Ultrashort peptides as exogenous second harmonic probes for bioimaging applications

Abstract

Various aspects of the present invention relate to a peptide based biomaterial for visualization by SHG microscopy. In particular the invention relates to the use of short peptides as a non-linear optical (NLO) material for second harmonic generation (SHG) microscopy. A preferred short peptide comprises LIVAGK (LK6) and contains a non-polar aliphatic tail (with decreasing hydrophobicity) and a polar head; and can self-assemble into hydrogels; wherein which the peptide forms a tunable fibrous structure for in vitro and in vivo imaging applications and is suitable 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), as well as drug delivery and/or wound dressings.

Claims

1. A second harmonic generation (SHG) microscopy method for imaging a sample, the method comprising: (a) contacting with a sample or a tissue a non-linear optical material that comprises peptides and/or peptidomimetics having the general Formula I:
Z.sub.a-(X).sub.b-(Y).sub.c-Z.sub.d (I) wherein Z is an N-terminal protecting group; a is 0 or 1; X is, at each occurrence, independently selected from the group consisting of aliphatic amino acids and aliphatic amino acid derivatives, and wherein the overall hydrophobicity decreases from N- to C-terminus; b is an integer selected from 1 to 7; Y is selected from the group consisting of polar amino acids and polar amino acid derivatives; c is 1 or 2; Z is a C-terminal protecting group; and d is 0 or 1, and b+c is at least 2; and (b) imaging the sample or tissue under a second harmonic generation (SHG) excitation wavelength to produce a SHG signature of the sample or tissue.

2. The method according to claim 1, wherein the non-linear optical material comprises a hydrogel, a fibrous structure, or a peptide particle wherein the hydrogel, or the fibrous structure is formed by dissolving the peptide or peptidomimetic in an aqueous solution or wherein the peptide particle is formed by evaporating the aqueous solution from the hydrogel, or the fibrous structure.

3. The method of claim 2, wherein the at least one peptide or peptidomimetic is dissolved in an aqueous solution at a concentration of 0.01 g/ml to 100 mg/ml.

4. The method of claim 2, further comprising exposing the peptide or peptidomimetic in aqueous solution to a temperature of 20 C. to 90 C.

5. The method of claim 2, wherein the aqueous buffer comprises phosphate buffered saline (PBS).

6. The method of claim 2, wherein the hydrogel, the fibrous structure or the peptide particle are used: (a) as probes for monitoring cells, in vitro and/or in vivo (bio-)imaging, as exogenous SHG probes or as drug or gene delivery vehicles; (b) for visualizing peptide-based tissue engineering scaffolds, comprising studying biological processes; or (c) as a diagnostic, for the diagnosis of diseases comprising or associated with the aggregation of peptide or protein structures of amyloidosis.

7. The method of claim 1, wherein the aliphatic amino acids and aliphatic amino acid derivatives and the polar amino acids and polar amino acid derivatives are either D-amino acids or L-amino acids.

