Patent Application
20180036432
The present invention is in the area of biodevices and diagnostics. The invention generally relates to second harmonic generation (SHG) microscopy for disease diagnosis and envisioning of tissue engineering scaffolds.
The following discussion of the background to the invention is intended to facilitate an understanding of the present invention. However, it should be appreciated that the discussion is not an acknowledgment or admission that any of the material referred to was published, known or part of the common general knowledge in any jurisdiction as at the priority date of the application.
Second harmonic generation (SHG) microscopy is a powerful imaging tool for disease diagnosis and envision of tissue engineering scaffolds. Second harmonic (SH) probes have been developed for in-vivo imaging in recent years as they possess many advantages over the classic fluorescent probes. SH probes are typically made of inorganic nanomaterials, such as barium titanate nanocrystals. Although these materials have achieved certain success in molecular imaging, they are concerned for their health and environmental issues.
In addition, second-harmonic generation (SHG) microscopy is a nonlinear optical (NLO) imaging technique that has emerged in recent years to visualize structures or functions of cells, tissues and organisms [1]. It has the advantages of being label-free, inherent three-dimensional (3D) resolution, near-infrared (NIR) excitation for superior optical penetration, lower photo-damage, and capable of providing quantitative information, which makes it an attractive tool for high resolution imaging [1-3]. It has been used to diagnose a wide range of diseases including cancer, liver fibrosis and skin damage [4]. It has also been used in tissue engineering and regenerative medicine [5] to envision three-dimensional biomaterial scaffolds and extracellular matrices that secreted by cells [6-7].
SHG is an optical frequency doubling process: when a non-linear optical (NLO) material interacts with light, two NIR incident photons can be converted into one emerging visible photon at exactly twice the energy (or half the wavelength) [1,4,9]. This NLO material requires a noncentrosymmetric structure. Examples include potassium niobate (KNbO3), lithium niobate (LiNbO3), barium titanate (BaTiO3), potassium titanyl phosphate (KTiOPO4, KTP), and zinc oxide (ZnO), Gallium nitride (GaN), Silicon carbide (SiC), noble metal particles, and quantum dots [8,9].
Inorganic nanomaterials, such as BaTiO3 nanocrystal has been used a second harmonic (SH) probe for in vivo imaging [13]. These SHG nanoprobes are superior to the existing fluorescent dyes in terms of dye bleaching, signal saturation and narrow emission spectrum (full width at half maximum (FWHM)<5 nm) [13, 14]. Although a report showed that BaTiO3 nanocrystals are non-toxic to zebrafish [13], inorganic nanomaterials have been questioned for their health and environmental issues which enormously limit their clinical applications [15-16].
The present invention provides a peptide based biomaterial for visualization by SHG microscopy. The present invention comprises the following features:
F1) Use of short peptides as a non-linear optical (NLO) material for second harmonic generation (SHG) microscopy.
F2) A preferred peptide of F1 is LIVAGK (L6), which contains a non-polar aliphatic tail (with decreasing hydrophobicity) and a polar head; and can self-assemble into hydrogels;
A. Wherein the peptide forms a tunable fibrous structure
B. For in vitro and in vivo imaging applications
C. In disease diagnostics such as amyloidosis, including 1) neuro-degenerative amyloidosis, e.g. Alzheimer's (AD), Parkinson's, Huntington's (PD), 2) non-neuropathic localized amyloidosis such as in Type II Diabetes, and 3) systemic amyloidosis that occurs in multiple tissues, e.g. cataracts and lattice corneal dystrophy (LCD).
In accordance with an aspect of the invention, the invention provides the use of a peptide and/or peptidomimetic, capable of self-assembling and forming a hydrogel, having the general formula I:
Za—(X)b—(Y)c—Z′d (I)
Preferably, said aliphatic amino acids and aliphatic amino acid derivatives, and said polar amino acids and polar amino acid derivatives are either D-amino acids or L-amino acids.
Preferably, said aliphatic amino acids are selected from the group consisting of alanine (Ala, A), homoallylglycine, homopropargylglycine, isoleucine (Ile, I), norleucine, leucine (Leu, L), valine (Val, V) and glycine (Gly, G), preferably from the group consisting of alanine (Ala, A), isoleucine (Ile, I), leucine (Leu, L), valine (Val, V) and glycine (Gly, G).
Preferably, all or a portion of said aliphatic amino acids are arranged in an order of decreasing amino acid size in the direction from N- to C-terminus, wherein the size of the aliphatic amino acids is defined as I=L>V>A>G.
Preferably, said aliphatic amino acids have a sequence selected from
Preferably, b is an integer from 2 to 7 or 2 to 6.
