Funcionalización de nanopartículas de oro con el péptido clpffd para potenciales aplicaciones en biomedicina
- Autores: Eyleen Araya Fuentes
- Directores de la Tesis: Marcelo Javier Kogan (dir. tes.), Fausto Sanz Carrasco (dir. tes.)
- Lectura: En la Universitat de Barcelona ( España ) en 2012
- Idioma: español
- Tribunal Calificador de la Tesis: Josep Samitier Martí (presid.), Olga López Serrano (secret.), Muriel Arimón Bedós (voc.)
- Materias:
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- Resumen
Resumen de la Tesis: The general goal of this work was the synthesis of gold nanoparticles (NPAu) conjugated with the peptide LPFFD that recognizes toxic aggregates of ß-amyloid protein with the purpose to destroy the mentioned aggregates that are present in brains of patients that suffer Alzheimer¿s disease. Additionally we evaluated the cytotoxicity of the conjugate and investigated the feasibility of the conjugate to accumulate in the brain. This work does not only provide a new tool for the treatment of this disease but also for other degenerative diseases classified as conformational diseases, characterized by the accumulation of amyloid deposits provided by a variety of proteins as Parkinson's disease and diabetes type II The specific goals of this thesis are: i) Synthesis of NPAu and their characterization.
ii) Functionalization of NPAu with the peptide LPFFD to increase their stability and recognize toxic aggregates of ß-amyloid .
iii) In vitro studies to destroy toxic aggregates of ß-amyloid in the presence of NPAu after irradiation with oscillating magnetic fields.
iv) Studies of cytotoxicity and accumulation of NPAu conjugated with the peptide LPFFD in the brain and in other organs.
Introduction Gold is a noble element and by nature, it is highly non-reactive. In its molecular form, gold compounds can fulfill various functions, ranging from the use in catalysis to other uses in medicine for arthritis treatment.
The term nano, derived from the Greek "nanos" meaning dwarf and is used to describe a property that occurs with the size of the nanometer scale (1-100 nm). Unlike bulk gold, gold nanoparticles have bright colors that could change according to their shapes and sizes (Figure 1). In recent years gold nanoparticles (NPAu) has been used for applications in chemistry, physics, and now in biomedicine. But beyond its beauty, NPAu have properties that are fundamentally different from other metals.
Figure 1: NPAu commonly used in biomedical applications. (a) gold nanorods, (b) Core-Shell Silica-gold (silica core nanoparticles and coated with gold) and (c) of gold nano-boxes. The intense color of these nanoparticles is due to the collective excitation of the conduction electrons or plasmon resonance, resulting in absorption of photons of wavelengths that vary with respect to (a) size, (b) thick and / or (c) in the displacement plating for gold.
Due to their size, these particles can accumulate preferentially at sites of inflammation in a growth or tumor and are able to penetrate into cells by mechanisms quite different and much faster than many small molecules, thus, can be used for targeting drugs to tumors efficiently. Moreover, due to the reactivity given by its surface features NPAu can be attached to active molecules as for example antibodies or other active molecules in order to address selectively the NPAu to therapeutic targets. Also, due to their photophysical properties can be used in assays for biodiagnosis as is the case of a pregnancy test (test marketed under First Response ® in the decade of the 90). In this connection it is interesting to note that the NPAu have the ability to absorb light energy and dissipate it locally to produce the so-called thermal ablation specifically for tumor or infected tissued. Furthermore, NPAu have the ability to absorb copious amounts of X-ray radiation that can be used to enhance radiation therapy in cancer treatment or to improve the image contrast of a computerized tomography.
The NPAu because their sizes are comparable to those of other macromolecules such as proteins and can selectively disrupt and alter cellular processes allowing them to act as drug carrier agents. For all the above features, the NPAu are interesting species to be used in biomedical applications, therefore, its use has been increasingly growing in recent years due to great interest in the development of nanotechnology in this field.
In this thesis we used NPAu to destroy beta-amyloid (ßA) peptide aggregates for the development of a new therapeutic strategy for Alzheimer¿s disease.
ßA and Alzheimer¿s disease ßA peptide received attention because of its importance in Alzheimer's disease (AD). This disease is characterized by a loss of short-term memory, problems with language, disorientation in time and space, changes in personality and other disorders. A physiological characteristic of AD is the deposition of ßA peptide aggregates that form amyloid fibrils and neurofibrillary tangles in the brains of individuals with the disease. These deposits are composed mainly of ßA in an aggregated form and of the tau protein phosphorylated at the intracellular level. One hypothesis of how are generated these deposits is that the transmembrane protein called amyloid precursor protein (APP) is cleaved extracellular and intracellularly by different secretases (¿ and ß) forming ßA. This last peptide aggregates to form oligomers and fibrils being the first more toxic with respect to the latter. This toxic aggregates lead to the formation of the amyloid plaque.
