Abstract:
The present invention provides novel Raman tags for exploring membrane interactions in cells. The tags comprise a phenyl diyne probe where in the dyine is capped with the phenyl group. Methods for using the tags are also provided.

Description:
[0001]    This invention was made with government support under CA182608 awarded by the National Institutes of Health. The government has certain rights in the invention. 
     
    
     TECHNICAL FIELD 
       [0002]    The present disclosure generally relates to tags for imaging molecules using Raman spectroscopy, and in particular to a method and composition that uses cholesterol mimics to track the location and movement of cholesterol. 
       BACKGROUND 
       [0003]    This section introduces aspects that may help facilitate a better understanding of the disclosure. Accordingly, these statements are to be read in this light and are not to be understood as admissions about what is or is not prior art. 
         [0004]    An important component of cellular membrane, cholesterol controls physical properties of the membrane and contributes to specific membrane structures such as lipid rafts. Inside cells, cholesterol plays an important role in various signaling pathways and serves as the precursor for signaling molecules, and modifies specific proteins, such as hedgehog, to control protein trafficking and activity. The distribution of cholesterol in a living cell is highly regulated. Intracellular cholesterol is stored in lipid droplets (LDs) in the form of cholesteryl ester to avoid the toxicity caused by free cholesterol. Dysregulation of cholesterol metabolism and/or trafficking has been linked to various diseases, including atherosclerosis, Niemann-Pick type C (NP-C) disease, and various cancers. 
         [0005]    Intracellular cholesterol transport and metabolism have been studied extensively using various reporter molecules, including cholesterol binding molecules and cholesterol analogs. Cholesterol binding molecules, such as cholesterol oxidase, filipin, and perfringolysin O derivatives, are commonly used to study steady-state distribution of cholesterol in fixed cells and tissues. Fluorescent cholesterol, including intrinsic fluorescent sterols (DHE for dehydroergosterol) and fluorophore-tagged analogs (NBD-cholesterol and BODIPY-cholesterol) are widely used in vitro and in vivo. Radiolabeled cholesterol or its precursors are used in biochemical studies of metabolism and trafficking of cholesterol. 
         [0006]    These current cholesterol assays have limitations. Cholesterol oxidase is commonly used in fluorometric or colorimetric assays to quantify total cholesterol in homogenized cells. Radiolabeled cholesterol has to be used in combination with separation methods to determine intracellular cholesterol distribution indirectly. For imaging purpose, filipin is the most commonly used molecule for visualizing distribution of free cholesterol, but it is only applicable to fixed cells or tissues with moderate specificity because filipin also labels other lipids. Fluorescent BODIPY-cholesterol is known to cause perturbations due to bulkiness of the fluorophore. DHE has the closest structure as cholesterol, but its fluorescence undergoes rapid photo-bleaching, which impedes real-time observation of cholesterol trafficking There exists a need for new technologies that allow for real time imaging of cholesterol transport and low toxicity in live cells. 
       SUMMARY OF THE INVENTION 
       [0007]    The present invention provides novel Raman tags for exploring membrane interactions in cells. The tags comprise a phenyl diyne probe where in the dyine is capped with the phenyl group. Methods for using the tags are also provided. 
     
    
     
       BRIEF DESCRIPTION OF THE FIGURES 
         [0008]      FIG. 1  depicts a synthesis scheme and some resulting cholesterol mimics. 
           [0009]      FIG. 2  depicts a spectral analysis of tagged cholesterol and SRS detection of PhDY-Chol. 
           [0010]      FIG. 3  is a graph illustrating the results of a cell-viability assay using various concentrations of some of the generated cholesterol mimics. 
           [0011]      FIG. 4  shows the SRS images of PhDY-Chol in live cells and what happens when PhDY-Chol storage in lipid droplets (LD) is blocked by an ACAT-1 inhibitor, a schematic of how ACAT-1 modifies the cholesterol mimics, and a graph showing a quantitative analysis of PhDY-rich and BODIPY-rich LDs in CHO cells before and after ACAT-1 inhibition. 
           [0012]      FIG. 5  shows the cholesterol mimics transport and storage restored in an M12 cells disease model using HPβCD, a graph showing quanititative analysis of PhDY-rich areas in the cells before and after treatment with HPβCD, and a graph showing quantitative analysis of PhDY-Chol storage in LDs in cells before and after treatment with HPβCD. 
           [0013]      FIG. 6  shows SRS images of PhDY-Chol visualized in various compartments of cholesterol storage in live  C. elegans , in the presence and absence of Cholesterol Uptake Protein-1 (ChUP-1). 
       