8. The method of claim 1, wherein the 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).

9. The method of claim 1, wherein all or a portion of the 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.

10. The method of claim 1, wherein the aliphatic amino acids have a sequence selected from the group consisting of TABLE-US-00002 (SEQIDNO:1) LIVAG, (SEQIDNO:2) ILVAG, (SEQIDNO:3) ILVAA, (SEQIDNO:4) LIVAA, (SEQIDNO:5) LAVAG, (SEQIDNO:6) IAVAG, (SEQIDNO:7) AIVAG, (SEQIDNO:8) GIVAG, (SEQIDNO:9) VIVAG, (SEQIDNO:10) ALVAG, (SEQIDNO:11) GLVAG, (SEQIDNO:12) VLVAG, (SEQIDNO:13) IVAG, (SEQIDNO:14) LIVA, (SEQIDNO:15) LIVG, (SEQIDNO:16) ILVA, (SEQIDNO:17) ILVG, (SEQIDNO:18) LVAG, IVA IVG, VIG, IVA, VIA, IV VI (SEQIDNO:19) LIVAGD, (SEQIDNO:20) ILVAGD, (SEQIDNO:21) ILVAAD, (SEQIDNO:22) LIVAAD, (SEQIDNO:23) LAVAGD, (SEQIDNO:24) IAVAGD, (SEQIDNO:25) AIVAGD, (SEQIDNO:26) LIVAGE, (SEQIDNO:27) LIVAGK, (SEQIDNO.28) ILVAGK, (SEQIDNO:29) ILVAAK, (SEQIDNO:30) IAVAGK, (SEQIDNO:31) AIVAGK, (SEQIDNO:32) LIVAGT, (SEQIDNO:33) ILVAAT, (SEQIDNO:34) IAVAGT, (SEQIDNO:35) AIVAGT, (SEQIDNO:36) LIVAD, (SEQIDNO:37) LIVGD, (SEQIDNO:38) ILVAD, (SEQIDNO:39) ILVGD, (SEQIDNO:40) LVAGD, (SEQIDNO:41) IVAD, (SEQIDNO:42) IVAK, (SEQIDNO:43) IVGD, (SEQIDNO:44) VIGD, (SEQIDNO:45) IVAD, (SEQIDNO:46) VIAD, (SEQIDNO:47) IVGK, (SEQIDNO:48) VIGK, (SEQIDNO:49) IVAK, (SEQIDNO:50) VIAK, (SEQIDNO:51) IIID, (SEQIDNO:52) IIIK, IVD, IVK, IID, LVE, IVE, LVD, VIE, VID, VIK, VLD, VLE, LLE, LLD, IIE, ID, IE, (SEQIDNO:53) LIVAGOrn, (SEQIDNO:54) ILVAGOrn, (SEQIDNO:55) ILVAAOrn, (SEQIDNO:56) IAVAGOrn, (SEQIDNO:57) AIVAGOrn, (SEQIDNO:58) LIVAGDab, (SEQIDNO:59) ILVAGDab, (SEQIDNO:60) ILVAADab, (SEQIDNO:61) IAVAGDab, (SEQIDNO:62) AIVAGDab, (SEQIDNO:63) LIVAGDap, (SEQIDNO:64) ILVAGDap, (SEQIDNO:65) ILVAADap, (SEQIDNO:66) IAVAGDap, (SEQIDNO:67) AIVAGDap, (SEQIDNO:68) LIVAOrn, (SEQIDNO:69) LIVGOrn, (SEQIDNO:70) ILVAOrn, (SEQIDNO:71) ILVGOrn, (SEQIDNO:72) LVAGOrn, (SEQIDNO:73) LIVADab, (SEQIDNO:74) LIVGDab, (SEQIDNO:75) ILVADab, (SEQIDNO:76) ILVGDab, (SEQIDNO:77) LVAGDab, (SEQIDNO:78) LIVADap, (SEQIDNO:79) LIVGDap, (SEQIDNO:80) ILVADap, (SEQIDNO:81) ILVGDap, (SEQIDNO:82) LVAGDap, (SEQIDNO:83) IVAOrn, (SEQIDNO:84) IVGOrn, (SEQIDNO:85) VIGOrn, (SEQIDNO:86) IVAOrn, (SEQIDNO:87) VIAOrn, (SEQIDNO:88) IVADab, (SEQIDNO:89) IVGDab, (SEQIDNO:90) VIGDab, (SEQIDNO:91) IVADab, (SEQIDNO:92) VIADab, (SEQIDNO:93) IVADap, (SEQIDNO:94) IVGDap, (SEQIDNO:95) VIGDap, (SEQIDNO:96) IVADap, (SEQIDNO:97) VIADap, IVOrn, IVDab, IVDap, VIOrn, VIDab, VIDap, and (SEQIDNO:98) ILVAGS.

11. The method of claim 1, wherein the 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.

12. The method of claim 1, wherein (Y).sub.c has a sequence selected from Asp, Asn, Glu, Gln, Ser, Thr, Cys, Met, Lys, Orn, Dab, His, Asn-Asn, Asp-Asp, Glu-Glu, Gln-Gln, Asn-Gln, Gln-Asn, Asp-Gln, Gln-Asp, Asn-Glu, Glu-Asn, Asp-Glu, Glu-Asp, Gln-Glu, Glu-Gln, 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, and Dap-Dap.