For some amyloidogenic peptides as ßA, the monomer has a disordered structure. The oligomers are globular aggregates that generally lack a well defined secondary structure. Some authors have proposed that the oligomers are similar to micellar structures with spherical or cylindrical shapes. The size and conformational characteristics of the oligomers are quite variable. Particularly, in the Aß were observed oligomers with a spherical shape and a diameter of 5 nm with molecular masses in the range of 20-50 kDa as well as oligomers with spherical with diameters of around 15 nm and masses close to one million Da. The protofibrils are linear aggregates that appear as early intermediates in the formation of amyloid plaque. ßA protofilaments and filaments typically have between 2.5 and 3.5 nm in diameter. The distinction between protofilaments and filaments is not well defined. The protofilaments are distinct from protofibrils to have structural characteristics similar to fully mature fibers (although not a universally distinction has not been used). The fibers have diameters between 7-12 nm and 1 micra or more in length with a defined ß-sheet structure. They typically appear in groups of 2 to 6 strands that can be rolled one with another forming a helix. fibril formation probably involves the cooperative association of protofilaments or filaments and possibly additional conformational changes. The fibers generally are linear but occasionally have some branching. Finally, the fibers tend to associate in bundles which ultimately lead to fibers amiloide.
Figure 2: shows a schematic representation of the general mechanism of aggregation of ¿A to form amyloid fibers. Proteins in whole or partially unfolded are associate to form small soluble aggregates that continue towards the formation of protofibrils or protofilaments (a) and after (b) mature fibers (electron microscope top). (c) The fibrils often accumulate in plaques or other structures, such as Lewy¿s bodies associated with disease like Alzheimer's or Parkinson's disease, respectively (optic microscope image, right). (d) Some of the aggregates, first aggregates are micelles that acquired ring forms with diameters of about 10 nm (electron microscope image).
Metal Nanoparticles (NPM) According to the glossary of terms used by the IUPAC (International Union of Pure and Applied Chemistry), a nanoparticle is a microscopic particle whose size is measured in nanometers, often restricted to nano-sized particles, ie with a diameter smaller than 100 nm, also called ultrafine particles.
The NPs can be manufactured with different metals (nickel, copper, cobalt, gold, silver, platinum, etc.) or with organic compounds, which may be of the type polymers. A typical and well known example is the synthesis of NPAu by reduction of a gold salt with sodium citrate. Figure 3 shows a photograph of transmission electron microscopy (transmission electron microscopy) of NPAu.
Figure 3: TEM micrograph corresponding to NPAu with an average size of approximately 10 nm.
NPAu in the presence of oscillating magnetic fields (CMO) or electromagnetic radiation can absorb energy and dissipate it locally producing a kind of molecular surgery destroying species that are near from them. Examples of this kind of surgery may be the breakdown of toxic protein aggregates or the destruction of tumor cells. A key factor for using NPM is that they should be address specifically to the site of action. The conjugation of NPM with different molecules as peptides for one hand, help to address them to selective targets and on the other hand they could prevent their precipitation or agglomeration.
Use of NPM for biomedical applications.
The NPs provide a number of interesting applications for biomedicine because the size of these NPs can vary from one to tens of nanometers, which places them at dimensions less than or comparable to a cell (10-100 microns) , a virus (20-450 nm), a protein (5-50 nm) or a gene (2 nm wide and 10-100 nm long) (Figure 4). The advantage of the sizes of the NPs is that they could approach to a biological target of interest and pass cell barriers through mechanisms such as endocytosis or by the enhanced increased permeability and retention (EPR) effect that are present in tumors.
Figure 4: Schematic comparison of the size of NPAu of 10 nm with respect to atoms, molecules, ß-amyloid aggregates, viruses, bacteria and cells.
One of the main applications of NPs is that they could actuate in diagnosis for molecular visualization. In particular, NPs with optical and/or magnetic properties may have important applications in the diagnosis in vivo or in vitro. The ability to visualize specific activities of enzymes using these NPs can achieve diagnostic applications such as a variety of diseases and evaluation of therapy in patients. The NPs attached to a suitable carrier such as an antibody or biomolecules capable of recognizing therapeutic targets are used to mark specific structures as for example microorganisms. Recently, immunoassay techniques have been developed in which the target, magnetically marked, can be detected by magnetic sensors.
An important point to the use of the NPs in therapy or diagnosis is that they should not be toxic, biocompatible and it must be stable in biological media. Moreover, an important feature is that the NPs can be coated with biological molecules that allow the interaction with a biological target, providing a "labeled" target. To ensure that NPs reach the desired goal, they could be capped with molecules as peptides or proteins that specifically recognize the therapeutic target. The peptides may be involved in ligand-receptor binding and molecular interactions of proteins with proteins 19 facilitating the molecular recognition. For example, the peptides are involved in molecular recognition of antibodies, which is relevant in the field of clinical diagnosis of infectious diseases and for the design of new drugs and vaccines.
Mainly for biomedical purposes, the peptides conjugated NPs are gold, gold-coated silica (core/shell) and iron oxide. The NPAu have the advantage of offering a high degree of stability and biocompatibility while the magnetic NPs are less stable and less biocompatible.
