In vivo Raman measurement of levofloxacin lactate in blood using a nanoparticle-coated optical fiber probe

: Monitoring drug concentrations in vivo is very useful for adjusting a drug dosage during treatment and for drug research. Specifically, cutting-edge “on-line” drug research relies on knowing how drugs are metabolized or how they interact with the blood in real-time. Thus, this study explored performing in vivo Raman measurements of the model drug levofloxacin lactate in the blood using a nanoparticle-coated optical fiber probe (optical fiber nano-probe). The results show that we were able to measure real-time changes in the blood concentration of levofloxacin lactate, suggesting that this technique could be helpful for performing drug analyses and drug monitoring in a clinical setting without repeatedly withdrawing blood from patients.


Introduction
Therapeutic drug monitoring could help clinicians individualize drug treatment and guide dosages such that systemic drug concentrations optimally reach levels associated with therapeutic efficacy, which in turn would reduce the risk of concentration-dependent adverse effects [1]. In pharmacokinetics, clinical pharmacology, and biopharmaceutical research, drug concentrations are mainly measured by chromatography, immunoassays, isotope labeling, and microbiological assays. Chromatography techniques mainly include colorimetric and fluorescence analysis, high performance liquid chromatography (HPLC) [2,3], and chromatography techniques that combine mass spectrometry [4,5]. Chromatography techniques that employ ultraviolet detectors are simple and fast, but they show poor selectivity and sensitivity, making them unsuitable for measuring low drug concentrations. By contrast, immunoassays, isotope labeling, and microbiological assays are accurate, but these techniques are time-consuming and laborious [5][6][7][8][9][10].
One technique that has considerable potential for rapid and sensitive detection is surfaceenhanced Raman scattering (SERS) [11], which provides detailed molecular-level information [12]. SERS is able to measure drug concentrations of furazolidone and malachite green at concentrations of 1 μg.g-1 and 200 ng.g-1, respectively [13], enrofloxacin at 0.01 mg.kg-1 [11], and blood glucose at 5 mmol/l [14]. In addition, SERS technology has been applied in biomedical imaging.
Thus, SERS technology could potentially be very beneficial for biopharmaceutical analyses, which require large amounts of data for clinical pharmacological analysis as well as fast, accurate, and highly selective and sensitive analysis methods. In addition, analysis methods that can be done in vivo and on-line are even more advantageous, as they offer the potential for real-time monitoring of drug concentrations. Therefore, this study investigated a SERS-based drug analysis method that can be performed in vivo and in real-time. Specifically, we measured concentrations of the model drug levofloxacin lactate (a fluoroquinolone that inhibits bacterial DNA replication, often used to prevent infectious diseases [15,16]) in vivo and on-line using a gold nanoparticle-coated optical fiber (optical fiber nano-probe). Resulting analyses of the Raman spectrum intensities corresponding to the in vivo drug concentrations indicate that these techniques provides useful information for pharmaceutical detection, and might help advance personalized drug treatment strategies.

Preparation of the optical fiber nano-probe
The optical fiber nano-probe was fabricated for Raman spectra measurements [17]. The optical fiber was pulled to form a tapered shape and etched with hydrofluoric acid (HF) to make the tip size less than 100 nm. Then, gold nanoparticles were coated onto the surface of the optical fiber tip using an electrostatic self-assembly technology. The fiber was cleaned with sulfuric acid and hydrogen peroxide for 30 min, then soaked in the mixture solution with 5%v/v deionized water, 5%v/v (3-Aminopropyl) trimethoxysilane (APTMS, 97%) and 90%v/v ethanol for 30min, and then incubated at 90° for 30 min. The fiber was immersed in the gold sol for 48h after the fiber washed thoroughly with deionized water and ethanol [18]. The optical fiber nano-probe was inserted into the needle and situated near the needle tip for protection ( Fig. 1).

Preparation of the in vivo experiments
The SD rat, about 326g weight, were obtained from Laboratory of Neuropharmacology and Neurotoxicology, Shanghai University. The rat was housed with temperature range from 20°C to 25 ° C. The rat was fed with commercial aseptic food and tap water ad libitum throughout the experimental period. The rat was anaesthetized with chloral hydrate. The optical fiber nano-probe (~12-cm long), protected by the syringe needle, was inserted into the tail vein of an anaesthetized rat; the other end of the fiber was connected to the confocal Raman spectrometer and then coupled to the 10 × objective lens. The model drug levofloxacin lactate (1 ml 0.1 g/ml) was injected into the rat by intraperitoneal injection and the concentration of the drug in blood was measured through the optical fiber.