13. The method of claim 1, wherein a is 1 and the 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.

14. The method of claim 1, wherein said N-terminal protecting group Z is an acetyl group, or a peptidomimetic molecule, 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.

15. The method of claim 1, wherein the C-terminal protecting group Z is: (a) an amide group; (b) an ester group; or (c) a peptidomimetic molecule or a natural or synthetic amino acid derivative thereof, the C-terminus of which can 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.

16. The method of claim 1, wherein the peptide or peptidomimetic comprises at least one additional compound selected from the group consisting of: small molecules, sugars, alcohols, hydroxy acids, amino acids, vitamins, biotin, L-Dopa, thyroxine, bioactive molecules or moieties, growth factors, cytokines, lipids, cell receptor ligands, hormones, prodrugs, drugs, vitamins, antigens, antibodies, antibody fragments, oligonucleotides, DNA, messenger RNA, short hairpin RNA, small interfering RNA, microRNA, peptide nucleic acids, aptamers, saccharides, label(s), dye(s), imaging contrast agents, pathogens, viruses, bacteria and parasites, microparticles, nanoparticles, and combinations thereof, wherein the at least one additional compound is covalently attached or coupled to the peptide or peptidomimetic, to the C-terminal group Z, amino acid side chain(s) and/or linker, and wherein the attachment or coupling can be carried out before, during or after self-assembly of the peptide or peptidomimetic.

17. The method of claim 1, wherein the peptide or peptidomimetic is present at a concentration in the range of from 0.1% to 30% (w/w), with respect to the total weight of the hydrogel or fibrous structure.

18. A second harmonic generation (SHG) microscopy method for imaging a sample, the method comprising: (a) contacting with a sample or a tissue a non-linear optical material that comprises peptides and/or peptidomimetics having the general Formula II:
Z.sub.a-M-Z.sub.d (II) wherein Z is an N-terminal protecting group; a is 0 or 1; M is an amino acid sequence selected from SEQ ID NOs: 99 to 102; Z is a C-terminal protecting group; and d is 0 or 1; and (b) imaging the sample or tissue under a second harmonic generation (SHG) excitation wavelength to produce a SHG signature of the sample or tissue.

19. The method of claim 6, wherein the biological processes are cell adhesion, migration or differentiation.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) Embodiments of the invention will now be described by way of example with reference to the accompanying drawings.

(2) FIG. 1: Fine structure and SHG activity of collagen and amyloid-like peptides.

(3) (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 LK.sub.6 peptide particles (inset, SHG image of LK.sub.6 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 (meanstandard deviation, n=5);

(4) SEM images of collagen fibers (b) and peptide particles (c).

(5) FIG. 2. LK.sub.6 peptides are SHG active.

(6) (a) SHG signal spectrum of LK.sub.6 peptide. Signal was ranging from 380 to 550 nm with excitation wavelength of 810 nm and peak emission wavelength was shown at 405 nm.

(7) (b) Spectral peaks at various excitation wavelengths spanning a broad spectral region (810-890 nm).

(8) (c) Emission -scan of the SHG signal (.sub.ex=850 nm) acquired from SHG imaging of LK.sub.6 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 1/2 relation to the spectral profile of the corresponding beam.

(9) (d) Log-log plot of the above SHG signal measurements demonstrating a log [I.sub.425]=0.45+2.01log [I.sub.850] dependence, quadratic to a good approximation, consistent with nonlinear second order optical upconversion.

(10) FIG. 3. Cellular uptake of biotin-conjugated LK.sub.6.

(11) Upper panel: HeLa cells exposed to biotin-conjugated LK.sub.6 peptide containing medium. Peptides in HeLa cells displayed pseudo red color (SHG) and green color (fluorescence).