¿ Cytotoxicity of NPs The degree of toxicity of NPs could be a problem for later use in the field of biomedicine. To overcome these drawbacks NPs can be biocompatibilized by capping with peptides in order to reduce their toxicity and increase their stability. Several toxicity studies have been carried out in different cell lines to determine the effects of coating of the NPs. Cytotoxicity of nanoparticles depends on different aspects: charge, hydrophobicity/hydrophilicity and size. NPs positively charged, for example, which are coated with the cationic surfactant bromide hexadecyl trimethyl ammonium bromide (CTAB) have cytotoxic effects on various cell lines. The presence of multiple positive charges can produce membrane disruption due to electrostatic interaction with the negatively charged cell membranes. Moreover, with regard to hydrophobicity have been shown that hydrophobic positively charged NPs accumulate in different organs resulting in the death of the fish Japanese Medaka. The size is also a crucial parameter. Small NPs (1-2 nm) have greater toxicity to NPs of 15 nm or with higher sizes. The toxicity of NPs can be related to the interaction with proteins culture medium which can favor the entry and to trigger cell toxic effects.
In this thesis we have evaluated the effects of NPAu on the cell line SHSY-5Y being important to note that there are not reported studies of effects on these cells.
Use of NPM and oscillating magnetic fields for local and remote heating.
The NPM offer attractive possibilities for biomedicine because they have controllable sizes ranging from a few nanometers to several microns and can reach the same biological entity of interest. In fact, NPM coated with molecules could interact or bind selectively with a biological entity if they are appropriately functionalized.
The NPM are species that under oscillating magnetic fields (CMO) of the type of microwave can effectively absorb energy and then dissipate it locally. The NPM selectively bind to the target can act as a antenna producing a local effect without affecting other molecules. This strategy represents a promising tool for treating certain tumors. Among the NPM, the magnetic have high magnetic susceptibility sensitively responding to the CMO at low frequencies (1000 MHz) instead of NPM which are not magnetic, such as gold, respond by application of higher frequencies (GHz). In addition, magnetic-type NPM may be attracted by an external magnetic field being retained in a given area of the body. This action at a distance combined with the penetration of the tissues of CMO opens the way for new applications involving the transport and/or immobilization of magnetic nanoparticles. For example, they could act delivering a dose of an anticancer drug to a tumor, and finally the NPM (both magnetic and non magnetic) may respond to a CMO, absorbing energy and dissipating it locally, making it possible to use them as agents for hyperthermia therapy of tumors. Furthermore, the local increase in temperature by the action of an CMO on NPs can control the hybridization of DNA, which enables the local handling of ADN.
Preparation, characterization of stability and NPAu Colloidal suspensions correspond to physical systems consisting of two phases, a continuous, fluid and other normally dispersed in a particulate form. Colloids cover a wide range of sizes ranging from a few nanometers to a few micrometers. Electrostatic interactions in these systems play a very important role for stabilization of the particles. The surface charge of the particles suspended in liquid, forms an electric double layer around them producing a repulsive effect between the particles. The NPAu are classified as lyophobic colloids that have low affinity for the dispersing medium. These kinds of colloids could be obtained through two categories of synthetic methodologies: (a) By condensation, in which subcolloidal particles are induced to aggregate reaching a colloidal size, and (b) By dispersion in which particles of greater sizes than colloidal are reduced in size.
In this manner and depending on the size of NPAu are to be generated, is that there are numerous protocols for synthesis. The traditional method for the synthesis of NPAu is a condensation method described in 1951 by Turkevich et al. which allows obtaining particle sizes of 6 nm or more. In this method the salt HAuCl4 is reduced with sodium citrate at reflux. The control the size of the NPAu is given by the variation in the ratio citrate/HAuCl4. Figure 5 shows the formation of a nucleating center, on which are adsorbed successive layers of gold atoms to lead to the desired size of the particle.
Figure 5: Nucleation Center theory. Is represented the growth of a gold nanocrystal to form the NPAu.
¿ Stability of NPAu.
The NPAu form a colloid or a colloidal dispersion, comprising two separate phases, the dispersed phase and the dispersing phase. Initially there is a nucleation center that is a lyophobic colloid that is thermodynamically unstable but in the case of NPAu there is an excess of citrate that capped the nanoparticle stabilizing the colloid. ¿ Surface plasmon Visible color of NPAu dispersion depends on their size, and is due to surface plasmon. Plasmon effect could defined as quanta of oscillations of surface charges produced by an external electric field. The first theoretical description of the plasmon effect on NPs was performed by Sun in a publication in 1908 and is in accordance with Mie theore where the Maxwell's equations, predicts the absorption and scattering in the visible length as the sum of the electric and magnetic oscillations. Spectroscopic observations of NPs correlate generally with the Mie theory, making some adjustments regarding the dispersion of sizes of the NPs, and the refractive index of the solvent. Importantly, the dispersion in the actual particle size (as opposed to the calculated) produces a widening of the absorption band in the visible region, as seen in Figure 6.
Figure 1.5: A) Comparison of the absorption spectrum calculated by Mie (blue) and real (red). B) Photography of a colloidal dispersion NPAu.
The absorption maximum is located around 520 nm for NPAu of sizes between 10-20 nm. The absorbed wavelength is green, and the transmitted radiation by the colloid is a dark red (Figure 6 B).
