Novel pH-Sensitive Cyclic Peptides

A series of cyclic peptides containing a number of tryptophan (W) and glutamic acid (E) residues were synthesized and evaluated as pH-sensitive agents for targeting of acidic tissue and pH-dependent cytoplasmic delivery of molecules. Biophysical studies revealed the molecular mechanism of peptides action and localization within the lipid bilayer of the membrane at high and low pHs. The symmetric, c[(WE)4WC], and asymmetric, c[E4W5C], cyclic peptides translocated amanitin, a polar cargo molecule of similar size, across the lipid bilayer and induced cell death in a pH- and concentration-dependent manner. Fluorescently-labelled peptides were evaluated for targeting of acidic 4T1 mammary tumors in mice. The highest tumor to muscle ratio (5.6) was established for asymmetric cyclic peptide, c[E4W5C], at 24 hours after intravenous administration. pH-insensitive cyclic peptide c[R4W5C], where glutamic acid residues (E) were replaced by positively charged arginine residues (R), did not exhibit tumor targeting. We have introduced a novel class of cyclic peptides, which can be utilized as a new pH-sensitive tool in investigation or targeting of acidic tissue.

might be formed only in a result of stacking of aromatic amino acids due to the cyclic peptide's aggregation to form tubular structures. Thus, we concluded that at the concentrations of the peptides used in this study, the formation of tubular structures is unlikely.
By monitoring shift of the position of the maximum of fluorescence spectra for the peptides in the result of the pH drop, we established apparent pK of peptide's partitioning into the bilayer. The pK for most cyclic and linear peptides varied in the range of 5.7-6.4, while the smallest pK value was observed for the cyclic Leu-containing peptide, c[(LE) 4 WC] (Fig. 2).
To establish localization of the peptides within a lipid bilayer of membrane, dual quenching assay 28 was employed ( Fig. 3 and Table S3). Effective quenching of fluorescence by acrylamide would occur only for tryptophan residues exposed to polar parts of the outer or inner leaflets of the bilayer. At the same time, tryptophan residues located in the middle of a membrane would be effectively quenched by 10-DN. The result of the dual quenching assay allows establishing if tryptophan residues were located in the middle of a membrane or close to the polar headgroups of the bilayer. However, it does not allow distinguishing between locations at the outer or inner leaflets of the bilayer. Therefore, we also performed Förster resonance energy transfer (FRET) assay 29 (Fig. 4). First, symmetrically-labelled by NBD dye, POPC liposomes were prepared. Then, membrane-impermeable dithionite was used to chemically modify exposed NBD, which led to the quenching of NBD fluorescence. Thus, NBD dyes located at the outer leaflet of the bilayer were deactivated and excess of dithionite was removed by gel filtration. As a result, asymmetrically-labelled liposomes with active NBD at the inner leaflet were obtained. FRET was monitored from tryptophan residues of the peptides to NBD. Energy transfer might occur only when both fluorophores are in proximity to each other (within 5-15 Å). Thus, when tryptophan residues located at the outer leaflet of the bilayer no significant energy transfer to NBD at the inner leaflet would occur (the distance is about 50-60 Å). The c[E 4 W 5 C] demonstrated the highest quenching by 10-DN and the highest FRET at pH 8 indicating the internal position of Trp residues within the bilayer of a membrane. All other peptides were located at the outer leaflet of the bilayer at pH 8. Drop of pH promoted partitioning of all cyclic peptides into bilayer, which highest FRET for the c[E 4 W 5 C] at pH 3. Among c[(WE) 3 WC], c[(WE) 4 WC] and c[(WE) 5 WC] peptides, the peptide with the smallest cycle, c[(WE) 3 WC], showed the deeper partitioning into the membrane. Linear peptide, l(CW(EW) 4 ), also demonstrated some partitioning into bilayer in the result of the pH drop.