    
    
     DETAILED DESCRIPTION 
       [0014]    For the purposes of promoting an understanding of the principles of the present disclosure, reference will now be made to the embodiments illustrated in the drawings, and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of this disclosure is thereby intended. The terms “I,” “we,” “our” and the like throughout the Detailed Description, Appendix-A, and Appendix-B do not refer to any specific individual or group of individuals. 
         [0015]    A novel composition and a method that allows bio-orthogonal imaging of cholesterol esterification, storage, and trafficking inside living cells and vital organisms. By rational design and chemical synthesis, we prepared a probe molecule, phenyl-diyne cholesterol (PhDY-Chol), which gives a 2,254 cm −1  Raman peak that is 122 times stronger than the endogenous C═O stretching band. Compared to alkyne-cholesterol mimic of which the IC 50  is 16 μM, the phenyl-diyne group is biologically inert and did not cause cytotoxicity after 16 h incubation at 50 μM. In live Chinese hamster ovary (CHO) cells, SRS imaging showed incorporation into plasma membrane, esterification of PhDY-Chol by acyl-CoA: cholesterol acyltransferase 1 (ACAT-1), and storage in lipid droplets (LDs). In a cellular model of NP-C disease, PhDY-Chol is selectively accumulated in lysosomes and is esterified and relocated to LDs after treatment with a cholesterol-mobilization drug. In live  C.elegans , SRS imaging of PhDY-Chol revealed a previously unnoticed compartment of cholesterol storage, regulated by the cholesterol uptake protein ChUP-1. These studies herald the potential of the method for unveiling intracellular cholesterol trafficking mechanisms and highly efficient screening of drugs that target cholesterol metabolism. 
         [0016]    The following describes compositions, methods of synthesis, and methods of use of these compositions in the study of cholesterol. These compositions may be used to study cholesterol localization and movement within live cells. The cholesterol mimics may be used to study and understand different changes in metabolism and organization by tracking the changes in localization and modification of cholesterol. The cholesterol mimics may be used to generate assays for screening lead compounds or treatments to prevent or treat a metabolic irregularity or disease such as cancer. In other aspects the cholesterol mimic may be used to study the lipid droplets with in a given cell, healthy or not healthy, to provide an analysis. The cholesterol mimcs may be used as a target in a drug delivery system, where the drug targets the mimic in a certain unmodified or modified state, specifically. The cholesterol mimics may be used to provide a diagnosis or monitor the health of a patient&#39;s cells before or during treatment for a disease. The cholesterol mimics may be used in vitro or in vivo (i.e. cells and tissues, whole organs, or live animals or humans), and may be analyzed in live or fixed cells. 
         [0017]    The cholesterol mimics may be provided for use in a powder or crystal form, or suspended in liquid. The cholesterol mimics may be supplied by several delivery means including but not limited to orally, intravenously, injection, inhalation, catheter, dermal absorption, or ingestion. 
         [0018]    In general, a cholesterol mimic is generated by replacing the aliphatic chain of in cholesterol with a group that changes the Raman spectra produced during Raman spectroscopy. Ideally, a peak is generated from this molecule that is differentiated from other signals produced by a cell or tissue. One aspect, is to replace the aliphatic chain of cholesterol with a group that will generate a peak between 1,800 and 2,800 cm −1  in a Raman scatter. One way to produce a signal in this region is to add a C≡C moiety to cholesterol, as shown in  FIG. 1 . In certain aspects a C≡C bond may be added one, two, three, four, five, or six times to increase signal. In certain aspects there are three C≡C bonds included in the cholesterol mimic. 
         [0019]    In certain aspects the PhDY-Chol mimic probe was generated by replacing the aliphatic chain in cholesterol with phenyl-diyne. The following chemical structures illustrate some of the mimics: 
         [0000]    
       
                 
         
             
             
         
       
       
                 
         
             
             
         
       
     
