1.

Introduction

Nowadays, biomedical photonics plays a significant role in the research of optical diagnosis, biomedical imaging, laser therapy, and so on. The constituents of biotissue not only include low-refractive-index (RI) interstitial fluid and cytoplasm,¹ but also some high-RI components, such as cell membrane, cell nucleus, organelles, and melanin. The RI mismatch between high-RI and low-RI components that limit the penetration depth of light in tissue^2,3 is the origin of tissue scattering. The optical clearing (OC) technique proposed by Tuchin et al.⁴ is an effective method to reduce the RI mismatches with a high-RI agent, which is commonly designed as an optical clearing agent (OCA). An OCA must meet the need of being a nontoxic for producing no side effects when it is applied on the biotissues. Glycerol,^5–8 polyethylene glycol 400 (PEG400),⁹ glucose,^10–14 propylene glycol,^15–17 and others have often been used in OC treatments of different biological tissues ^3–8 and other tissues, such as skull and cartilage, and so on.^18–20 OC begins with OCA application to the tissues. OCAs usually create a transparency effect on tissues by the combination of some reported mechanisms such as the dehydration mechanism,^17,21 the refractive index matching mechanism,^{3,7,10,13,22–27} and in some cases collagen solubility.^13,28–31 Meanwhile, to detect the efficiency of OCAs on OC treated tissue, the integrating sphere technique,^9,32–36 the derivative total reflection method,^37–40 the resolution target imaging,⁴¹ CCD microimaging,⁴² and optical coherence tomography (OCT) ^15,43,44 have been proposed.

Stratum corneum (SC), the outermost layer of the epidermis, is the main component that hinders OCAs from infiltrating into the skin. To weaken the inhibition of skin, some chemical agents that can either modify the SC’s structure reversibly or reinforce the fluidity of SC are applied on the surface of skin, such as azone,^33,45 thiazone,^33,43,44,46 oleic acid,^47,48 and dimethyl sulfoxide (DMSO).^49–52 Thiazone, one of the derivatives of azone, has been reported as an effective permeation enhancer to the OC treatment of skin. The infiltration capacity of thiazone is nearly 2.99 times higher than that of azone.⁵³ It was reported that the efficiency of OC on human dorsal hand skin increased roughly 33.33% when treated with 0.25% thiazone PEG400 solution.⁴⁴

Numerous studies have been done to promote the skin’s OC while decreasing the limit of SC. However, the SC thickness varies from 10 to $40\mu \mathrm{m}$,⁵⁴ even though the epidermis thickness is nearly 0.1 mm.^55,56 It has been indicated that the $Z_{\text{threshold}}$, the depth at which the OCT signal dropped below a threshold signal of ${10}^{-5}$ reflectance, increased from 0.219 to 0.275 mm for rat skin during a 15-min application of 5% thiazone-PEG400,⁴³ and the shallow $1/e$ light penetration depth that was detected by OCT increased from 0.35 to $0.47\pm 0.02\mathrm{mm}$ for human skin during a 60-min treatment of 0.25% thiazone-PEG400.⁴⁴ This reveals that thiazone mixed with OCAs can easily enhance diffusion through the skin and quickly have an effect on the subcutaneous muscle tissue. Although several reports have discussed the impact of OC on muscle treated with different OCAs,^{7,11,13,23–25,39,40} an understanding of the influence of the clearing on muscle exposed to permeation enhancers, such as thiazone and azone, has remained incomplete.

In this study, we use the integrating sphere technique and a CCD microimaging system to investigate the OC efficiency of different concentrations of thiazone–propylene glycol solution on in vitro porcine muscle and skin, respectively. The efficiency of OC on skin fits well with previous studies,^44–46 whereas the result on muscle is quite different from others.¹¹ We discover that thiazone induced decrease of OC efficiency on muscle tissue and the remarkable inhibition effect of 10% thiazone–propylene glycol solution.

2.

Materials and Methods

2.3.