Since all peptides exhibited pH-dependent interactions with the lipid bilayer of membrane and no cytotoxicity was observed ( Figure S3), we proceeded to the experiments on cultured cancer cells. Symmetric, c[(WE) 4 WC], and asymmetric, c[E 4 W 5 C], cyclic peptides were evaluated for their ability to move polar cargo across the membrane. The experiments on live cells were performed at physiologically relevant pHs, such as pH 7.4 (extracellular pH of normal cells) and pH 6.0 (extracellular pH in the vicinity of cancer cells 12 ). As a polar cargo, we used amanitin, which is a cell-impermeable cyclic peptide of molecular mass similar to the masses of investigated pH-sensitive cyclic peptides. Amanitin is a deadly toxin, which inhibits RNA polymerase II if transferred across the lipid bilayer of the plasma membrane 30 . The pH-and concentration-dependent cell death was observed after treatment  (Fig. 5a,b). The calculated IC 50 values for symmetric cyclic peptide amanitin construct, c[(WE) 4 WC]-S-S-amanitin, at pH 7.4 and 6.0 were 3.39 ± 0.12 μ M and 1.06 ± 0.02 μ M, respectively. The calculated IC 50 values for asymmetric cyclic peptide amanitin construct, c[E 4 W 5 C] -S-S-amanitin, at pH 7.4 and 6.0 were 1.68 ± 0.13 μ M and 0.74 ± 0.08 μ M, respectively. Previously we showed that amanitin alone does not induce cell death at the concentrations used in this study and for the duration of treatment of 2-4 hours 31 . We also tested construct, where amanitin was conjugated to the asymmetric c[E 4 W 5 C] cyclic peptide via non-cleavable bond (c[E 4 W 5 C]-GMBS-amanitin) (Fig. 5c). The cytotoxic effect for the non-cleavable construct was reduced significantly at both pHs. It might indicate that the peptide-amanitin construct remained within the membrane and cleavage of amanitin from the peptide was required to allow amanitin to reach RNA polymerase II in the nucleus. Alternatively, if peptide-amanitin was translocated into the cytoplasm, the cleavage of amanitin might be required, since affinity of the peptide-amanitin to the RNA polymerase II might be reduced compared to the affinity of free amanitin to the RNA polymerase II.  Based on the obtained results, we proposed that at high/normal pH cyclic peptides were located at the outer leaflet of the bilayer. Triggered by pH drop the protonation of Glu residues enhanced peptide's hydrophobicity and induced partitioning of the pepetides into the bilayer of the membrane. This assumption was further confirmed by quenching of fluorescence of FITC-labelled asymmetric cyclic peptide by cell impermeable Trypan Blue (Fig. 5d). Trypan Blue is used to quench fluorescence of FITC located in the extracellular space 32 . Cells treated with the FITC-labelled peptide at low pH followed by Trypan Blue quenching showed higher fluorescent signal on cells opposed to the cells treated with the FITC-labelled peptide at normal pH followed by Trypan Blue quenching. It indicates that at normal pH FITC is more exposed to extracellular space (to Trypan Blue) in contrast to low pH treatment. Since FITC fluorescence is a pH-senstive and significantly quenched at low pH (pH 5.0-5.5) in endosomal compartment, we concluded that the FITC-labelled peptide was localazed at the inner leaflet of the plasma membrane rather than in endosomes. Also, the cellular uptake of Alexa546-labelled c[E 4 W 5 C] peptide at normal and low pHs in presence and absence of FBS was investigated (Fig. 5e). We observed statistically significant difference in cellular uptake of peptide at normal and low pHs, and no reduction of the uptake in the presence of FBS.
Next we proceeded to animal studies to identify the lead peptide demonstrating best tumor targeting. The murine 4T1 xenograft model, which closely mimics stage IV of human breast cancer 33,34 , was used in our study. Small 4T1 tumor (tumor volume < 150 mm 3 ) generates a significant level of lactate and serves as a good model of an aggressive, acidic tumor 35 . All peptides were covalently conjugated with Alexa546-malemide. The fluorescent constructs were given as a single IV injection, and at 4 hours after administration, animals were euthanized followed by necropsy. The mean fluorescence of tumor, muscle, kidney, liver and lungs were recorded and analyzed (Fig. 6). The least targeting was observed for Leu-containing peptide, c(LE) 4 CW. At the same time, the highest tumor targeting was monitored for linear peptide, l(CW(EW) 4 ), symmetric, c[(WE) 4 WC], and asymmetric, c[E 4 W 5 C], cyclic peptides.
To prove pH-dependent tumor targeting of WE cyclic peptides we tested positively-charged asymmetric cyclic peptide, c[R 4 W 5 C], where Glu residues were replaced by Arg residues. This WR-peptide interacted with the lipid bilayer of the membrane at high pH ( Figure S4) due to the presence of Trp residues, similar to WE-peptide. At the same time, it does not exhibit pH-dependent changes in interaction with the lipid bilayer of membrane, thus it can serve as a pH-insensitive control for pH-sensitive WE-cyclic asymmetric peptide, c[E 4 W 5 C].