(12) Lower panel: HeLa cells exposed to normal medium without peptide (control). Scale bars: 50 m.

(13) FIG. 4. Interface of LK.sub.6 peptide particles and animal fat tissue.

(14) (a) Schematic illustration of experiment arrangement: a low intensity ultrafast infrared laser penetrating glass cover slip and rat fat tissue (thickness50 m) and viewing LK.sub.6 peptide particles sandwiched in between the glass slide and the glass cover slip.

(15) (b) Three dimensional image of LK.sub.6 peptide particles in the entire volume of (2252254.5 m.sup.3). SHG z-stack was generated with 0.5 m step. Total nine images were taken (z=4.5 m).

(16) FIG. 5. SHG images of human corneal biopsy samples.

(17) (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 m.

(18) FIG. 6: SHG images of four pristine peptides with natural amyloidogenic core sequences.

(19) 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 KE.sub.7>GA.sub.6>DF.sub.5>NL.sub.6. Scale bar=50 m.

(20) FIG. 7: Cytotoxicity studies of LK.sub.6 peptide treated HDF and HeLa cells.

(21) (a) Cell viability at 48 h of HDF and HeLa cells incubated with cell culture media containing LK.sub.6 peptide solutions, as determined by the MTS assay (meanstandard deviation, n=9). Concentrations ranged from 0.1 to 1000 g/mL.

(22) (b) Cytotoxicity determined by Calcein AM/EthD-1 (live/dead, green/red) staining method after 48 h treated with 1 and 1000 g/mL LK.sub.6 peptide solutions using non-treated cells as control.

(23) Scale bars: 100 m.

(24) 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.

DETAILED DESCRIPTION

(25) 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].

(26) 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.

(27) 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) (SEQ ID NO: 27) was investigated.

(28) A hexamer peptide Ac-LIVAGK-NH.sub.2 (LK6) (SEQ ID NO: 27) 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.

(29) 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.

(30) 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 micrometers. 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, , was measured by Hyper Rayleigh Scattering (HRS). The first hyperpolarizability of these trimers was about 0.08710-30 esu (esu is the unit of the first hyperpolarizability) [19]. However, the first hyperpolarizability of collagen I was found to be (125020)10-30 esu [19], which could be viewed as ten thousand trimers combining together.

(31) 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.

(32) 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, FIG. 1c). The particle size was ranging from submicron to 10 microns and the particle shape was irregular. Peptide particles also tended to form larger aggregates during drying (FIG. 2d, FIG. 1a inset and FIG. 4b). Currently, we are manipulating preparation parameters to achieve better control over the size and the shape of peptide particles.

(33) 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 (FIG. 2a). Illuminating the LK6 peptide particles at 810 nm with a conventional two-photon microscope yielded an SHG signal profile with a discrete peak at 405 nm.

(34) SHG image (green) of LK6 peptide particles was shown in FIG. 1a (inset). LK6 peptide particles showed bright SHG signals. The size and shape of particles can be visualized, consistent with FE-SEM image (FIG. 1a). We then examined the excitation wavelength tunability of SHG signals from these peptide particles. As shown in FIG. 2b, the exciting wavelength was increased from 810 to 890 nm in a conventional two-photon laser-scanning microscopy, the wavelength of the SHG signals from the LK6 peptide particles increased from 405 to 450 nm respectively. Excitation wavelength tunability is one of the advantages of SHG. This enables us freely tune the excitation wavelength to best match the optical properties of the sample.

(35) 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. FIG. 2c shows the emission wavelength data of the SHG signal (ex=850 nm). The data in the graph were fitted to a Gaussian curve exhibiting a maximum at 425 nm, which is exactly half the excitation wavelength of 850 nm. The full width at half-maximum of Gaussian distribution (10 nm) obeys a relationship to the corresponding wavelength profile of the fundamental beam (15 nm). Moreover, the dependence of the output signal on irradiation intensity was measured by varying the 850 nm excitation intensity. The linear regression was applied to the log-log plots revealed a quadratic power dependence of SHG intensity to the power intensity, as shown in the FIG. 2d.
log [I425]=0.45+2.01*log [I850]

(36) This confirms the two-photon nature of the emission from LK6 ultrashort peptide particles.