Spectral Measurement

A schematic diagram of the total transmittance measurement system is shown in Fig. 1. The sample was held between two slides of glass and then fixed by a plastic holder with a circle window. White light from a xenon lamp was transported through an aperture A before arriving the converging lens L. Collimated light coming out from L was divided into two parts after being transported through a splitter M. One part irradiated the detector D as the monitor and the other illuminated the sample. The transmittance was collected by a single integrating sphere and a spectrometer (Ocean Optics HR4000). Different agents and similar processes were applied on one side of the muscle and the epithelial surface of the skin. The residual agents on the surface of the sample were removed before measurements. Light was illuminated from the side that has been applied by agents.

Fig. 1

Schematic diagram of the total transmittance measurement system.

The tendency of the OC effect could be qualitatively evaluated from the total transmittance spectra, yet the quantitative information could not be obtained. We defined the increment of total transmittance ($\mathrm{\Delta }T$) at 563.5 nm to quantitatively calculate the transmittance. Total transmittance spectra were taken at a time interval of 5, 10, 15, 20, 30, 40 min, respectively. Each experiment was measured three times. The expression of $\mathrm{\Delta }T$ with different time intervals was given by⁴⁴

Eq. (1)

$$\mathrm{\Delta }T=\frac{T_x-T_0}{T_0}\times 100\%,$$

where $T_0$ and $T_x$ are the measured total transmittances at 563.5 nm for each group at the time intervals of 0 and $x$ min, respectively.

2.5.

Collimated Transmittance Measurement

PG, 1%T/PG, 3%T/PG solutions were mixed with water at a ratio of $1:9$, respectively, and experiments were divided into four groups: water, PG/W, 1%T/PG/W, and 3%T/PG/W. Mixtures were injected into a cuvette with a thickness of 1 mm. The schematic diagram of the collimated transmittance measurement system is shown in Fig. 2. White light from a xenon lamp was transported through an aperture A1 before arriving to the converging lens L. Collimated light coming out from L illuminated the cuvette (1 mm). The collimated transmittance was collected by a spectrometer (Ocean Optics HR4000) behind another aperture A2. The total attenuation coefficient was calculated using the Lambert–Beer law.

Fig. 2

Schematic diagram of the collimated transmittance measurement system.

3.

Results

3.1.

Measured Total Transmittance Spectra

3.1.1.

In vitro porcine muscle

The transmittance spectra of samples treated with different solutions were measured by an integrating sphere and presented in Fig. 3.

Fig. 3

Total transmittance spectra of fresh in vitro porcine muscle at a time interval of 5, 10, 15, 20, 30, and 40 min relative to (a) control group, (b) PG group (b), (c) 1%T/PG group, (d) 3%T/PG group, and (e) 10%T/PG group.

Due to the natural dehydration, a slight increment appears on the total transmittance of the control group [Fig. 3(a)]. In comparison with the control group, the total transmittance with PG treated increases absolutely during 40 min [Fig. 3(b)]. Interestingly, as illustrated in Fig. 3(c), the total transmittance of the 1%T/PG group at 5 min induces a similar increase compared with that of 1%T/PG group at 10 min. Meanwhile, the total transmittance of 3%T/PG group decreases at a time interval of 5 min [Fig. 3(d)]. For the sample treated with 10%T/PG, total transmittance reduces after agent treatment [Figs. 3(e) and 4], and an obvious decrease can be observed with a longer treatment.

Fig. 4

Increment in total transmittance at 563.5 nm of fresh in vitro porcine muscle with the treatment of nothing, pure PG, 1%T/PG, 3%T/PG,10%T/PG, at a time interval of 5, 10, 15, 20, 30, and 40 min, respectively.

To quantitatively analyze the effect of thiazone on the OC of fresh in vitro porcine muscle, $\mathrm{\Delta }T$ is evaluated by Eq. (1) and the results are shown in Fig. 4.