         [0020]    In certain aspects of the technology, the mimic is viewed by Raman spectroscopy. Those of skill in the art will understand the set up and laser sources to use given the chemical nature of the cholesterol mimic. For illustrative purposes only, one may look to the contents of Appendix-A, herein incorporated by reference in its entirety, to understand one example of a setup for performing Raman spectroscopy using the disclosed cholesterol mimics. 
         [0021]    Cholesterol mimics were synthesized and a few specific embodiments will now be described. Referring now to  FIG. 1 , alkyne cholesterol (A-Chol, 5), phenyl-alkyne cholesterol (PhA-Chol, 6), phenyl-diyne cholesterol (PhDY-Chol, 7), and cyano cholesterol (CN-Chol, 8). Initially, synthesis commenced with cholenic acid 3. Using a sequence of THP-protection, LiA1H 4  reduction, Dess-Martin oxidation and Seyferth-Gilbert-Bestmann homologation, cholenic acid 3 was converted to compound 4 intermediate with a terminal alkyne group. Removal of the THP-protecting group gave probe A-Chol 5. PhA-Chol 6 and PhDY-Chol 7 were prepared from compound 4 intermediate via a palladium-catalyzed Sonogashira reaction and a copper-catalyzed Cadiot-Chodkiewicz reaction, respectively, followed by acidic removal of THP group. Additionally, CN-Choi 8 was prepared from cholenic acid 3 via standard transformations. 
         [0022]    The following are specific examples and embodiments, and not meant to be limiting in any way. 
         [0023]    Design and Synthesis of Composition 
         [0024]    Referring to  FIG. 1 , in order to design a probe molecule that not only maintains physiological functions of cholesterol, but also has a large Raman scattering cross section, we chose to replace the aliphatic side chain of cholesterol with a cyano or alkynyl groups. These groups have small size, which could minimize structural perturbation of the molecule of interest, in this case, cholesterol. These groups produce strong Raman scattering peaks in a cellular silent region (1,800-2,800 cm −1 ), and can be used for Raman imaging in a low-concentration condition. It has been reported that as the chain length increases, the hyperpolarizability increases in polyynes. Also, aromatic ring capped alkyne was shown to give stronger Raman signals than terminal alkyne. To design cholesterol mimic with very strong Raman intensity, we calculated the Raman cross section of potential tags—alkyne, phenyl-alkyne, diyne, phenyl-diyne using the Q-Chem and GAMES S electronic structure packages to provide insight of the relation between molecular structure and Raman intensity. The results showed that the localized polarizabilities on each C≡C moiety increase with the number of conjugated triple bonds, as well as with addition of a phenyl ring. 
         [0025]    The total polarizability of the molecule increases as a result of the additive effect as well as non-linear boost in the polarizability of conjugated bonds. The phenyl ring serves as both a donor and an acceptor of π-electrons from the neighboring triple bonds, further escalating polarizabilities of neighboring conjugated bonds. Taking into account that the Raman intensity is proportional to squares of polarizability derivatives, the additional three-fold enhancement of the total polarizability due to conjugation results in a ˜10-fold boost in Raman intensity. Together, the Raman intensity increases further by adding a phenyl group to the terminal alkyne, and increases even further by conjugating a phenyl group and another alkyne. 
         [0026]    Raman Spectral Analysis of Stimulate Raman Scattering of Cholesterol Mimics 
         [0027]    Referring now to  FIG. 2 , to determine the Raman shift of the C≡C stretching vibrational mode and to compare the level of Raman signals from the cholesterol mimics, 50 mM of each compound was prepared in cyclohexanone and confocal Raman spectral analysis was performed. The signal from CN-Chol was too weak to be detected. A-Chol showed its peak for C≡C vibrational mode at 2,122 cm −1 ; PhA-Chol at 2,239 cm −1 ; PhDY-Chol at 2,254 cm −1 . Comparing the Raman peak of each tag to the 1,714 cm −1  C═O vibrational peak from the solvent (9.7 M for pure cyclohexanone), the alkyne, PhA, and PhDY groups were 11 times, 16 times, and 122 times stronger in Raman intensity, respectively. This showed that the PhDY tag produces a spectrally-isolated peak, which is stronger than the C═O vibrational mode by two orders of magnitude. 