When the wavelength passes through the particle surface its polarized oscillating in phase with the light frequency (¿) causing a coherent oscillation as shown in Figure 7.
Figure 7: Diagram of the interaction of electromagnetic radiation with the surface NPAu, showing an absorption band at ~ 520 nm.
¿ Zeta potential In general, the particles dispersed in an aqueous system acquire a surface charge. In the case of this is given by NPAu atoms gold (I) surface, and citrate adsorption of conferring to the resulting negative charge, Modifing the surface charge distribution of ions surrounding, resulting in an area around the particle which is electrically different from the bulk of the solution (Figure 8). This zone can be divided into two parts: the inner layer strongly adhered to the particle and it is called the Stern layer and an outer region, which is diffused with increasing distance from the surface. In this region there is a conceptual boundary beyond which the ions do not move along the particle. This border is known as a hydrodynamic shear plane. The zeta potential (¿) is the electric potential in the cutting plane, and usually expressed in millivolts (mV). Is a measure of the magnitude of the electric repulsion or attraction between the particles comprise the colloid.
Figure 8: Zeta potential. The cutting plane is represented by the circle gray, and the letter C. The red line represents the radius of the particle. On green line, K-1 is the Debye length, and the zeta potential is the potential at the distance C.
¿ Characterization of NPAu UV-visible spectroscopy One of the most useful techniques to characterize NPAu is UV-visible spectrophotometry, which provides information on concentration on the size and homogeneity of particle size. Thus the concentration is related to the absorbance at the maximum absorption peak (surface Plasmon peak), the size is related to the peak position, while the size homogeneity with the width of the signal. The gold colloids, which are characterized by their intense red color, absorb light in the blue-green region of spectrum. This absorption depends on such factors as the size of NPAu, state of aggregation and frequency surface plasmon resonance characteristic of the NPM.
Transmission Electron Microscopy (TEM) Another technique usually used in the characterization of NPAu is transmission electronic microscopy (TEM), which allows to obtain mainly information about the size and shape of the NPAu because the sample is irradiated by an electron beam, thereby allowing a contrast enhanced image product of the electrons retained, absorbed or scattered by the NPAu. Additionally other techniques as atomic force microscopy (AFM) could be used.
NPAu functionalization with peptides to address them to a specific target.
¿ NPAu functionalized with peptides In the literature there are two main strategies to link peptides with biological activity to NPAu. The first strategy involves the conjugation of a peptide by means of the spontaneous reaction of a thiol contained for example in the cysteine (C) belonging to the sequence of the peptide with the surface of the NPAu. By contrast in second strategy NPAu binds to the biologically active peptide via a linker molecule. This linker molecule is a bifunctional molecule that contains in one extreme a thiol group, which allows binding to the surface of gold, and in the other extreme a functional group (eg carboxyl group), which allows the binding to the peptide. (Figure 9) ¿ Stability of the Au NP-conjugated peptides In some cases, the colloidal stability of NPAu when they are functionalized with peptides, increases due to repulsive interactions between the peptide molecules which protrude from the particles. Thus van der Waals and stabilization occurs due to steric repulsion as described for other adsorbed molecules which are adsorbed on NPs. One example is the increased stability of the NPAu by conjugation with CLPFFD peptide used for a potential therapy for Alzheimer's disease, in which the particles are stabilized by steric effects. Additionally, the charge of the peptide molecules can play an important role in the stability of the colloid as for example at pH=7.4 the peptide LPFFD is negatively charged and may increase the stability the colloid whilce by change the pH the charge also change.
Figure 9: Representation of the conjugate-CLPFFD NPAu. Note that the peptide can change its charge depending on the pH ionization occurring amino or carboxyl.
Synthesis of peptides with biomedical applications ¿ Synthesis of solid phase peptide Peptide synthesis in solid phase (SPPS) was first described by Merrifield in 1963 and today is the most common and easy to obtain peptides. This method relies on the binding of the carboxy-terminal amino acid (C-terminal) to an insoluble support and subsequent elongation sequential amino acid after amino acid of the peptide chain. This method of solid phase synthesis has many advantages over the synthesis of peptides in solution. Yields are good because when working with the peptide bound to a solid support may be employed excesses of reagents which are easily removed after simple filtration and washing processes. Mechanical losses do not occur since the peptide solid support to which is attached the peptide remains in the same vessel throughout the process. Furthermore, washing and filtering operations are simple and amenable to automation.
The growth of the peptide chain has always place the carboxyl by successive additions of amino acids that have both the ¿-amino end and the side chains of amino acid conveniently protected.
Assays to evaluate the cytotoxicity of the conjugates of peptide NPAu While the use of NPs for biomedical applications is very promising, all these materials are subject to an evaluation process that involves preclinical absorption, distribution, metabolism, excretion and toxicity, commonly known as the ADMET profile. The factors that determine ADMET profiles of NPs are not well defined. For example, although it is known that colloidal NPAu are biologically inert, there is a noticeable variation in the cytotoxicity and the absorption of them in relation with their size, shape and capping.
In this thesis we evaluate the neurotoxicity of the conjugate NPAu-CLPFFD on neuroblastoma SHSY-5Y cell line because this conjugate is addressed to the brain where are located the ¿A aggregates. Table 1: Summary of methods used for evaluate the cytotoxicity.