We performed side-by-side 4T1 tumor targeting and biodistribution investigation of asymmetric pH-sensitive, c[E 4 W 5 C], and pH-insensitive, c[R 4 W 5 C], peptides. A significant difference in tumor targeting was observed at all time points after IV administration of the constructs (Figs 7a and S5). It resulted in a significant difference between tumor/organ ratios for pH-sensitive and insensitive peptides ( Fig. 7b and Table S4). The accumulation of the pH-insensitive peptide in tumor and other organs was very minimal and varied in the range of 0.8 to 1.6 values, which could be attributed to the passive diffusion with blood flow.

Discussion
We have introduced a novel class of cyclic peptides, which demonstrate pH-sensitive interaction with lipid bilayer of membrane of liposomes, cultured cancer cells and tumors in animal model. We tested six cyclic peptides of three different cycle sizes, and different localization of Trp, Glu, Leu and Arg residues within the cycles. The size of investigated peptides is an optimal for the straightforward synthesis. The significantly smaller or larger sized cycles are more challenging to synthesize. The interaction of cyclic peptides with lipid bilayer of membrane has different mechanism compared to the action of linear pH-sensitive peptides such as pHLIP ® peptides. Negatively charged Glu residues of cyclic peptides, especially asymmetric one, which showed the best pH-dependent performance, are exposed to the aqueous solution at normal pH. At the same time, indole rings of Trp residues most probably interact with the lipid headgroups (Figs 8 and S6). It was demonstrated previously that aromatic Trp residues have high affinity to lipid headgroups 11,36-39 . Our biophysical titration data indicate that c[E 4 W 5 C] cyclic peptide partitions into the lipid bilayer at pH 8. About 7.3 kcal/mol of free energy is released during the process ( Figure S7), which indicates on strong interactions of the peptide with the membrane. The high affinity of the peptides to the cellular membranes at normal pH might explain long time of circulation in mice. When pH is lowered, Glu residues are protonated. The pK of Glu residue protonation in the vicinity of the hydrophobic membrane is higher compared to the pK in a solution. Protonation leads to the increase of hydrophobicity of the peptide, which promotes partitioning of the peptide into the bilayer. As a result, about 0.7 kcal/mol of additional free energy is released at low pH ( Figure S7). This energy could be used to move cell-impermeable cargo, such as amanitin, across membrane and target acidic tumors. Because Trp residues have higher affinity to the headgroups region compared to the central hydrophobic part of the bilayer, the peptide is equilibrated in the regions of headgroups between inner and outer leaflets of the bilayer. We and others showed that pH equilibrates fast inside a liposome 40,41 . Thus, an equal amount of the peptide molecules is distributed between both leaflets of the liposomal membrane with low pH outside and inside of it ( Figure S6). However, in the case of live cells, pH inside a cell is normal (7.2-7.4), while bulk extracellular pH is slightly lower (6.5-6.8) [42][43][44] . However, the pH is at its lowest at the surfaces of cells 9 and increases with distance from the cellular membrane, becoming normal in the vicinity of blood vessels 45 . Recently, we were able to measure in vitro and in vivo pH in the vicinity of plasma membrane of cancer cells, the pH values drops to 6.0-6.5 at the surface of cancer cells 12 . The most aggressive cancer cells are the most acidic. Thus, peptides reaching inner leaflet of the bilayer could expose their Glu residues to the cytoplasm, where they are expected to be de-protonated and became charged again. It would reduce the rate of the peptide diffusion back into the membrane and should lead to the shift of the equilibrium toward the accumulation of the peptides at inner leaflet of bilayer of the plasma membrane of cells (Fig. 8).