Preferably, said polar amino acids are selected from the group consisting of aspartic acid (Asp, D), asparagine (Asn, N), glutamic acid (Glu, E), glutamine (Gln, Q), 5-N-ethyl-glutamine (theanine), citrulline, thio-citrulline, cysteine (Cys, C), homocysteine, methionine (Met, M), ethionine, selenomethionine, telluromethionine, threonine (Thr, T), allothreonine, serine (Ser, S), homoserine, arginine (Arg, R), homoarginine, ornithine (Orn), lysine (Lys, K), N(6)-carboxymethyllysine, histidine (His, H), 2,4-diaminobutyric acid (Dab), 2,3-diaminopropionic acid (Dap), and N(6)-carboxymethyllysine,
wherein said polar amino acid is preferably selected from the group consisting of aspartic acid, asparagine, glutamic acid, glutamine, serine, threonine, methionine, lysine, ornithine (Orn), 2,4-diaminobutyric acid (Dab), and 2,3-diaminopropionic acid (Dap).
Preferably, c is 2 and said polar amino acids are identical amino acids, or wherein c is 1 and said polar amino acid comprises any one of aspartic acid, asparagine, glutamic acid, glutamine, serine, threonine, cysteine, methionine, lysine, ornithine, 2,4-diaminobutyric acid (Dab) and histidine, preferably lysine, ornithine, 2,4-diaminobutyric acid (Dab) and 2,3-diaminopropionic acid (Dap).
Preferably, (Y)b has a sequence selected from Asp, Asn, Glu, Gln, Ser, Thr, Cys, Met, Lys, Orn, Dab, His, Asn-Asn, Asp-Asp, Glu-Glu, Gin-Gin, Asn-Gln, Gln-Asn, Asp-Gln, Gln-Asp, Asn-Glu, Glu-Asn, Asp-Glu, Glu-Asp, Gin-Glu, Glu-Gin, Asp-Asn, Asn-Asp Thr-Thr, Ser-Ser, Thr-Ser, Ser-Thr, Asp-Ser, Ser-Asp, Ser-Asn, Asn-Ser, Gln-Ser, Ser-Gln, Glu-Ser, Ser-Glu, Asp-Thr, Thr-Asp, Thr-Asn, Asn-Thr, Gln-Thr, Thr-Gln, Glu-Thr, Thr-Glu, Cys-Asp, Cys-Lys, Cys-Ser, Cys-Thr, Cys-Orn, Cys-Dab, Cys-Dap, Lys-Lys, Lys-Ser, Lys-Thr, Lys-Orn, Lys-Dab, Lys-Dap, Ser-Lys, Ser-Orn, Ser-Dab, Ser-Dap, Orn-Lys, Orn-Orn, Orn-Ser, Orn-Thr, Orn-Dab, Orn-Dap, Dab-Lys, Dab-Ser, Dab-Thr, Dab-Orn, Dab-Dab, Dab-Dap, Dap-Lys, Dap-Ser, Dap-Thr, Dap-Orn, Dap-Dab, Dap-Dap.
Preferably, (X)a-(Y)b has a sequence selected from the group consisting of
In accordance with an aspect of the invention, the invention provides the use of a peptide having the general formula II:
Za-M-Z′d (II)
wherein
The peptides with amino acid sequences selected from SEQ ID Nos. 99 to 102 are four natural amyloidogenic core sequences (KED, GA6, DF5, NL6).
See below for further details, e.g. Table 1.
In both general formulas I and II:
Preferably, a is 1 and said N-terminal protecting group Z has the general formula —C(O)—R, wherein R is selected from the group consisting of H, unsubstituted or substituted alkyls, and unsubstituted or substituted aryls, wherein. R is preferably selected from the group consisting of methyl, ethyl, propyl, isopropyl, butyl and isobutyl.
Preferably, said N-terminal protecting group Z is an acetyl group.
Preferably, said N-terminal protecting group Z is a peptidomimetic molecule, including natural and synthetic amino acid derivatives, wherein the N-terminus of said peptidomimetic molecule may be modified with a functional group selected from the group consisting of carboxylic acid, amide, alcohol, aldehyde, amine, imine, nitrile, an urea analog, phosphate, carbonate, sulfate, nitrate, maleimide, vinyl sulfone, azide, alkyne, alkene, carbohydrate, imide, peroxide, ester, aryl, ketone, sulphite, nitrite, phosphonate, and silane.
Preferably, said C-terminal protecting group Z′ is an amide group, wherein, preferably, the C-terminus has the formula —CONHR or —CONRR′, with R and R′ being selected from the group consisting of H, unsubstituted or substituted alkyls, and unsubstituted or substituted aryls.