SERS measurement setup
Once the optical fiber nano-probe was in place, a Renishaw Raman microscope employing a 10 × objective and 633-nm excitation was used to obtain all of the spectra. The SERS spectra were acquired with a 10-s integration time and in the range of 900 -1800 cm −1 . A 10 × objective lens (numerical aperture, NA = 0.25) was close to the tip of the multimode optical fiber (core diameter of 50 μm, NA = 0.20). The experimental setup for in vivo experiments is shown in Fig. 2.

The Raman spectra measurements of levofloxacin lactate in vivo
The in vivo Raman spectrum of the drug detected by the optical fiber nano-probe is shown in Fig. 3, where curve a represents the raw data, while curve b shows the resulting curve once the baseline is subtracted. The in vivo spectra obtained by the optical fiber nano-probe show Raman peak positions for levofloxacin lactate at 1404 cm −1 , 1574 cm −1 , and 1621 cm −1 . Among them, the 1404 cm −1 and 1621 cm −1 are the characteristic peaks of levofloxacin lactate [17]. Other characteristic peaks of levofloxacin lactate could have potentially been shifted by an interaction between the drug and the albumin in the blood, which may affect the drug's molecular conformation [19,20]. After the intraperitoneal injection of levofloxacin lactate, the Raman spectra were measured in vivo and in real-time using the optical fiber. The resulting Raman signals of levofloxacin lactate are shown in Fig. 4. The Raman intensity of characteristic peak at 1621 cm −1 was the strongest at the 21st minute, but then gradually became weaker with time. After the 1621 cm −1 peak disappeared, the Raman intensity of peak 1604 cm −1 appeared, after which it became stronger and then decreased with time. By 25 minutes, the peak at 1621 cm −1 was decreasing gradually, but the peak at 1604 cm −1 had been increasing, and reached its maximum. Thus, the Raman peak of levofloxacin lactate in blood seems to have shifted from 1621 cm −1 to 1604 cm −1 over time. This indicates that the drug may happen to interact with blood components such as the proteins. The peak at 1621 cm −1 disappeared by the 25th minute, which indicates that the drug may be cleared from the body's circulatory system at that time.
The Raman peak at 1404 cm −1 indicates the symmetric stretching vibrations of O-C-O. The stronger levofloxacin lactate peak at 1621 cm −1 is attributed to C = C stretching and C = N stretching.
From Fig. 4, it seems that the optical nano-probe may be associated with some time-delay in the measurements, since the drug peak only appeared at the 10th minute, and the strongest peaks were reached at the 20th minute. Raman spectroscopy measurements with optical fiber nano-probes could potentially be in vivo as a diagnostic tool, which would be helpful for performing real-time analyses in current clinical procedures and avoiding long time waiting. In vivo Raman spectroscopy may fulfil important roles in a variety of different medical applications [21]; for example, optical fiber probes have been used for Raman measurements of inner arterial plaque depositions [22] and for imaging cancer in vivo, where nanoparticles are used as the Raman tags [23,24]. In addition, a clinical Raman system using an optical fiber probe has been used to perform in vivo Raman spectroscopy of the esophageal and peripheral arteries [25][26][27]. Thus, Raman spectra can be applied to in vivo imaging using multimode and multifunctional nanoparticles or probes.

Conclusions
The concentration of levofloxacin lactate in the blood was measured in vivo and in real-time using an optical fiber nano-probe. In this experiment, the typical levofloxacin lactate peaks changed in intensity over time. Because these results showed that the optical fiber nano-probe was able to detect the typical Raman spectra peaks of levofloxacin lactate, this system may represent a useful tool for measuring drug concentrations in vivo and in real-time and potentially allowing for rapid analyses. Such rapid analyses would be useful for predicting an individual's drug exposure level and the corresponding clinical effects, and may also be helpful for studying the in vivo interactions that occur between drugs and components of the blood. Overall, this experiment showed that nanoparticle-coated optical fibers have the potential possibility to be used in real-time detection of the model drug levofloxacin lactate, indicating that this technique might represent a new and effective measurement method for pharmaceutical investigation of some drugs with stronger SERS spectra signals in the future.