         [0028]    To determine the SRS imaging sensitivity for PhDY-Chol, we used a femtosecond stimulated Raman loss (SRL) microscope reported elsewhere. Cyclohexanone solutions of PhDY-Chol were prepared by serial dilution, and SRS images of PhDY-Chol were recorded with the laser beating frequency tuned to be resonant with C≡C vibration at 2,252 cm −1 . In solutions without PhDY-Chol, a residual background was detected, caused by cross phase modulation. The SRS contrast, defined as (S—B)/B, where S and B denote SRS signal and background, was calculated as a function of PhDY-Chol molar concentration. At the speed of 200 μs as per pixel, a linear relationship was observed and 13% and 4% contrasts were reached at 313 μM and 156 μM, respectively. To increase the detection sensitivity, we chirped the femtosecond lasers to 0.8 picosecond with a SF-10 glass rod. This spectral focusing approach maintained 85% of the SRS signal while reduced the cross phase modulation background level by 3 times, to a level of 6.3×10 −7  in terms of modulation depth. As a result, the SRS contrast became 14% at 31 μM, corresponding to ˜1,800 molecules in the excitation volume. We also depicted the modulation depth (ΔI/I) as a function of molar concentration (Appendix-B), which is used for estimating the molar concentration of PhDY-Chol inside cells in following studies. 
         [0029]    Cytoxicity Analysis of the Cholesterol Mimics 
         [0030]    Referring now to  FIG. 3 , the cytotoxicity of cholesterol mimics was evaluated by MTT cell-viability assays after treating CHO cells with cholesterol mimic. Various concentrations of cholesterol mimic were added to the culture media and the cells were incubated for 48 hours before the assays were conducted. A-Chol was found to be toxic to the cells with IC 50  of 16 μM. Adding a phenyl group reduced the cytotoxicity. To directly visualize the toxic effect, we stained the cells with propidium iodide for late apoptosis and necrosis. Cells incubated with A-Chol showed reduced density and extensive apoptosis, whereas both PhA and PhDY caused minimum cell death (Appendix-B). This result presents another important role of the phenyl group, which is to reduce the toxicity caused by terminal alkyne. 
         [0031]    Membrane Incorporation of Cholesterol Mimics 
         [0032]    Referring now to  FIG. 4 , CHO cells which are commonly used for cholesterol trafficking and metabolism studies, were used in these studies. To enhance cellular uptake of PhDY-Chol, the cells were pre-incubated in medium supplemented with lipoprotein-deficient serum to deplete medium cholesterol, after which the cells were incubated with 50 μM PhDY-Chol for 16 hours. By tuning the laser beating frequency to be resonant with C≡C vibration (2,252 cm −1 ), SRL signals arose from PhDY-Chol. We also tuned the laser to be resonant with C—H vibration (2,885 cm −1 ) and obtained signals from C—H-rich lipid structures, such as LDs. 
         [0033]    To show the incorporation of PhDY-Chol into the plasma membrane, the cells were trypsinized and performed spectral-focusing SRS imaging of the rounded live CHO cells with 10 μs per pixel speed. PhDY-Chol in the membrane was detected in the on-resonance image, and the contrast disappeared in the off-resonance image. The membrane incorporation was confirmed by filipin staining of free cholesterol and Raman spectral analysis (Appendix-B). By focusing at the filipin-stained membrane, we have obtained the Raman spectrum showing the C═C band from filipin, the amide I band from protein, and the C≡C band from the PhDY (Appendix-B). Inside live CHO cells, PhDY-Chol was colocalized with LDs found in the C—H vibrational region, as shown in  FIG. 4 . This colocalization was confirmed by two-photon-excited fluorescence (TPEF) imaging and Raman spectral analysis of BODIPY-stained LDs in fixed CHO cells. (Appendix-B). The Raman spectrum of the BODIPY-labeled LDs showed the C═C band from BODIPY, 702 cm −1  peak from cholesterol ring, and the C≡C band from the PhDY (Appendix-B), which further supports the localization of PhDY-Chol in LDs. 
         [0034]    It is important to note that PhDY-Chol-rich structures inside the CHO cells could not be stained by filipin (Appendix-B), indicating that it is not in the free form. It is hypothesized that PhDY-Chol is converted into PhDY-cholesteryl ester, by ACAT-1, the enzyme responsible for cholesterol esterification, as diagrammed in  FIG. 4 . To confirm the esterification of PhDY-Chol, we inhibited ACAT-1 with avasimibe for 24 h before addition of PhDY-Chol. After blocking cholesterol esterification, the amount of PhDY-Chol found in CHO cells significantly decreased ( FIG. 4   c ). Although LDs were still visible, the amount of PhDY-Chol signal found inside LDs reduced by 4 times ( FIG. 4   d ). ACAT-1 knockdown by shRNA was also conducted to specifically inhibit the enzyme. Similarly, we found decreased amount of PhDY-Chol in ACAT-1 knocked down CHO cells, and the amount of PhDY-Chol in LDs reduced significantly (Appendix-B). To determine where PhDY-Chol accumulates after ACAT-1 inhibition, we stained the cells with LysoTracker for lysosomes. Our results indicated that after ACAT-1 inhibition, PhDY-Chol partially located in lysosomes (Appendix-B). Collectively, these results show that PhDY-Chol can be transported into cells, converted into PhDY-cholesteryl ester by ACAT-1, and stored in LDs following the normal metabolic pathway of cholesterol. To emphasize the physiological compatibility of our PhDY tag, we treated CHO cells with BODIPY-cholesterol. The amount of BODIPY-cholesterol incorporated into LDs did not change after ACAT-1 inhibition ( FIG. 4   d  and e), indicating that BODIPY-cholesterol directly labels the LDs without metabolic conversion into cholesteryl ester. 
