oxidation of the secondary amines. Consequently these were modified to incorporate additional oxygen donor atoms. This modification led to greater resistance to demetallation and ligand oxidation, providing a better platform for further development of copper macrocycles as redox sensors for use in biological systems.

An in-vitro-in-vivo model for the transdermal delivery of cholecalciferol for the purposes of rodent management

The natural selection of anticoagulant resistant rats has resulted in a need for an alternative to anticoagulant rodenticides which differs in both active ingredient and in the method of dosing. Cholecalciferol toxicity to rodents using the dermal route is demonstrated using a variety of penetration enhancing formulations in two in-vitro models and finally in-vivo. A 1 ml dose of 50/50 (v/v) DMSO/ethanol containing 15% (v/v) PEG 200 and 20% (w/v) cholecalciferol was judged as 'sufficiently effective' in line with the European Union's Biocidal Products Regulation (No. 528/2012) during in-vivo studies. This dose was found to cause 100% mortality in a rat population in 64.4 h (±22 h).

Multiparameter flow cytometric analysis of C -reactive protein binding to human-derived cell lines and peripheral blood monocytes

C-reactive protein (CRP), a normally occurring human plasma protein may become elevated as much as 1,000 fold during disease states involving acute inflammation or tissue damage. Through its binding to phosphorylcholine in the presence of calcium, CRP has been shown to potentiate the activation of complement, stimulate phagocytosis and opsonize certain microorganisms. Utilizing a flow cytometric functional ligand binding assay I have demonstrated that a monocyte population in human peripheral blood and specific human-derived myelomonocytic cell lines reproducibly bind an evolutionarily conserved conformational pentraxin epitope on human CRP through a mechanism that does not involve its ligand, phosphorylcholine. ^ A variety of cell lines at different stages of differentiation were examined. The monocytic cell line, THP-1, bound the most CRP followed by U937 and KG-1a cells. The HL-60 cell line was induced towards either the granulocyte or monocyte pathway with DMSO or PMA, respectively. Untreated HL-60 cells or DMSO-treated cells did not bind CRP while cells treated with PMA showed increased binding of CRP, similar to U-937 cells. T cell and B-cell derived lines were negative. ^ Inhibition studies with Limulin and human SAP demonstrated that the binding site is a conserved pentraxin epitope. The calcium requirement necessary for binding to occur indicated that the cells recognize a conformational form of CRP. Phosphorylcholine did not inhibit the reaction therefore the possibility that CRP had bound to damaged membranes with exposed PC sites was discounted. ^ A study of 81 normal donors using flow cytometry demonstrated that a majority of peripheral blood monocytes (67.9 ± 1.3, mean ± sem) bound CRP. The percentage of binding was normally distributed and not affected by gender, age or ethnicity. Whole blood obtained from donors representing a variety of disease states showed a significant reduction in the level of CRP bound by monocytes in those donors classified with infection, inflammation or cancer. This reduction in monocyte populations binding CRP did not correlate with the concentration of plasma CRP. ^ The ability of monocytes to specifically bind CRP combined with the binding reactivity of the protein itself to a variety of phosphorylcholine containing substances may represent an important bridge between innate and adaptive immunity. ^

Conservação de material genético de espécies silvestres do bioma caatinga utilizando a manipulação oócitos inclusos em folículos ovarianos pré antrais (MOIFOPA)

The objective of the present thesis was to use the manipulation of oocytes enclosed in preantral follicles (MOEPF) as a tool for the female gametes rescue and optimization, from wild species of Caatinga biome. The thesis was divided into 4 experiments. At first experiment, it was performed the estimative and description of the agouti (Dasyprocta leporina) preantral follicles (PF) histologic and ultrastructural features, in which it was estimated 4419.8 ± 532.26 and 5397.52 ± 574.91 follicles for the right and left ovary, respectively, and the majority (86,63%) belonged to the primordial follicles category (P<0.05). Most of the population consists of morphologically normal follicles (70.78%), presenting a large and central nuclei and uniform cytoplasm. At ultrastructural evaluation it was verified the presence of a great number of round mitochondrias associated to lipid droplets. In the second experiment, it was performed the estimative and description of yellow-toothed cavies (Galea spixii) PF characteristics, also, the evaluation of the effect of solid surface vitrification (SSV) on the in situ PF morphology. The total of 416.0 ± 342.8 PF was estimated for the ovary pair and the presence of a large quantity of primary follicles (P<0.05) was evidenced. Most of the PF was morphologically normal (94.6%), in which the oocyte nuclei presented condensed granules of heterochromatin. Round or elongated shaped mitochondria constituted the most abundant organelles. In regard of the SSV, the protocol using the dimethylsulfoxide (DMSO) 3M possibility the preservation of 69.5% of morphologically normal PF, which was evidenced by the light and transmission electronic microscopy. At third experiment, the evaluation of the SSV procedure on the morphology and viability in situ PF form collared peccaries (Pecari tajacu) was performed. No differences were observed among treatments, in which the use of DMSO, ethylene glycol (EG) and dimethylformamide (DMF) as cryoprotectants, regardless its concentration, promoted the morphology preservation of much than 70% of PF. Concerning the PF viability, the DMSO and EG promoted the best preservation. The fourth experiment aimed to evaluate the effect of α MEM+ or TCM199 associated or not to 50 ng of FSHr on the morphology, activation and growth of collared peccaries PF, in vitro cultured (IVC) during 1 or 7 days and the effect on the extracellular matrix (ECM). After 7 days of IVC only the use of TCM199/FSH maintained the proportion of intact PF, similar to day 1(63.2%), however, no differences were observed among treatments (P>0.05). Also, an improvement of the proportion of intact growing PF was verified (P>0.05). By the Ag-NOR analysis it was observed that only the treatment using TCM199/FSH promoted the maintenance of cell proliferation similar to day 1 (P>0.05). The picrosirius red stain revealed that ECM remained intact in all treatments (P>0.05). Thus, as the general conclusion, the use of MOEPF in the refereed species allowed the knowledge of aspects related to its reproductive morphology and physiology, enabling the germplasm conservation, with the possibility of germplasm bank formation, as the elucidation of mechanisms related to the PF survive and in vitro development.

Encapsulamento de solventes em nanotubos de haloisita para promoção de auto-reparo em polímeros

Micro cracking during service is a critical problem in polymer structures and polymer composite materials. Self-healing materials are able to repair micro cracks, thus their preventing propagation and catastrophic failure of structural components. One of the self-healing approaches presented in the literature involves the use of solvents which react with the polymer. The objective of this research is to investigate a procedure to encapsulate solvents in halloysite nanotubes to promote self-healing ability in epoxy. Healing is triggered by crack propagation through embedded nanotubes in the polymer, which then release the liquid sovent into the crack plane. Two solvents were considered in this work: dimethylsulfoxide (DMSO) and nitrobenzene. The nanotubes were coated using the layer-by-layer technique of oppositely charged polyelectrolytes: cetyltrimethylammonium bromide (CTAB) and sodium polyacrylate. Solvent encapsulation was verified by X-ray diffraction (XRD), Fourier transform infrared (FTIR), analysis thermogravimetry (TGA), adsorption and desorption of nitrogen and scanning electron microscopy (SEM). The introduction of the solvent DMSO into the cavity of the nanotubes was confirmed by the techniques employed. However, was not verified with nitrobenzene only promoted clay aggregation. The results suggest that the CTAB reacted with the halloystite to form a sealing layer on the surface of the nanotubes, thus encapsulating the solvent, while this was not verified using sodium polyacrylate.