Thus, cyclic peptides could be considered as a weak acid with multiple protonatable groups, which can diffuse across the bilayer. For weak acids, the intracellular-extracellular distribution, C i /C e , should be calculated according to the following equation: where pH i and pH e are the intracellular and extracellular pH values, respectively. Since the cyclic peptides have affinity to the membrane, we consider C i and C e as the concentrations of the peptide on inner and outer leaflets, respectively. We established the pKa of membrane partition for the asymmetric cyclic peptide, which equals to 5.7. The calculation shows that at pHe = 7.4 and pHi = 7.2 the concentration ratio at the inner and outer leaflets for asymmetric cyclic peptides is 0.6. However, the same ratio increases to 4.5, 7.8, and 10.9 if extracellular pHe would be 6.5, 6.2 and 6.0, respectively. We assume that the symmetrical WE peptides have the same mechanism of action as an asymmetric peptide, however they have less favorable localization of Trp and Glu residues, which reduces their ability to accumulate at the inner leaflet of bilayer of cellular membranes and target acidic tumors. Leu-containing peptides are less advantageous due to their reduced affinity to the headgroup part of the bilayer and high affinity to the center of the membrane. A novel class of pH-sensitive cyclic peptides containing tryptophan and glutamic acid residues have potential applications for targeting tumors and translocation of polar cargo molecules across cellular membrane. These peptides might have application not only in targeting of acidic diseased tissue, they might find very interesting applications in cosmetics. It is well known that the natural skin surface pH is on average below 5 46 . Thus, topical application of short (8-10 residues long), very stable cyclic peptides might open an opportunity to tether various cosmetic and skin care products to the skin surface.  Methodology of Peptide Synthesis. In brief, the linear protected peptide was first assembled. Direct cleavage of peptide-attached resin in the presence of AcOH/TFE/DCM (1:2:7 v/v/v) generated the linear peptide. Cyclization in dilute condition using DIC/HOAt in DMF/DCM solution for 12 hours, followed by the deprotection of the side chain by using cleavage cocktail (TFA:thioanisole:anisole:EDT (90:5:2:3 v/v/v/v) afforded the cyclic peptide. The peptides were synthesized by employing the N-(9-fluorenyl)methoxycarbonyl (Fmoc)-solid phase chemistry using PS3 automated peptide synthesizer (Rainin Instrument Co., Inc.) at room temperature. The peptide sequence was assembled on preloaded amino acid on 2-chlorotriyl resin using coupling, activating, and deprotecting reagents using HBTU, N-methylmorpholine (0.4 M), and piperidine in DMF (20% v/v), respectively. The amino acids in the peptide sequence were coupled using coupling reagents and activating reagent in DMF for 1 hour followed by washing with DMF 3 times. The deprotection was carried using piperidine (20%, v/v) in DMF for 2 times, 10 minute for each time, followed by washing with DMF (3 times). The appropriate sequence of linear protected peptide was assembled using the synthesizer. N-to C-terminal cyclization of peptide were achieved by cleavage of protected peptidyl resin by stirring the peptidyl resin in freshly prepared cleavage cocktail of AcOH/TFE/DCM (1:2:7, v/v/v) for 1 hour at room temperature followed by washing the resin with TFE:DCM (2:8 v/v, 2 times). The collected filtrate was evaporated using a rotary evaporator followed by azeotropic removal of acetic acid by addition of hexane and dichloromethane to afford high viscous liquid or solid-protected linear peptide. The crude linear protected peptide was dissolved in excess of solvents DMF:DCM (4:1 v/v) followed by the addition of HOAt/DIC (1:1.1 equiv) for cyclization for 12-48 hours confirmed by MALDI TOF-TOF mass spectrometry. The solvent was evaporated under high reduced pressure in a rotatory evaporator at 40-45 °C to remove DMF. The final cleavage of side chain protection from the peptide were carried out after confirming the peptide cyclization by MALDI mass spectrometer data by shaking the cyclized peptide mixture in cleavage cocktail reagent R (TFA/thioanisole/anisole/EDT (90:5:2:3 v/v/v/v, 10-15 mL) for 2-4 hours followed by precipitation of peptide using cold ether, centrifugation at 2500 rpm and washing with excess of cold ether at 25 °C for 5 min. The crude peptide was purified with semi preparative reversed phase high performance liquid chromatography (RP-HPLC) by using Hitachi L-2455 on a C18 Phenomenex Prodigy reversed-phase column (10 μ m, 250 cm × 21.2 cm). The pure peptide was eluted at 15.0 mL/min using a gradient of binary solvent system using water and acetonitrile with 0.1% TFA for 0-100% over 60 min. The pure collected peptide fractions were pooled and