Statistics reveal that, compared with the control group, the $\mathrm{\Delta }T$ of PG group increases about 4 times after a 40-min treatment. Yet, in contrast with PG group, there is no such huge increase in the $\mathrm{\Delta }T$ of the 1%T/PG and 3%T/PG groups. The values of $\mathrm{\Delta }T$ in response to the PG, 1%T/PG, and 3%T/PG groups within a 40-min increase from 32.48%, 21.03%, and 0.58% to 127.24%, 57.12%, and 36.00%, respectively. The increasing rates of $\mathrm{\Delta }T$ of PG, 1%T/PG, and 3%T/PG groups are 2.71%, 1.03%, and 1.01% per min, respectively. Obviously, the thiazone-treated groups have a lower increase rate of $\mathrm{\Delta }T$. In the case of 10%T/PG group, $\mathrm{\Delta }T$ drops to $-0.07\%$ within the first 5 min, and shows a continually negative increase all the way between 10 and 40 min. It reveals that thiazone inhibits the OC efficiency of PG and increases the scattering of muscle. The 10%T/PG group induces the largest increase of scattering, whereas the 1%T/PG group induces the lowest. The experimental results support the inhibition effect of thiazone on OCA treatment of muscle, what is more, the extent of inhibition increases with the concentration of thiazone.

During the experiments of the 1%T/PG group, 3%T/PG group, and 10%T/PG group, we discovered that some white crystals emerged in muscle when agents were applied and the amount of crystals aggrandized with the increase of concentration of thiazone. To analyze the components of these crystals, CCD microimaging and x-ray single crystal diffractometer were preformed, which will be discussed later.

3.1.2.

In vitro porcine skin

To compare the OC effect of thiazone for muscle and skin, we measured the increment of total transmittance on in vitro porcine skin at 563.5 nm.

As shown in Fig. 5, for all chemical agent groups, $\mathrm{\Delta }T$ increases much faster than that of the control group. Statistics show that the PG group, 1%T/PG group, 3%T/PG group, and 10%T/PG group induce 14.66%, 18.96%, 21.30%, and 26.59% increases in $\mathrm{\Delta }T$ at 5 min, respectively, whereas only a 3.02% change was observed in the control group. Meanwhile, similar increases in OC were obtained for the 10%T/PG group past 5 min and control group past 30 min. This indicates that thiazone promotes the efficiency of OC on skin, and the efficiency of OC increases with the concentration of thiazone. The tendency of the OC effect on thiazone-treated skin agrees well with previous reports.^44,46 Compared with the effect of thiazone on OC or fresh porcine muscle, the effect on skin is quite different from that on muscle. Thiazone has no effect on the OC of muscle and even produces an inhibition effect at high concentrations, whereas it effectively promotes the OC efficiency of in vitro porcine skin. Hence, thiazone is not suitable for the OC treatment on muscle.

Fig. 5

Increment in total transmittance at 563.5 nm of fresh porcine skin in vitro with the treatment of nothing, pure PG, 1%T/PG, 3%T/PG, 10%T/PG, at a time interval of 5, 10, 15, 20, 30, and 40 min, respectively.

3.2.

CCD Microimaging and X-Ray Single-Crystal Diffractometer

Microimages of samples obtained by the CCD microimaging system at the time interval of 40 min are shown in Fig. 6, corresponding to (a) sample B of control group, (b) sample B of PG group, (c) sample A of 3%T/PG group, and (d) sample B of 3%T/PG group.

Fig. 6

The microimages of in vitro fresh porcine muscle by CCD camera at the time interval of 40 min. (a)–(d) Sample B of control group, sample B of PG group, sample A of 3%T/PG group, and sample B of 3%T/PG group, respectively.

For sample B of the control group and that of the PG group, though no crystal has been found, sample B of the PG group has a greater transmittance than that of the control group. Compared with the result of PG group, large quantities of crystals were observed on the surface of both samples A and B with the treatment of 3%T/PG. Thus, it indicates that thiazone-PG can easily penetrate through a 1 mm sample and crystals appear on both the outside and the inside of the muscle, which largely increases the scattering of samples.