Preferably, said C-terminal protecting group Z′ is an ester group, wherein, preferably, the C-terminus has the formula —CO2R, with R being selected from the group consisting of H, unsubstituted or substituted alkyls, and unsubstituted or substituted aryls.
Preferably, said C-terminal group Z′ is a peptidomimetic molecule, including natural and synthetic amino acid derivatives, wherein the C-terminus of said peptidomimetic molecule may be modified with a functional group selected from the group consisting of carboxylic acid, amide, alcohol, aldehyde, amine, imine, nitrile, an urea analog, phosphate, carbonate, sulfate, nitrate, maleimide, vinyl sulfone, azide, alkyne, alkene, carbohydrate, imide, peroxide, ester, aryl, ketone, sulphite, nitrite, phosphonate, and silane.
Preferably, b+c is at least 2, preferably 2 to 9, more preferably 3 to 7 or 2 to 7.
Preferably, the peptide and/or peptidomimetic is visualized under a second harmonic generation (SHG) microscopy or by SHG imaging without additional dye(s) and/or label(s).
Preferably, the peptide and/or peptidomimetic comprises further compound(s), such as
Preferably, the at least one peptide and/or peptidomimetic font's a fibrous structure, which is preferably tunable, such as
Preferably, the fibrous structure is a fibrous scaffold resembling the collagen fiber in extracellular matrix.
Preferably, the at least one peptide and/or peptidomimetic as defined herein is stable in aqueous solution at physiological conditions at ambient temperature for a period of time in the range from 1 day to at least 6 months, preferably to at least 8 months more preferably to at least 12 months.
Preferably the at least one peptide and/or peptidomimetic as defined herein is stable in aqueous solution at physiological conditions, at a temperature up to 90° C., for at least 1 hour.
Preferably, the peptide and/or peptidomimetic is present at a concentration in the range of from 0.1% to 30% (w/w), preferably 0.1% to 20% (w/w), more preferably 0.1% to 10% (w/w), more preferably 0.1% to 5% (w/w), even more preferably 0.1% to 3% (w/w), with respect to the total weight of said hydrogel or fibrous structure.
In accordance with another aspect of the invention there comprises a method of preparing a hydrogel or fibrous structure, the method comprising dissolving at least one peptide and/or peptidomimetic—as defined herein with general formula I—of the first aspect of the invention in an aqueous solution.
Preferably, the dissolved peptide and/or peptidomimetic in aqueous solution is further exposed to temperature, wherein the temperature is in the range from 20° C. to 90° C., preferably from 20° C. to 70° C.
Preferably, the at least one peptide and/or peptidomimetic is dissolved at a concentration from 0.01 μg/ml to 100 mg/ml, preferably at a concentration from 1 mg/ml to 50 mg/ml, more preferably at a concentration from about 1 mg/ml to about 20 mg/ml.
In accordance with another aspect of the invention, a method of preparing peptide particles is provided, the method comprising dissolving at least one peptide and/or peptidomimetic as defined herein with general formula I in an aqueous solution, preferably phosphate buffered saline (PBS), and solvent evaporation from said aqueous solution, such as by vacuum drying.
In another aspect of the invention, the invention provides a use according to the present invention or the use of a hydrogel, fibrous structure or peptide particle obtained by a method according to the present invention, as probes for (bio-)imaging, preferably as exogenous SHG probes.
In another aspect of the invention, the invention provides a use according to the present invention or the use of a hydrogel, fibrous structure or peptide particle obtained by a method according to the present invention, for monitoring cells, in vitro and/or in vivo (bio-)imaging.
In another aspect of the invention, there comprises a use according to the present invention or the use of a hydrogel, fibrous structure or peptide particle obtained by a method according to the present invention for visualizing peptide-based tissue engineering scaffolds, preferably comprising studying biological processes, such as cell adhesion, migration and differentiation.
In accordance with another aspect of the invention, the invention provides the use of a hydrogel, fibrous structure or peptide particle obtained by a method according the present invention as diagnostics, preferably for the diagnosis of diseases comprising or associated with the aggregation of peptide or protein structures, such as amyloidosis, including 1) neuro-degenerative amyloidosis, e.g. Alzheimer's (AD), Parkinson's, Huntington's (PD), 2) non-neuropathic localized amyloidosis such as in Type II Diabetes, and 3) systemic amyloidosis that occurs in multiple tissues, e.g. cataracts and lattice corneal dystrophy (LCD).
In another aspect of the invention, the invention provides the use of a hydrogel, fibrous structure or peptide particle obtained by a method according to the present invention as drug or gene delivery vehicle.