lyophilized to provide solid powder in purity of ≥ 98%. All peptides were characterized by using high resolution time of flight AXIMA-performance MALDI TOF-TOF mass spectrometer (Shimadzu). The above mentioned protocol was applied for the synthesis of all cyclic peptides. The concentration of the peptides was calculated spectrophotometrically by measuring absorbance at 280 nm. The extinction coefficients, ε 280 , M −1 cm −1 , for the peptides are the following: c[(WE) 4  Synthesis of Cyclic Peptides. The linear peptide sequence was synthesized on PS3 automated synthesizer as described above in the scale of 0.3 mmol. H-Trp(Boc)-2-chlorotrityl resin (384.6 mg, 0.3 mmol, 0.78 mmol/g) was swelled in DMF, followed by coupling and deprotection cycles to assemble respective amino acids on the peptidyl resin using respective amino acids, such as Fmoc-Glu(OtBu)-OH (382.9 mg, 0.9 mmol), Fmoc-Trp(Boc)-OH (473.9 mg, 0.9 mmol), Fmoc-Cys(Trt)-OH (527.1 mg, 0.9 mmol), and HBTU (341 mg, 0.9 mmol) as the coupling reagent. Fmoc group of N-terminal in the peptidyl resin was removed using deprotection cycle, and the resin was transferred to 100 mL round bottom flask. The linear protected peptide was cleaved by shaking peptidyl resin in cleavage cocktail AcOH/TFE/DCM (1:2:7 v/v/v, 50 mL) for 1 hour followed by washing the resin using TFE:DCM (2:8 v/v, 10 mL, 2 times). The combined filtrate was evaporated to dryness with the subsequently addition of hexane (50 mL × 3) and DCM (10 mL × 3) to remove acetic acid, which provided solid white crude protected peptide ready for cyclization. The cyclization was carried out by dissolving the solid peptide in anhydrous DMF/DCM (250 mL, 4:1 v/v) under nitrogen using DIC (155.0 μ L, 0.99 mmol) and HOAt (122.5 mg, 0.9 mmol) with stirring at room temperature for 24 hours. The cyclized product was confirmed by taking a small aliquot of the reaction mixture and cleavage with reagent R and using MALDI. After cyclization was confirmed, the solvents were evaporated under high reduced pressure, and the side chain protections were removed by addition of cleavage cocktail of reagent R, TFA/thioanisole/anisole/EDT (15 mL, 90:5:2:3 v/v/v/v), and shaking at room temperature for 3 hours. The peptides were precipitated, centrifuged, and washed with cold diethyl ether to yield the crude white solid peptide. The peptides were dissolved in H 2 O/CH 3 CN with 0.1% TFA and purified using RP HPLC. Then, the pure fractions were collected, concentrated and lyophilized to afford pure solid white powder of c[(WE) 4 Figure 8. The cyclic peptide molecules distribution between outer and inner leaflets of the lipid bilayer of plasma membrane. At neutral and high pHs, Glu residues are negatively-charged (red circles). Trp residues (green circles) interact with polar headgroups. Cys residue (yellow circle) could be directed into bilayer or away depending on cargo hydrophobicity conjugated with Cys. The majority of cyclic peptides could be found on the outer bilayer of plasma membrane of normal cells compared to the inner bilayer due to the small pH gradient (pHe = 7.4 and pHi = 7.2). A drop of a pH leads to the protonation of Glu residues (blue circles), which enhances peptides hydrophobicity and induces partition into the bilayer.  4 ). The linear peptide was assembled as described above using H-Trp(Boc)-2-chlorotrityl resin (384.6 mg, 0.3 mmol 0.78 mmol/g) in reaction vessel. The peptide sequence was assembled using the appropriate amino acid building blocks Fmoc-Glu(OtBu)-OH (382.9 mg, 0.9 mmol), Fmoc-Trp(Boc)-OH (473.9 mg, 0.9 mmol), Fmoc-Cys(Trt)-OH (527.1 mg, 0.9 mmol), and HBTU (0.9 mmol, 341 mg) as the coupling reagent. The final N-terminal Fmoc group was deprotected. The peptide was cleaved from the resin and side chain was deprotected by reaction of the peptidyl resin with freshly prepared cleavage cocktail reagent R, TFA/thioanisole/anisole/EDT (15 mL, 90:5:2:3 v/v/v/v), for 3 hours at room temperature. The linear protected peptide was precipitated, centrifuged, and purified by using RP-HPLC as mentioned above to yield l(CW(EW) 4  Labeling of Peptides with Fluorescent Dyes. Peptides were conjugated with Alexa546-and Fluorescein-5-maleimide (Life Technologies) in DMF at a ratio of 1.2:1 and incubated at room temperature for about 6 hours and then at 4 °C until the conjugation reaction was completed. 50 mM of sodium phosphate/150 mM NaCl buffer pH 7.0 (saturated with argon) was added to the reaction mixture (1/10 of the total volume). The reaction progress was monitored by the reverse phase HPLC. The products were purified by the reverse phase HPLC, lyophilized and characterized by SELDI-TOF mass spectrometry. The concentration of the constructs was determined by absorbance at 556 and 494 nm using molar extinction coefficients of 93,000 M −1 ·cm −1 for Alexa546 and 68,000 M −1 ·cm −1 for Fluorescein-5 (FITC).