In this thesis we used the MTS assays [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy methoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] and the trypan blue assay to evaluate the effects of NPs on a neuron cell line (neuroblastoma SHSY-5Y). We also evaluated the morphology of the cells incubated with NPAu.
MTS is a colorimetric method used for determining the number of viable cells in proliferation assays or cytotoxicity. The reagent is reduced by the cells to a brown color compound, which is soluble in the culture medium and absorbs at 490 nm wavelength. This reduction reaction is carried out by the NADH or NADPH dehydrogenase enzymes produced in metabolic active cells, therefore, the amount of the reduced compound produced is directly proportional to the number of live cells in culture.
Moreover, we evaluated the exclusion cell of the trypan blue dye. Viable cells tend to exclude the dye while the damaged cells incorporate the dye which in the nucleus interacts with DNA giving a blue staining of the cells.
Crossing biological barriers Because future applications of the peptide conjugates NPAu in therapies addressed to the central nervous system (CNS) is very important to assess whether NPAu can cross the blood brain barrier (BBB) to reach the nervous tissue in considerable amounts. The BBB represents an impenetrable barrier for the vast majority of drugs including anticancer agents, antibiotics, peptides and other drugs (Figure 10). The limited access of these drugs to the brain is due to the tight junctions "tight junctions" between the endothelial cells lining the blood vessels of the brain, as well as the existence of drug transport systems are very active, for example, so-called ABC transporters "ATP Binding Cassette" (such as Pgp) in the luminal membrane of these cells.
Figure 10: Illustration of the BBB. The BBB is formed by the tight junctions between endothelial cells of the blood vessels in the brain, protecting it from harmful substances.
Peptide inhibitors capable of retarding the aggregation kinetics Aß Naslund et al., conducted a sweep of the native protein positional Aß to identify the fragments that allow the recognition and binding between two strings of Aß, directly demonstrating the ability of recognition having the sequence. In the same work demonstrated the ability of a peptide molecule derived from the Ac-OKLVFF Aß-NH2, to prevent formation of amyloid fibers in vitro. Soto et al., replaced some residues of the original sequence (15-25) of Aß by proline to reduce its tendency to form ß-type structures. With these premises designed and synthesized peptide called iAß1 (RDLPFFPVPID), observing that it was able to reduce the formation of fibers also producing Aß disaggregating preformed amyloid fibers. A drawback to this molecule is large in size. By increasing the size, transport through the blood brain barrier is imparted and also the risk of an immune response is present. Therefore Soto et al., developed the iAß5 (LPFFD), a smaller molecule than iAß1 and with a similar activity. It is postulated that iAß5 act by blocking the growth of the aggregates, via interactions with the hydrophobic core residues of ßA se quence (residues 17-21) (Figure 11).
Figure 11: Representation of an aggregate of Aß (protofilament according to Li et.al.) Aß molecules are forming ß-sheets (yellow) highlighting the hydrophobic residues of phenylalanine (F) (in purple). LPFFD peptide blocks the growth of amyloid fibers by interactions with residues in the region 16-21 (KLVFFA) of Aß.
Recognition of aggregates involved in AD and its impartment to form less toxic species.
The so-called conformational diseases, among which we find the EA, are mainly characterized by protein misfolding due to changes in secondary and/or tertiary structure. This conformational change may cause a direct toxic effect or merely block the function of the protein in the physiological conditions. LPFFDNH2 peptide designed by Soto et al., is pointed to the region 17-20 (LVFF) of ßA (Figure 1.11).
Figure 1.11: This figure shows the primary sequence of ßA1-42. In red is represented the hydrophobic amino acids that form the core of aggregation. The sequence contains two different domains: the extracellular part of APP (polar) and the other one starting from the region of APP transmenbranal (hydrophobic). This amphipathic nature makes ßA having a great tendency to selfaggregate.
In this thesis have been conjugated gold nanoparticles with the peptide LPFFD in order to address them to toxic aggregates of ßA to manipulate its process of aggregation and to potentially reduce toxicity by irradiation with electromagnetic radiation. The peptide LPFFD, which in this case acts as an agent for the recognition of aggregates Aß, is used in concentrations much lower than those used to inhibit the aggregation process. Therefore, in the following chapters will discuss the preparation of the conjugates, its characterization, its application in vitro and its biological behavior.
Gold Nanoparticles and Microwave Irradiation Inhibit Beta-Amyloid Amyloidogenesis We discuss the use of oscillating magnetic fields (microwaves) to produce disaggregation of amyloid fibers and to inhibit the amyloidogenic process of Aß in the presence of NPAu-CLPFFD by dissipation of energy at the local level and remotly. ßA aggregates were selectively attached to NPAu-CLPFFD and the complexes Aß-NPAu-CLPFFD were irradiated with microwaves. This treatment produces dramatic effects on mature aggregates of Aß leading to their breakdown. Moreover, this type of treatment may inhibit the amyloidogenic process of preformed intermediaries of amyloidogenic Aß (PIAA).