In another aspect of the invention, the invention provides a peptide particle comprising the peptide and/or peptidomimetic as defined herein, preferably obtained by a method of the present invention (according to claim 28).
Preferably, the peptide particle has a particle size in the range from submicron to 10 microns.
In accordance with another aspect of the invention, the invention provides a drug or gene delivery vehicle comprising the peptide and/or peptidomimetic as defined herein, and a drug or gene to be delivered.
In accordance with another aspect of the invention, the invention provides a peptide-based tissue engineering scaffold comprising the peptide and/or peptidomimetic as defined herein, preferably obtained by a method according to the present invention.
In accordance with another aspect of the invention, the invention provides a biosensor or bioimaging device comprising the peptide and/or peptidomimetic as defined herein.
In accordance with another aspect of the invention, the invention provides a wound dressing comprising the peptide and/or peptidomimetic as defined herein.
Other aspects and features of the present invention will become apparent to those skilled in the art upon review of the following description of specific embodiments of the invention in conjunction with the accompanying figures.
Embodiments of the invention will now be described by way of example with reference to the accompanying drawings.
(a) Comparison of the SHG signal intensity from endogenous collagen I (inset, in vivo SHG image of the normal capsular region of rat liver, left) to LK6 peptide particles (inset, SHG image of LK6 peptide particles, right). Instrument settings for both SHG imaging were the same. Five spots from each image were chosen and the SHG signal intensity was normalized to collagen I (mean±standard deviation, n=5); SEM images of collagen fibers (b) and peptide particles (c).
(a) SHG signal spectrum of LK6 peptide. Signal was ranging from 380 to 550 nm with excitation wavelength of 810 nm and peak emission wavelength was shown at 405 nm.
(b) Spectral peaks at various excitation wavelengths spanning a broad spectral region (810-890 nm).
(c) Emission λ-scan of the SHG signal (λex=850 nm) acquired from SHG imaging of LK6 peptide particles. The solid spheres represent back scattering SHG data and the solid line represents a Gaussian fit. The full width at half-maximum of the fitted curve bears a 1/√{square root over (2)} relation to the spectral profile of the corresponding beam.
(d) Log-log plot of the above SHG signal measurements demonstrating a log [I425]=0.45+2.01×log [I850] dependence, quadratic to a good approximation, consistent with nonlinear second order optical upconversion.
Upper panel: HeLa cells exposed to biotin-conjugated LK6 peptide containing medium. Peptides in HeLa cells displayed pseudo red color (SHG) and green color (fluorescence). Lower panel: HeLa cells exposed to normal medium without peptide (control). Scale bars: 50 μm.
(a) Schematic illustration of experiment arrangement: a low intensity ultrafast infrared laser penetrating glass cover slip and rat fat tissue (thickness˜50 μm) and viewing LK6 peptide particles sandwiched in between the glass slide and the glass cover slip.
(b) Three dimensional image of LK6 peptide particles in the entire volume of (225×225×4.5 μm3). SHG z-stack was generated with 0.5 μm step. Total nine images were taken (z=4.5 μm).
(a) Sample from a normal person (control; (b)) and a patient who has Lattice corneal dystrophy (LCD) disease. Collagen fibrils can be seen under SHG (green). Arrow indicates the amyloid deposit. Scale bar=20 μm.
The whole window was covered with pristine peptides. However, only part of the peptides can be visualized under SHG microscopy. The SHG intensity follows the order of KE7>GA6>DF5>NL6. Scale bar=50 μm.
(a) Cell viability at 48 h of HDF and HeLa cells incubated with cell culture media containing LK6 peptide solutions, as determined by the MTS assay (mean±standard deviation, n=9). Concentrations ranged from 0.1 to 1000 μg/mL.
(b) Cytotoxicity determined by Calcein AM/EthD-1 (live/dead, green/red) staining method after 48 h treated with 1 and 1000 μg/mL LK6 peptide solutions using non-treated cells as control.
Scale bars: 100 μm.
Other arrangements of the invention are possible and, consequently, the accompanying drawings are not to be understood as superseding the generality of the preceding description of the invention.
Peptide/protein based biomaterials can also be assembled into large, ordered noncentrosymmetric structures, which make them possible to produce second harmonic signals [1]. Famous example include collagen I [10], diphenylalanine (FF, a dimer peptide) [11], elastin and muscle myosin [12]. Among them, collagen I has been commonly used as an endogenous SHG probe for disease diagnosis [4].