(37) 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 (FIG. 1a, left inset). Interestingly, our hexamer shows similar intensity to that of collagen (FIG. 1a). This suggested that our hexamer can be used as SHG probes for bioimaging.

(38) 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 g/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 g/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. FIG. 7b shows the representative live/dead images of cells in response to LK6 solutions (1 and 1000 g/mL). LK6 shows no toxicity to both HDF and HeLa cells, in agreement with results of MTS assays.

(39) Biological Application

(40) 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 (FIG. 4a). Peptide particles (green, FIG. 4b) can still be seen clearly as a laser penetrates the tissue. This is a proof of the concept for using LK6 as exogenous SH probes for bioimaging.

(41) 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.

(42) 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.

Further Preferred Embodiments

(43) Abstract

(44) 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.

(45) 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 (GA.sub.6) (SEQ ID NO: 100) and KLVFFAE (KE.sub.7) (SEQ ID NO: 99), can self-assemble into amyloid-like aggregates [30, 31]. These two peptides are also known as the core sequence of amyloid- (A). A 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 (ID.sub.3) and LIVAGD (LD.sub.6) (SEQ ID NO: 19), have shown similar behavior [31, 17]. Both these natural and synthetic peptides are amyloid-like peptides. They all form cross- 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].

(46) 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.

(47) For further details, see Example.

Key Features of the Invention

(48) A novel class of peptides, which only consists of 3-7 amino acids, possesses non-linear optical properties, which can be viewed under second harmonic generation (SHG) microscopy. An interesting mechanism of self-assembly into fibrous scaffolds, which resembles the collagen fiber in extracellular matrix. Based on the non-linearity of the intrinsic peptide bonds, such supramolecular assembly can generate second harmonic signals Peptide particles demonstrate the SHG excitation wavelength tunability. When the exciting wavelength was increased from 810 to 890 nm in a conventional two-photon laser-scanning microscopy, the wavelength of the SHG signals from the LK6 peptide particles increased from 405 to 450 nm respectively. Peptide particles demonstrate the two-photon nature of the emission. The emission was fitted nicely to a Gaussian curve and the SHG output signal obeyed a quadratic power dependence of SHG intensity to the power intensity. Peptide particles generate strong SHG signals, similar as that of endogenous collagen. This suggested that this class of peptides could be used as SHG probes for bioimaging. The peptides are biocompatible and biodegradable. No toxic effects of these peptides were detected by MTS assay and live/dead staining. All-peptide based contract agents can be visualized under SHG microscope without additional dyes or chemicals. Therefore, these peptides can be used for monitoring cells and in vivo imaging. Peptide-based tissue engineering scaffolds can be directly visualized under SHG microscope. Fundamental biological processes including cell adhesion, migration and differentiation on these peptides made matrices can be revealed. Visualization of peptide aggregation can be used for disease diagnostics. Diseases include Alzheimer's and Parkinson's disease. Peptides can be easily functionalized to introduce additional functions. Peptide particles can be used as drug or gene delivery vehicles. Peptide-based nanowires can be used as biosenors.

Examples

(49) 1. Materials and Methods

(50) 1.1 SHG Imaging:

(51) 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 nm. To achieve spectral analysis and detect the SHG signal, we used the META detector with 32-gated photon counting module.

(52) 1.2 Ultrashort Amyloid-Like Peptides:

(53) 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].

(54) 1.3 Peptide Particles:

(55) 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 m (FIG. 4S).

(56) 1.4 Cell Culture:

(57) We cultured both HeLa and human dermal fibroblasts in DMEM supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (Life technologies, Singapore).

(58) 1.5 Human Corneal Biopsy Samples:

(59) 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).

(60) 2. Results and Discussion

(61) 2.1 A Hexamer Peptide is Collagen-Like and SHG-Active.