This new approach offers a new strategy to destroy, locally and remotely, ßA toxic aggregates which could have application in the therapy of Alzheimer's disease. We carried out experiments with two systems: fibrils and PIAA that were incubated with NPAu-CLPFFD selectively attached to the aggregates. In order to form a stable binding of the peptide LPFFD to the gold surface of NPAu a Cys residue was added to the secuence at the N-terminal extreme of the peptide.
In the case of fibrils we incubated the Aß monomer with NPAu-CLPFFD to form immature fibrils (16 hs) or mature fibrils (7 days). After irradiation was possible to redissolve the amyloid deposits and also to interfere with their growth, using the local heat dissipated by NPAu selectively attached to the aggregates and irradiated with low gigahertz electromagnetic fields (microwaves). Simultaneous tagging and manipulation by NPAu of ¿A at different stages of aggregation allow both, noninvasive exploration and dissolution of molecular aggregates. We analyzed the samples before and after irradiation by TEM , Thioflavien fluorescence and Size exclusion chromatography. By TEM was observed a destruction of fibrils after the treatment (Figure 12).
Figure 12: TEM of the solutions before and after irradiation. (top). Images of the NPAu-CLPFFD bound to Aß aggregates after incubation of NPAu-CLPFFD with 10 ¿M Aß1-42 for 48 h. (bottom) Images of the previous NPAu-CLPFFD bound to Aß1-42 after 8 h of irradiation. Horizontal arrows indicate the presence of dimers and trimers, vertical arrows the presence of isolated particles, and circles the presence of small fibers. Bars are 500 nm.
On the other, Aß PIAA in the presence of NPAu-CLPFFD-NH2 form complexes that were irradiated and incubated in order to evaluate the amylodiogenic process determining the formation of amyloid fibers by TEM and Thioflavine test. Figure 13 shows TEM micrographs of the sample ßA PIAA/AuNP-CLPFFD complex and controls before and after irradiation for 30 min and further incubation (right), and non-irradiated and incubated sample and controls (left). The complex ßA PIAA/NPAu-CLPFFD (Figure 13b) was irradiated (Figure 13c) and after incubation no fibril formation was observed (Figure 3d). In contrast, in control experiments with irradiation, the fibril formation process was not interrupted (Figure 13: h, l, and p). We also carried out control experiments to determine whether the presence of NPAu-CLPFFD, bare NPAu, or CLPFFD alone interfered with the normal fibrillogenic process of ßA PIAA in the absence of irradiation (Figure 13, left). In these cases, fibril formation was not inhibited (Figure 13: a, e, i, m). After irradiation of ßA PIAA/NPAu-CLPFFD samples, noncharacteristic structures corresponding to typical ßA PIAA were visualized by TEM (Fig. 13c). Irradiation in the presence of NPAu-CLPFFD produced dramatic effects on ßA PIAA and consequently on the amyloidogenesis.
Figura 13: TEM micrographs of Aß PIAA/AuNP-CLPFFD sample and controls (starting conditions: b, f, j, and n, respectively). Sample and controls after irradiation for 30 min (c, g, k, and o, respectively) and incubated for 48 h at room temperature (d, h, l, and p, respectively). Nonirradiated sample and controls incubated for 48 h (a, e, i, and m, respectively). Bars represent 200 nm In summary, the microwave and the use of NPAu-CLPFFD-NH2 selectively attached to the Aß-PIAA highly amyloidogenic, may irreversibly inhibit its normal aggregation, resulting in non-amyloidogenic structures. On the other hand by this method is possible to destroy amyloid fibrils obtaining species with non amyloidogenic capacity. Our approach provides a new strategy to irreversibly inhibit the amyloidogenic processing of Aß which is a potential tool for the design of a new therapeutic strategy for Alzheimer's disease.
How Changes in the Sequence of the Peptide CLPFFD-NH2 Can Modify the Conjugation and Stability of Gold Nanoparticles and Their Affinity for ß-Amyloid Fibrils.
NPAu functionalization with the peptide CLPFFD allows the selective binding of the same with different types of amyloid aggregates such as fibers and smaller species such as amiloesferoides and protofibrils. The application of radiofrequency energy corresponding to the absorption and leads to a local dissipation of energy that leads to the destruction of amyloid fibers and inhibition of amyloidogenic process. After treatment of the samples, in no case were changes in the same temperature (bath temperature) for which the effects are due to local and remote phenomena.
Influence of the peptide sequence on the conjugation with NPAu, the stability of the conjugates and their affinity for ßA fibers.
An important in the field of nanomedicine is to know how the change in the peptide sequence could affect to the colloidal stability and affinity for the biological target. The coating of nanoparticles by peptide molecules may contribute to increase stability and reduce the toxicity of them. However, it is important to know the structure of molecules once bound to the surface because it is necessary to maintain the molecular recognition toward the target. Factors such as the peptide sequence, the steric effect on the colloids, the charge and the arrangement of the hydrophilic and hydrophobic residues are crucial parameters for designing a conjugate NPAu-peptide as a potential tool for biomedical applications.
In this thesis we study the effects of changing the sequence CLPFFD-NH2 (obtaining the isomers CDLPFF and CLPDFF) on the degree of functionalization of NPAu, on the colloidal stability of the NPAu-peptide and on their affinity for ßA fibers.