Peptide/protein based biomaterials appear to be a promising candidate for a SH probe due to their biocompatibility and biodegradability. A peptide-based biomaterial, i.e. a ultrashort peptide biomaterial is presented that can be directly visualized by second harmonic generation microscopy. It shows excitation wavelength tenability, similar second harmonic signal intensity as endogeneous collagen I, and cytocompatibility with two human cell lines.
Based on the above, the inventors concluded that the purely synthetic, peptide-based material holds the potential to be used as a future SH probe for bioimaging applications.
In particular the Supramolecular assembly of collagen in tissues has been visualized by SHG microscopy for more than a decade. It was discovered by the inventors that striking similarity exists between ultrashort peptides and collagen fibers. Inspired by such findings, the non-linear optical (NLO) properties of a hexemer peptide, Ac-LIVAGK-NH2 (LK6) was investigated.
A hexamer peptide Ac-LIVAGK-NH2 (LK6) was designed and synthesized. This hexamer peptide can self-assemble into hydrogels through hydrophobic interactions, ionic interactions, hydrogen bonding and van de Waals forces. It contains a non-polar aliphatic tail and a polar head. The non-polar aliphatic tail was designed to have a decreasing hydrophobicity. This type of arrangement favours a parallel-antiparallel stacking. The nanostructured peptide aggregates then form a fibrous scaffold, which resembles the collagen fiber in extracellular matrix [17-18]. In this work, the ultrashort peptide is submitted as a novel NLO material for SHG imaging. Results suggest this purely synthetic, peptide-based material holds the potential to be used as a future SH probe for bioimaging applications.
Supramolecular assembly of collagen in tissues has been visualized by SHG microscopy for more than a decade [4]. In particular, collagen I (in blood vessels, bone, cornea, kidney, liver, lung, ovary, skin and tendon), and collagen II (in cartilage) have been found to efficiently produce SHG [4]. Since diseased and normal tissues show different distribution patterns of collagen fiber alignment and intensity, their SHG images can be used for disease diagnosis.
Success has been achieved in many clinical applications, for example cancer delineation and liver fibrosis [4]. Collagen is a triple-helix molecule and can self-assemble into fibers with diameters up to a few micrometres. A recent study [19] showed that the origin of SHG signals from collagen fibers possibly lies in their peptide bonds. The building blocks of collagen fiber were mimicked by tri-amino acid peptides PPG and GGG (P and G are the one letter code for Proline and Glycine respectively) and a molecular-level property of the nonlinearity, i.e., the first hyperpolarizability, β, was measured by Hyper Rayleigh Scattering (HRS). The first hyperpolarizability of these trimers was about 0.087×10-30 esu (esu is the unit of the first hyperpolarizability) [19]. However, the first hyperpolarizability of collagen I was found to be (1250±20)×10-30 esu [19], which could be viewed as ten thousand trimers combining together.
Previous studies from our lab showed striking similarity between the ultrashort peptides and collagen fibers [20]. Inspired by these findings, we investigated the NLO properties of our hexamer peptide, LK6. Based on the non-linearity of the intrinsic peptide bonds, we hypotheses that our hexamer peptide, like collagen (which could be viewed as GX1X2, with X1 and X2 corresponding mainly to proline and hydroxyproline), should be able to generate SHG signals.
Peptide particles were prepared by solvent evaporation. Phosphate buffered saline (PBS) was chosen as the solvent. After vacuum drying, the various sizes and shapes of peptide particles were formed (FE-SEM image,
To test our hypothesis, we directly examined LK6 peptide particles using SHG imaging. The SHG signal from LK6 peptide particles was strong and spectrally well defined (
SHG image (green) of LK6 peptide particles was shown in
Biological samples, such as cells and tissues, are known to have auto-fluorescence. With the excitation wavelength tunability, we can avoid auto-fluorescence. In addition, we can further increase penetration depth in tissues by simply increasing excitation wavelength.
log [I425]=0.45+2.01*log [I850]
This confirms the two-photon nature of the emission from LK6 ultrashort peptide particles.
As mentioned previously, collagen fibres produce much stronger SHG signals compared to their building blocks (ten thousand times higher). It is interesting to compare our hexamers to endogenous collagen in terms of SHG signal intensity. In this case, we chose the normal capsular region of rat liver. From in vivo imaging, we observed bright SHG signals from its collagen (
In order to use LK6 for bioimaging applications, such as monitoring cells and in vivo imaging, we assessed the cytotoxicity of LK6 by the MTS test. The MTS test was applied to two human cell lines, HeLa and primary human dermal fibroblasts (HDF). The toxicity of LK6 was measured at 0.1, 1, 10, 100 and 1000 μg/mL. Cells were exposed to cell culture media containing LK6 solution for 48 h. Cell viability is reported in FIG. 7a. Cell viability at various LK6 concentrations was either equivalent to or even higher than that of untreated cells over the whole concentration range which was tested in this study (0.1 to 1000 μg/mL). To further confirm these results, we stained cells with and without peptide treatment with solutions containing a mixture of calcein AM and ethidium homodimer-1 (EthD-1). Calcein AM can penetrate cell membrane and be converted to cell membrane impermeable green fluorescent calcein, while red fluorescent EthD-1 is excluded by the intact cell membrane of live cells.