(62) To test whether the amyloid-like peptides of the present invention are SHG-active, we first investigated a hexamer peptide, Ac-LIVAGK-NH.sub.2, (LK.sub.6) (SEQ ID NO: 27). 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 (FIG. 1a). The fine structures of collagen and peptide particles can be clearly seen under SHG and scanning electron microscopy (SEM, FIG. 1a insert, 1b and 1c).

(63) We further characterized LK.sub.6 peptide. It exhibits typical SHG characteristics (FIG. 2). When excited at 810 nm, LK.sub.6 emitted a sharp SHG peak at 405 nm (FIG. 2a). We then examined the excitation wavelength tunability of SHG signals from LK.sub.6 peptide. In FIG. 2b, as the excitation wavelength was increased from 810 to 890 nm, the wavelength of the SHG signals from the LK.sub.6 peptide particles increased from 405 to 445 nm respectively. Biological samples, such as cells and tissues, are known to have auto-fluorescence. With excitation wavelength tunability, we can better avoid auto-fluorescence. In addition, we can further increase penetration depth in tissues by simply increasing excitation wavelength. FIG. 2c shows the emission wavelength data of the SHG signal (.sub.ex=850 nm). The data in the graph were fitted to a Gaussian curve exhibiting a maximum at 425 nm, which is exactly half the excitation wavelength of 850 nm. The band width (full width at half-maximum of Gaussian distribution, FWHM) was narrow (10 nm) and it obeys a relationship to the corresponding wavelength profile of the fundamental beam (15 nm). Moreover, we measured the dependence of the output signal on irradiation intensity by varying the 850 nm excitation intensity. The linear regression, applied to the log-log plots, revealed a quadratic power dependence of SHG intensity to power intensity, as shown in the FIG. 2d.
log [I.sub.425]=0.45+2.01log [I.sub.850](1)

(64) 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 LK.sub.6 peptide.

(65) 2.2 Comparison of Synthetic and Natural Amyloid-Like Peptides Under SHG Microscopy

(66) Next, we examined several other amyloid-like peptides, including both natural amyloidogenic core sequences (NL.sub.6, DF.sub.5, GA.sub.6, and KE.sub.7) and designed synthetic peptides (LD.sub.6, IS.sub.6 and IK.sub.3). The peptide sequences are listed in Table 1.

(67) TABLE-US-00001 TABLE 1 Powder SHG Efficiencies of Various Amyloid-like Peptides and Human Collagen Subtypes. Amyloid-like Peptides/ Human Collagens SEQ ID NO. I.sup.2/I.sup.2(sucrose).sup.a) IVK (IK.sub.3) 3.31 0.57 LIVAGD (LD.sub.6) 19 3.06 0.65 ILVAGS (IS.sub.6) 98 1.96 0.58 LIVAGK (LK.sub.6) 27 0.95 0.19 KLVFFAE (KE.sub.7) 99 3.18 0.67 GGVVIA (GA.sub.6) 100 1.52 0.43 DFNKF (DF.sub.5) 101 0.89 0.33 NFGAIL (NL.sub.6) 102 0.76 0.21 Type I 1.23 0.36 Type II 0.41 0.13 Type III N.D..sup.b) Type IV N.D..sup.b) Type V N.D..sup.b) .sup.a)Value = Average standard deviation; .sup.b)N.D. = not detected.

(68) 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 LD.sub.6 and IK.sub.3 were even three times higher than that of sucrose. These may due to their molecular structures. LD.sub.6 is known to form hydrogel at much lower concentrations compared to LK.sub.6. The trimer IK.sub.3 has larger dipole moment compared to LK.sub.6. 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.

(69) We have previously showed that NL.sub.6, DF.sub.5, GA.sub.6, and KE.sub.7 as well as LD.sub.6, IS.sub.6, LK.sub.6 and IK.sub.3 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, LD.sub.6 (negatively charged), IS.sub.6 (neutral) and LK.sub.6 (positively charged) to investigate the effect of the charged head groups. The SHG efficiency of these three hexamers followed the order of LD.sub.6>IS.sub.6>LK.sub.6.