         [0035]    Lysosomal accumulation and relocation to Lipid Droplets in NP-C animal disease model discovery using cholesterol mimics. 
         [0036]    Referring now to  FIG. 5 , the potential of PhDY-Chol for studying cholesterol transport in NP-C disease, a disorder featured by abnormal cholesterol accumulation in late endosome/lysosome caused by mutation in NPC1 or 2 gene, was explored. M12 cells, mutant CHO cells that contain a deletion of the NPC1 locus, were established as a cellular model of the NP-C disease. By combining SRL imaging of PhDY with TPEF imaging of filipin, we observed that, unlike wildtype CHO cells, the PhDY-Chol-rich structures were stained by filipin, indicating that these PhDY-Chol molecules were located in lysosomes. (Appendix-B). Moreover, we observed some filipin labeled structures that do not contain PhDY-Chol. This result is reasonable given that filipin has been shown to label other lipid molecules, such as glycosphingolipids. As additional evidence, we incubated M12 cells with PhDY-Chol and stained the cells with LysoTracker. It was found that all PhDY-Chol-rich areas were localized in LysoTracker-stained organelles (Appendix-B). Collectively, these results showed that PhDY-Chol can selectively represent the lysosomal storage of cholesterol in the NP-C disease model. 
         [0037]    We then treated the PhDY-Chol-labeled M12 cells with a cholesterol-mobilizing drug, hydroxypropyl-β-cyclodextrin (HPβCD). This drug is known to mediate lysosomal escape of cholesterol, and promote storage of excess cholesterol into LDs. After treating with HPβCD, the amount of PhDY-Chol in M12 cells decreased by half ( FIG. 5   b  and  c ). Interestingly, we observed that some PhDY-Chol-rich areas were not labeled by filipin after HPβCD treatment ( FIG. 5   b , arrow heads). These areas likely represent PhDY-cholesteryl ester stored in LDs. To confirm this possibility, we stained the cells with BODIPY for localization of LDs. The result clearly showed that PhDY-Chol moved into LDs after HPβCD treatment, and the number of PhDY-rich LDs increased significantly ( FIGS. 5   d  and  e ). Together, these data indicate that PhDY-Chol can be used as a reliable probe molecule to study cholesterol mobilization inside living cells. 
         [0038]    Cholesterol mimics identify cholesterol storage compartments in animal model. 
         [0039]    Referring to  FIG. 6 , to demonstrate the capability of monitoring cholesterol uptake and distribution in vivo, we fed N2 wildtype  C. elegans  with PhDY-Chol-labeled  E. coli  and imaged PhDY-Chol storage in the worms using our SRL microscope at speed of 40 μs per pixel. PhDY-Chol was found to be stored in the intestinal cells inside the wildtype worms ( FIG. 6   a , upper panels). To confirm the uptake of PhDY-Chol by intestinal cells, we fed mutant  C. elegans , in which dietary cholesterol uptake is inhibited by ChUP-1 deletion, with PhDY-Chol. We did not observe PhDY-Chol inside this strain ( FIG. 6   a , lower panels), which indicates that the PhDY tag did not affect the cholesterol uptake process. Then, we tuned the laser to be resonant with C—H vibration for lipid-rich LDs. Unlike wildtype CHO cells, the PhDY-Chol-rich compartments were found to be distinguished from LDs in wildtype worms ( FIG. 6   a , upper panels). To explore the nature of these compartments, we used hjIs9 worms that contains GFP targeted to lysosome-related organelles (LROs) in intestinal cells. Dual-modality SRS and TPEF imaging showed that PhDY-Chol is stored in the LROs ( FIG. 6   b ). Collectively, these results suggest that dietary PhDY-Chol uptake is through a ChUP-1 mediated process, and unlike mammalian CHO cells,  C. elegans  stores cholesterol in LROs, but not in LDs in the intestine. 
         [0040]    Chemical structure (V) or Compound S8. The mixture of 4 (22 mg, 0.05 mmol), PdCl  2  (PPh 3 ) 2  (1.4 mg, 0.002 mmol), CuI (0.4 mg, 0.002 mmol), and B (12.3 mg, 0.06 mmol) in THF (0.5 mL) was bubbled with Argon gas for ten minutes. To the mixture, DIPEA (0.02 mL) was added at room temperature. After stirring for 2 h at room temperature, the reaction was quenched with saturated NH 4 Cl aqueous solution and extracted with ethyl acetate. The organic layer was washed with brine, dried over MgSO 4 . The solvent was removed under vacuum, and the residue was purified by chromatography (Hexane/EtOAc, 40:1) to give S8 (18 mg, 65%) as a white solid. 
         [0000]    
       