Biological Application
The ability to manufacture SH probes entirely out of LK6 peptides offers additional opportunities as they are biocompatible, biodegradable. A possible application for all-protein/peptide spectra elements such as LK6 is to use them within biological tissues to provide exogenous spectral signatures. These structures can be visualized as all-peptide based contract agents that do not require dyes or chemicals. This is validated by performing an ex vivo experiment in which LK6 peptides are placed under slices of rat fat tissues (
As our ultrashort peptides show striking similar to collagen (an extracellular matrix protein, SHG-active molecule and fibrous structures), we can study the interaction between cells and these ultrashort peptides using SHG imaging. Many fundamental biological processes including cell adhesion, migration and differentiation on these ultrashort peptides made matrices can be directly visualized by SHG. The three-dimensional architecture of the tissue engineering scaffolds made of these ultrashort peptides can be viewed in situ and without labelling. The degradation of these scaffolds can be monitored and traced. It also opens the possibility to study the interaction between scaffolds and seeded cells in vitro prior implantation, as well as to monitor the interplay between the scaffolds with the surrounding native tissue in vivo.
In conclusion, we demonstrated that our hexamer peptide, LK6 shows strong SHG signals, similar to those of endogenous normal capsule collagen of rat liver. This ultrashort peptide is biocompatible and biodegradable, which makes it attractive as a future candidate of SH probe for bioimaging.
Abstract
Amyloid-like peptides are an ideal model system to study the mechanisms of amyloidosis which may lead to many human diseases, such as Alzheimer's. Recently, amyloid-like peptides were also used as a new type of biomaterials as tissue engineering scaffolds and drug delivery vehicles. Here we report that these amyloid-like peptides show strong second harmonic generation (SHG) effect, signals equivalent to or even higher than those of endogenous collagen fibers. Several amyloid-like peptides (both synthetic and natural) were examined under SHG microscopy and shown they are SHG-active. These peptides can also be viewed under thick fat tissues (ex vivo) and inside cells (in vitro). This interesting property makes these amyloid-like peptides second harmonic probes for bioimaging applications. Furthermore, SHG microscopy provides us a simple and label-free approach to detect amyloidosis. Lattice corneal dystrophy (LCD) was chosen as a model disease of amyloidosis. Both normal and diseased human corneal biopsy samples were examined under SHG microscopy. Morphological difference between these two can be easily recognized without adequate medical knowledge. Therefore, SHG can be a useful tool for disease diagnosis.
Amyloidosis is responsible for more than fifty human diseases, including Alzheimer's disease (AD), Parkinson's disease (PD), type II diabetes, cataracts and lattice corneal dystrophy (LCD). Inhibition of amyloidosis is a promising strategy for the development of therapeutic agents to treat such diseases. Better understanding of amyloidosis mechanisms may avoid using a trial and error method in searching for amyloid inhibitors, eventually designing or even predicting molecules with inhibitory effects to amyloids. However, the mechanism of amyloidosis is still largely unclear. One logical mechanistic approach is to study how these amyloid-like peptides/proteins fold or misfold [29]. Ultrashort peptides with only three to seven amino acids provide us an ideal model system for mechanistic studies. For example, GGVVIA (GA6) and KLVFFAE (KE7), can self-assemble into amyloid-like aggregates [30, 31]. These two peptides are also known as the core sequence of amyloid-β (Aβ). Aβ is the major component in the amyloid aggregates responsible for AD. Besides ultrashort peptides that can be found in natural peptides/proteins, several designed synthetic ultrashort peptides, such as IVD (ID3) and LIVAGD (LD6), have shown similar behavior [31, 17]. Both these natural and synthetic peptides are amyloid-like peptides. They all form cross-β peptide structure at molecular level. Morphologically they are usually micrometers in length but only 7-10 nm in diameter [32]. Mechanically they are rigid, with strength comparable to steel [33]. Ultrashort amyloid-like peptides can be not only useful for searching amyloid inhibitors [17], but also as a new type of biomaterials with desirable properties such as biocompatibility and biological activities [31, 34].