In order to address the NPAu to the ßA toxic aggregates, we obtained the conjugate NPAu-CLPFFD-NH2, which selectively binds to the fibrils.
For pharmaceutical applications of conjugates NPAu-peptide is important to consider the colloidal stability and the capacity of them to recognize the therapeutic target that in this case are ßA fibers. To maintain the colloidal stability after conjugation is important that the load NPAu remains in solution, while avoiding compression of the double layer that stabilizes the colloid. It is also important to consider the arrangement in the conjugates of peptide molecules on the surface which is related to the steric stabilization of the nanoparticle and the load to be exhibited to give rise to the double layer.
Moreover, to address the NPAu-peptide conjugates for recognize the ¿A fibers is important that the hydrophobic residues (L, F, and F) are accessible for binding to ßA. Therefore, in this work we studied the influence that the peptide sequence has in the molecular recognition, for which the conjugates CLPFFD-NH2, CLPDFF-NH2 and CDLPFF-NH2 were obtained.
If the peptide adopts an extended conformation CLPFFD, the residue aspartic acid (D) will be orthogonal to the surface holding the group D away from the surface, whereas for the case of conjugate NPAu-CDLPFF-NH2, D is the residue over near the surface of the NPAu (Figure 14). In the case of the conjugate NPAu-CLPDFF-NH2, there is an intermediate situation. In NPAu-peptide conjugates, the stability of the colloid is modulated by the exposure of the peptide charge that is forming the double layer and also lead to steric repulsion. Moreover, the affinity of the conjugated for the fibers ¿A is dependent on the structure of the peptides and the exposure of hydrophobic residues L, F and F.
Figure 14: Scheme of the conjugates NPAu-peptides. The residue D, represented in red, possesses a negative charge at pH=7.4. Hydrophobic residues are represented in blue (LFF). The arrows indicate the relative distances between the gold surface and the residue D assuming an extended conformation of the peptide.
NPAu-peptide conjugates were prepared by mixing the colloidal solution of NPAu with an excess of peptide to ensure a full functionalized NPAu. With the aim of removing excess peptide a dialysis was carried out. NPAu-peptide conjugates were characterized by using different techniques UV-Vis spectrophotometry, EELS, XPS, amino acid analysis, gel electrophoresis and CD. Figure 15 shows the UV-Vis spectra of citrate NPAu and NPAu-peptide conjugates. In the spectra is observed a shifr of the surface plasmon resonance band from 519 nm (NPAu) to 527 nm (conjugated) which indicate the nanoparticles are capped with the peptides. This shift in absorption maximum is related to a change in refractive index which in turn generates changes in plasmon.
Figure 15: UV-absorption spectra NPAu visible in citrate and NPAu-conjugated peptides.
The three conjugates NPAu-peptide (CLPFFD-NH2, CDLPFF-NH2, and CLPDFF-NH2) were more stable than NPAu. Also, the stability was evaluated at 4 °C and pH=7.4 observing that NPAu-CLPFFD-NH2 and NPAu-CLPDFF-NH2 were stable for 1 year but the conjugate NPAu -CDLPFF-NH2 aggregated.
In order to evaluate the stability of the conjugates NPAu-peptide UV-Vis spectra were obtained for different conditions. The colloidal stability of the NPAu and their conjugates were studied after rapid freezing and defreezing of the sample (Figure 16 A) and after lyophilization and reconstitution in water (Figure 16 B). The conjugates NPAu-CLPFFD-NH2 and NPAu-CLPDFF-NH2 were more stable than the conjugate- NPAu-CDLPFFNH2 and that NPAu citrate under the same conditions. The lower stability of NPAu-CDLPFFNH2 with respect to NPAu-CLPFFD-NH2 and NPAu-CLPDFF-NH2 could be attributed to an increased exposure of hydrophobic residues phenylalanine (F) and phenylalanine (F) to solvents. Therefore, after freezing, hydrophobic interactions of the residues in the conjugate NPAu -CDLPFF-NH2 lead to aggregation and subsequent colloidal flocculation.
Figure 16. Photographies of colloidal solutions of bare AuNP and conjugated AuNP after: (A) freezing and defreezing and reconstitution in water (AP: 0.09 ± 0.03 for AuNP-CLPFFD-NH2 and 0.06 ( 0.01 of AuNP-CLPDFF-NH2, 0.45 ± 0.06 for AuNP CDLPFF-NH2, and 5.37 ± 0.24 for bare AuNP). (B) After freezing and freeze-drying the colloidal solutions and reconstitution in water (AP: 0.10 ± 0.05 for AuNP-CLPFFD NH2 and 0.14 ± 0.16 for AuNP-CLPDFF-NH2, 0.35 (0.13, for AuNP-CDLPFF-NH2 and for bare AuNP the AP could not be determined because the sample was totally precipitated).