(70) We also investigate the effect of the peptide chain length by choosing IK.sub.3, a trimer, compared to the other three hexamers. It followed the order of IK.sub.3 (trimer)>LD.sub.6 (hexamer). Both findings are helpful for designing a superior amyloid-like peptide.

(71) We then investigated the SHG efficiency of four natural amyloidogenic core sequences. They followed the order of KE.sub.7>GA.sub.6>DF.sub.5>NL.sub.6 (Table 1 and FIG. 6). KE.sub.7 (KLVFFAE) (SEQ ID NO: 99), containing its diphenylalanine (FF) motif, showed the highest SHG efficiency among these four peptides. However, its SHG efficiency is similar as that of IK.sub.3. It is noteworthy that IK.sub.3 is an aliphatic peptide. Aromatic residues such as tyrosine and tryptophan are known to be used as an endogenous molecular probe of peptides and proteins for SHG at the air-water interface [25]. However, our results indicated that aromatic residues are not necessary for higher SHG efficiency. LD.sub.6, IS.sub.6, LK.sub.6 and IK.sub.3 are all aliphatic and lacking aromatic residues, clearly makes them a unique type of peptides for SHG applications. These peptides are rational designed by Hauser and colleagues [17, 20]. They are amphiphilic, consisting of an aliphatic amino acid tail of decreasing hydrophobicity and a hydrophilic head. They can self-assembly via parallel-antiparallel -helical pairs and subsequent stacking into -turn fibrils, which show striking similarity to collagen fibers [20].

(72) 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).

(73) In addition, a molecular-level property of the nonlinearity, i.e., the first hyperpolarizability, , was measured by Hyper Rayleigh Scattering (HRS). The first hyperpolarizability of these trimers was about 0.08710.sup.30 esu (esu is the unit of the first hyperpolarizability) [19]. However, the first hyperpolarizability of collagen I was found to be (125020)10.sup.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.

(74) 2.3 Cytocompatibility of a Hexamer Peptide

(75) In order to use LK.sub.6 for bioimaging applications, such as in vitro monitoring cells and in vivo imaging the whole animal, we assessed the cytotoxicity of LK.sub.6 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 LK.sub.6 solutions was reported in FIG. 7. LK.sub.6 shows no toxicity to either HDF or HeLa cells.

(76) 2.4 A Hexamer Peptide as a Second Harmonic Probe for In Vitro Cell Imaging

(77) A possible application for LK.sub.6 is cell imaging. To this end, HeLa cells were incubated with biotin-conjugated LK.sub.6 for 4 h and then fixed for immunostaining. LK.sub.6 peptides were visualized by both SHG and confocal fluorescence microscopes. In FIG. 3, biotin-conjugated peptides displayed green color as we added DyLight 488-conjugated NeutrAvidin (Thermo Scientific, Singapore). Meanwhile, peptides can be detected under SHG, displaying pseudored color.

(78) 2.5 A Hexamer Peptide as a Second Harmonic Probe for Ex Vivo Deep Imaging

(79) Another possible application for LK.sub.6 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 LK.sub.6 peptide samples are placed under a slice of fat tissue (great omentum removed from a sacrificed rat, thickness50 m). The results are shown in FIG. 4 and confirm the ability to detect SHG signals under sub-millimeter thick tissue.

(80) 2.6 Diseased and Normal Human Corneal Biopsy Samples Examined by SHG Microscopy

(81) More interestingly, when we examined the human corneal biopsy samples (FIGS. 5a and 5b), both amyloids (indicated by arrow) and collagen can be seen. Under SHG, the normal corneal sample showed collagen fibrils aligned as parallel straight lines in stroma. By contrast diseased samples showed collagen fibrils became curved and thicker. The thickened collagen fibrils could be due to amyloid deposits. Thus, SHG provides us a new diagnostic tool for lattice corneal dystrophy (LCD) that is superior to the existing histological staining methods: SHG is label-free (no staining such as Congo red is required).

(82) 2.7 Other Applications

(83) 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.

CONCLUSION

(84) 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.

(85) 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.

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