Here we report that ultrashort amyloid-like peptides exhibit strong SHG effect. SHG is a nonlinear optical (NLO) effect, first demonstrated in 1961 [35]. Recently, microscopy based on SHG effect has emerged as a powerful bioimaging technique [4]. It provides many significant advantages over the conventional fluorescence imaging techniques for its deeper optical penetration, lower photo-damage and longer observation time [4, 1]. Several amyloid-like peptides (both synthetic and natural), examined under SHG microscopy, revealed they are SHG-active. This interesting property makes these amyloid-like peptides second harmonic probes for bioimaging applications. Furthermore, SHG microscopy provides a simple and label-free approach to detect amyloid-like peptides, which can be a useful tool for disease diagnosis, especially for LCD.
For further details, see Example.
Key Features of the Invention
1. Materials and Methods
1.1 SHG Imaging:
We acquired SHG images using a commercial laser scanning microscopic imaging system (Zeiss LSM 510 META, Jena, Germany) coupled to a mode-locked femtosecond Ti: sapphire laser (Mai-Tai broadband, Spectra-Physics), tunable from 710 nm to 990 mm. To achieve spectral analysis and detect the SHG signal, we used the META detector with 32-gated photon counting module.
1.2 Ultrashort Amyloid-Like Peptides:
We purchased peptides from the American Peptide Company (purity≧95%). The peptide sequences were confirmed by liquid chromatography-mass spectrometry (LC-MS). Net peptide content varied between 70% and 85%. All peptides were acetylated at the N terminus. Peptide handling and hydrogel preparation were done as reported previously [17].
1.3 Peptide Particles:
We prepared peptide particles by hydrodynamic focusing. Details can be found in elsewhere [21]. The particle size was determined by scanning electron microscopy, showing a grain size of around 5 μm (
1.4 Cell Culture:
We cultured both HeLa and human dermal fibroblasts in DMEM supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (Life technologies, Singapore).
1.5 Human Corneal Biopsy Samples:
Singapore General Hospital provided biopsy samples. The normal tissue was from a seventy-eight year-old Caucasian male. The diseased tissue was from a sixty-year-old female who has lattice corneal dystrophy (LCD).
2. Results and Discussion
2.1 A Hexamer Peptide is Collagen-Like and SHG-Active.
To test whether the amyloid-like peptides of the present invention are SHG-active, we first investigated a hexamer peptide, Ac-LIVAGK-NH2, (LK6). Peptide particles deposited on a glass microscope slide were examined under an SHG imaging system. Instrument details can be found in our previous work [3]. During the whole experiments, only the backscattered geometry was employed because it is the only suitable configuration for in vivo imaging [1]. We chose collagen I as a control. For more than a decade it has been shown that SHG microscopy can visualize supramolecular assembly of collagen in tissues [1]. Both collagen I and peptide particles showed SHG signals with comparable intensity (
We further characterized LK6 peptide. It exhibits typical SHG characteristics (
log [I125]=0.45+2.01×log [I850] (1)
The intensity of SHG signal is proportional to the square of the incident laser intensity. This confirmed the two-photon nature of the emission from LK6 peptide.
2.2 Comparison of Synthetic and Natural Amyloid-Like Peptides Under SHG Microscopy
Next, we examined several other amyloid-like peptides, including both natural amyloidogenic core sequences (NL6, DF5, GA6, and KED) and designed synthetic peptides (LD6, IS6 and IK3). The peptide sequences are listed in Table 1.
a)Value = Average ± standard deviation;
b)N.D. = not detected.
All these peptides are SHG-active. We used a powder technique developed by Kurtz and Perry [22] to evaluate the SHG efficiency of these second-order nonlinear optical materials. Sucrose, having a relatively modest nonlinearity, was chosen as a standard to evaluate second harmonic generation efficiencies [23]. In Table 1, all amyloid-like peptides that have been tested showed higher or equivalent SHG efficiency compared to sucrose. The efficiency of LD6 and IK3 were even three times higher than that of sucrose. These may due to their molecular structures. LD6 is known to form hydrogel at much lower concentrations compared to LK6. The trimer IK3 has larger dipole moment compared to LK6. Human collagen subtypes I to V were examined (also listed in Table 1). Only type I and II showed SHG activity, III, IV and V did not. This agrees with the previous findings [4, 24]. In Table 1, all the tested amyloid-like peptides were SHG-active with some showing even higher SHG efficiency than collagen subtypes. This provides a broad spectrum of materials as second harmonic probes.