In the colloids there are several interactions that determine their stability. The combination of attractive forces of van der Waals forces and steric type are the basis of the theory of colloidal stabilization. Small particles tend to attract each other due to the attractive forces of van der Waals which may be neutralized through the addition of charge and/or molecules on the surface of the nanoparticle. In the first case, the stabilization occurs due to electrostatic repulsion, while in the second case, the stabilization occurs due to the repulsive steric forces. Based on the above identified two parameters related to the charge of nanoparticles, the zeta potential and migration in gel electrophoresis were determined an on the other hand Circular Dichroism (CD) spectra were taken to obtain information related to the peptide structure on the nanoparticle surface. In addition we determined the number of peptide molecules per nanoparticle by UV-Vis and analysis of aminoacids. In addition we determined the affinity of the three conjugates by ¿A fibrils.
In agreement with our experiments DC we proposed a hypothetical model (Figure 17) for the structure that the three different isomers. Are located on the nanoparticle surface. In CLPFFD-NH2 peptide is placed orthogonally on the surface of the NPAu, allowing accommodation of a greater number of peptide molecules in relation to isomer CLPDFF-NH2. CLPFFD-NH2, present primary ampnhipaticity where D hydrophilic residue is away from the surface of the NPAu and the peptide tends to adopt an extended conformation in ß sheet secondary structure (Figure 17).
Figure 17: Representation hypothetical of the disposition of peptide molecules on the NPAu surface based on CD spectra. The hydrophobic groups are represented in blue and the hydrophilic groups in red. In the case of peptide CLPFFD-NH2 they are oriented orthogonally to the gold surface by adopting a ß secondary structure, whereas the peptide CLPDFF-NH2 acquires a disordered structure. The scale of the molecules does not keep the proportion with NPAu.
According to the hypothetical model proposed in Figure 17, a higher number of peptide molecules of CLPFFD on the surface are located producing a steric repulsion that increase stability of the colloid. In contrast, the peptide CLPDFF-NH2 adopts a more disordered structure decreasing the interaction of molecules and reducing the degree of functionalization and decreasing the colloidal stability and affinity for the fibrils Aß.
The results obtained provide information on how the bioactive peptides might interact, with the surface of the NPAu which have influence on the stability and with the interaction with the biological target.
Biodistribution studies of the conjugate-CLPFFD NPAu-NH2 for improved outreach to the brain After we demonstrate that the conjugate NPAu-CLPFFD is stable in physiological conditions and that them could be used to destroy ¿A fibers and inhibit the amiloidogenic process we focused on the study of the accumulation of the conjugate NPAu-CLPFFD in the brain al also in different rat organs. The accumulation of them in the brain is crucial for therapeutic uses.
The dependence between the size of NPAu and biodistribution in different organs has been previously reported by various authors. NPAu between 10 and 15 nm have a widespread distribution in various organs, but the concentration varies in each organ, eg, in liver and spleen concentration is higher than in lungs, kidneys, heart and brain. Previously we showed that after a subronic intraperitoneal administration of NPAu of 12 nm in diameter them were accumulated in different organs including the brain depending on the administered doses.
One factor that prevents the release of NPAu to the brain is the retention of them by the reticule endothelial system (RES). This system capture NPAu, preventing it from reaching the desired organ. In plasma, the NPAu can be coated by proteins that promote the opsonization process which the capping of the particle with plasma proteins forming the called ¿Protein Corona¿. The opsonisn could be recognized by receptors on phagocytes and consequently be retained by the RES. A decrease in negative charge of the particle would help to reduce this process.
There are several strategies to favor NP permeability through blood brain barrier (BBB) to the brain, as for example, conjugation with amphipathic molecules that enhance transport across the BBB, or reduction of the negative charge of NP by functionalization with biocompatible molecules such as polyethylene glycol reducing the retention indirectly by RES.
It is well established that LPFFD cross the BBB in this sense we determined whether this peptide could act as a shuttle for NPAu 12 nm to cross the BBB. NPAu conjugation with peptide CLPFFD-NH2 reduces the negative charge of the colloid by the replacement of citrate molecules present on the surface of the NPAu increasing the colloidal stability as was mentioned above (Figure 18).
Figure 18: Effect of conjugation with the peptide NPAu-CLPFFD-NH2 on the charge and stability. NPAu citrate prior to conjugation is in the left and the effect of the replacement of citrate by peptide molecules after conjugation is represented in the right. The sulfur atoms, oxygen and carbon are represented in yellow, red and blue, respectively.
NPAu of 12 nm conjugated with the peptide CLPFFD-NH2 increase the in vivo to the rat brain. CLPFFD-NH2.The peptide could be actuating as a shuttle to cross BBB by recognizing specific receptors. Furthermore, the conjugation of NPAu with CLPFFDreduces the negative charge which may in turn reduce the particle retention by the SRE in a manner to increase the bioabailavility to the CNS.
The conjugation of the amphipathic peptide CLPFFD to the NPAu helps to increase their penetration into the brain, possibly by transcytosis mediated by the recognizing of receptors such as RAGE that play an important role in the influx of ¿A to the brain, or by the recognizing of the lipoprotein receptor (LDL) in endothelial cells. The increased penetration also could be due to an increase in the lipophilic character of the particles functionalized and/or a reduction in the retention of particles by the RES increasing the bioavailability of the particles to the central nervous system.