                 
         
             
             
         
       
     
         [0041]      1 H NMR (500 MHz, CDCl 3 ): δ 7.50 (d, J=7.0 Hz, 2H), 7.38-7.35 (m, 1H), 7.33-7.30 (m, 2H), 5.35 (t, J=6.5 Hz, 1H), 4.72 (m, 1H), 3.93-3.90 (m, 1H), 3.55-3.47 (m, 2H), 2.44-2.20 (m, 4H), 2.01-1.96 (m, 2H), 1.88-1.83 (m, 4H), 1.74-1.70 (m, 2H), 1.62-1.42 (m, 12H), 1.30-1.06 (m, 7H), 1.01 (s, 3H), 0.93 (d, J=6.5 Hz, 3H), 0.69 (s, 3H);  13 C NMR (100 MHz, CDCl 3 ): δ 141.2, 141.1, 133.1, 129.6, 128.6, 121.7, 121.6, 121.4, 108.8, 97.2, 97.0, 83.3, 76.2, 75.5, 74.8, 67.6, 65.6, 63.1, 63.0, 59.6, 56.9, 56.0, 53.6, 50.3, 50.3, 42.6, 40.4, 40.0, 38.9, 37.6, 37.4, 37.0, 36.9, 35.4, 34.5, 32.0, 31.4, 29.8, 28.3, 28.1, 25.6, 24.4, 21.2, 20.3, 20.2, 19.5, 18.3, 16.8, 12.0; IR (film): 2958, 2925, 2326, 2125, 1643, 1457, 1379, 1016 cm −1 ; MS (ESI): m/z 585 [M+Na] + . 
         [0000]    
       
                 
         
             
             
         
       
     
         [0042]    Additional disclosure is found in Appendix-A, Appendix-B, and Appendix-C filed herewith, entirety of each of which is incorporated herein by reference into the present disclosure. 
         [0043]    Those skilled in the art will recognize that numerous modifications can be made to the specific implementations described above. The implementations should not be limited to the particular limitations described. Other implementations may be possible. 
         [0044]    While the inventions have been illustrated and described in detail in the drawings and foregoing description, the same is to be considered as illustrative and not restrictive in character, it being understood that only certain embodiments have been shown and described and that all changes and modifications that come within the spirit of the invention are desired to be protected.