We have previously showed that NL6, DF5, GA6, and KE7 as well as LD6, IS6, LK6 and IK3 self-assembled into hydrogels at 12 mM [18]. However, in this study, the gelation speed does not correlate to that of SHG efficiency (data not shown). We chose three rational designed hexamers, LD6 (negatively charged), IS6 (neutral) and LK6 (positively charged) to investigate the effect of the charged head groups. The SHG efficiency of these three hexamers followed the order of LD6>IS6>LK6.
We also investigate the effect of the peptide chain length by choosing IK3, a trimer, compared to the other three hexamers. It followed the order of IK3 (trimer)>LD6 (hexamer). Both findings are helpful for designing a superior amyloid-like peptide.
We then investigated the SHG efficiency of four natural amyloidogenic core sequences. They followed the order of KE7>GA6>DF5>NL6 (Table 1 and
We reason that the origin of these amyloid-like peptides' SHG activity comes from their nanostructures. A recent study showed that the origin of SHG signals from collagen fibers possibly lies in their peptide bonds [19]. The collagen fiber building blocks were mimicked by tri-amino acid peptides PPG and GGG (P and G are the one letter code for Proline and Glycine respectively).
In addition, a molecular-level property of the nonlinearity, i.e., the first hyperpolarizability, β, was measured by Hyper Rayleigh Scattering (FIRS). The first hyperpolarizability of these trimers was about 0.087×10−30 esu (esu is the unit of the first hyperpolarizability) [19]. However, the first hyperpolarizability of collagen I was found to be (1250±20)×10−30 esu [19], which could be viewed as ten thousand trimers combining together. Based on the non-linearity of the intrinsic peptide bonds and their aggregation capability, these amyloid-like peptides can generate SHG signals, just like collagen.
2.3 Cytocompatibility of a Hexamer Peptide
In order to use LK6 for bioimaging applications, such as in vitro monitoring cells and in vivo imaging the whole animal, we assessed the cytotoxicity of LK6 by the MTS test and live/dead assay with two human cell lines, human epithelial carcinoma cells (HeLa) and primary human dermal fibroblasts (HDF). Cell viability and the representative live/dead images of cells in response to LK6 solutions was reported in
2.4 A Hexamer Peptide as a Second Harmonic Probe for In Vitro Cell Imaging
A possible application for LK6 is cell imaging. To this end, HeLa cells were incubated with biotin-conjugated LK6 for 4 h and then fixed for immunostaining. LK6 peptides were visualized by both SHG and confocal fluorescence microscopes. In
2.5 A Hexamer Peptide as a Second Harmonic Probe for Ex Vivo Deep Imaging
Another possible application for LK6 is to use them within biological tissues to provide exogenous SHG signatures. These structures can be envisioned without any dyes or chemicals, even in a highly scattering environment such as living tissue. This is validated by performing an ex vivo experiment in which LK6 peptide samples are placed under a slice of fat tissue (great omentum removed from a sacrificed rat, thickness˜50 μm). The results are shown in
2.6 Diseased and Normal Human Corneal Biopsy Samples Examined by SHG Microscopy
More interestingly, when we examined the human corneal biopsy samples (
2.7 Other Applications
In our laboratory, we have explored the biomedical applications of ultrashort amyloid-like peptides in many aspects. In one study, an anti-cancer drug, oxaliplatin, was tagged to our peptides by click chemistry [26]. Peptides were then injected into mice tumor sites. In vivo studies showed significant tumor growth inhibition. In another study, we used ultrashort peptides as a wound dressing. It heals the burn wounds much faster in a rat model [27]. Because our peptides are SHG-active, we can visualize these processes with SHG microscopy when we apply our peptides as drug delivery vehicles or wound dressings. Click chemistry enables adding therapeutic molecules to the ultrashort peptides. Thus, ultrashort peptides can also be used as theranostic agents that provide imaging and therapy at the same time. Recently, SHG microendoscopy has been developed [28]. Therefore, we could expect for more bioimaging applications by using amyloid-like peptides as a second harmonic probe.
We demonstrated for the first time that amyloid-like peptides are nonlinear optical materials showing strong SHG signals. Amyloid-like peptides can be viewed under thick fat tissues and inside cells which make them suitable as second harmonic probes. Amyloid-like peptide nanomaterials hold great potential in nanotechnology and nanomedicine. Their nonlinear optical properties hold promise for new bioimaging applications.
It is to be understood that the described embodiment(s) have been provided only by way of exemplification of this invention, and that further modifications and improvements thereto, as would be apparent to persons skilled in the relevant art, are deemed to fall within the broad scope and ambit of the present invention described herein.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10201403924Q | Jul 2014 | SG | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/SG2015/050204 | 7/8/2015 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 20170196993 A1 | Jul 2017 | US |