where R is the gas constant and T is the temperature in Kelvin.
Dual Quenching. POPC liposomes without and with 10% of the lipids replaced by 10-doxylnonadecane To observe quenching of tryptophan fluorescence by 10-DN or acrylamide, the tryptophan fluorescence was recorded as described above. The appropriate POPC blanks were measured and subtracted from the measured spectra before analysis. The percentage of quenching was calculated.
Next, 1.2 mL of 6 mM of symmetrically NBD-labelled POPC liposomes were incubated with 150 μ L of 1 M freshly prepared membrane-impermeable dithionite in buffer at pH 8.0 to chemically deactivate of NDB only at the outer leaflet of the bilayer and obtain asymmetrically NBD-labelled POPC liposomes. The decrease of NBD fluorescence occurring in the result of quenching of NBD by dithionite was monitored at the excitation of 463 nm and emission at 530 nm. The dithionite quenching led to the reduction of about 60-65% of NBD fluorescence signal corresponding to the quenching of NBD at the outer leaflet of the bilayer. Next, POPC solution was passed through a G-10 sephadex (Sigma-Aldrich) fast spin column to remove the excess of dithionite. Asymmetrically labelled POPC liposomes were incubated with peptides at concentrations indicated above, and FRET from tryptophan residues to NBD at the inner leaflet of bilayer was monitored at 280 nm excitation wavelength, and emission was recorded from 310 to 580 nm. Proliferation Assay. HeLa cells were loaded in the wells of 96-well plates (~5,000 cells per well) and incubated overnight. Growth medium was replaced with the medium without FBS pH 6.0 or pH 7.4 containing increasing amounts of peptide-amanitin construct (0.5, 1, 2, and 4 μ M). The construct was removed from cells after 3 hours. After 48 hours of cell incubation in standard growth medium, a colorimetric reagent (Cell Titer 96 AQ ueous One Solution Assay, Promega) was added for 1 hour followed by measurements of absorbance at 490 nm to assess cell viability. All samples were prepared in triplicate. 5 times at pH 7.4 and 0.4% Trypan Blue was added for 5 min (1/10 of the total volume). Fluorescent images were acquired with a Retiga CCD camera (Qimaging) mounted to an inverted Olympus IX71 microscope (Olympus America, Inc.). Cellular uptake. HeLa cells (150,000 cells per sample of total volume of 500 μ L) were incubated with 5 μ M of Alexa546-labelled peptide in Leibovitz's (L15) medium at pH 6.2 and 7.4 in the presence or absence of 4% of fetal bovine serum (FBS) for 6 hours, followed by extensive washing with L15 medium. The cellular uptake of the constructs was measured by fluorescent signal from cells counted using cellometer (Cellometer Vision CBA, Nexcelom). 4% of FBS was used to mimic the amount of albumin in whole blood, which contains 45% of red blood cells, white blood cells and platelets suspended in plasma (about 55% of volume). Plasma is composed of about 92% of water, 1% of vitamins, sugars, salts, minerals, hormones and 7% of vital proteins including albumin, gamma globulins and other clotting factor. Thus, the amount of albumin in whole blood is expected to be less than 3.6%.

Fluorescent
Ex Vivo Fluorescence Imaging. All animal studies were conducted according to the animal protocol Statistical analysis. Statistically significant differences were determined by two-tailed unpaired Student's t-test (p-level < 0.05 was taken as significant).