To determine the type of crystals, a small amount of crystals is extracted and analyzed by x-ray single-crystal diffractometer (Rigaku 007 Saturn 70). Chemical ingredients, unit cell dimensions (Table 2), the structure refinement, and bond lengths show that the crystal appearing in the muscle is the single crystal of thiazone.

Table 2

Part of the report of crystals by x-ray single crystal diffractometer.

Formula	${\mathrm{C}}_{11}{\mathrm{H}}_{13}{\mathrm{NO}}_3\mathrm{S}$
Formula weight	239.28
Wavelength	0.71073 A
Unit cell dimensions	$a=7.107(4)$ A $\text{alpha}=103.824(7)\mathrm{deg}$
	$b=7.431(4)$ A $\text{beta}=91.717(8)\mathrm{deg}$
	$c=11.447(6)$ A $\text{gamma}=108.655(9)\mathrm{deg}$
Volume	$552.4(5){\mathrm{A}}^3$
$Z$, calculated density	2, $1.438\mathrm{Mg}/{\mathrm{m}}^3$
Absorption coefficient	$0.284{\mathrm{mm}}^{-1}$
$F$ (000)	252
Crystal size	$0.20\times 0.18\times 0.12\mathrm{mm}$
Limiting indices	$-9\le h\le 9$, $-9\le k\le 9$, $-14\le l\le 14$

3.3.

Calculation of the Total Attenuation Coefficient

To further prove the critical role of water content of muscle on the crystallization of thiazone, we perform the collimated transmittance measurement (Fig. 2). The attenuation coefficient of each group over the range from 400 to 800 nm is calculated using Lambert–Beer law. As shown in Fig. 7, the PG/W group and water group induce similar values. Compared with the PG group, the 1%T/PG/W group and 3%T/PG/W group induce an obvious increase in the attenuation coefficient, respectively. Statistics indicate that the attenuation coefficients of the 1%T/PG/W group and 3%T/PG/W group are about 64.7 and 122.3 times greater than that of the water group, respectively. Otherwise, we found that water became turbid when mixed with thiazone PG solution during the experiments. This reveals that crystals of thiazone appear when the T/PG solution was mixed with water. The water content of muscle tissue is of fundamental importance for the extraction of thiazone, which mainly induces the increase of the attenuation coefficient of the mixture.

Fig. 7

The total attenuation coefficient of water group, PG/W group, 1%T/PG group, and 3%T/PG group.

4.

Discussion

From analyzing the transmittance of fresh skin and muscle during the treatment with PG, we discover that PG can obviously reduce the scattering of muscle and skin. As shown in Fig. 5, the increment of total transmittance of skin treated with PG is nearly five times greater than that of the control group at a time interval of 5 min. This results from the hydrogen bonding between the hydrophilic hydroxyl of PG and water molecules in the muscle. Moreover, the dehydration effect of the PG group induces a much more effective penetration of agents into tissue and leads to a better RI matching environment. Our experimental results show that PG can effectively improve the effect of OC on both skin and muscle.

According to the results of the in vitro porcine skin experiment with different concentrations of thiazone-PG solution, the application of thiazone promotes the effect of PG on OC. On one hand, the usage of thiazone increases the fluidity of the lipid bilayer and improves the penetration of PG. On the other hand, PG that has been introduced into skin partly replaces the interstitial fluid and diminishes the RI mismatch in tissue. This suggests the synergistic effect of thiazone and PG. Our results agree well with Zhu’s correlation between the thiazone PEG400 solution and the OC of fresh in vitro porcine skin.⁴⁶

Yet, total transmittance spectrum of 10%T/PG group of muscle indicates that thiazone induces a decrease in OC on muscle [Fig. 3(e)]. Similarly, the CCD microimages show that large quantities of thiazone crystals are observed in muscle, which increase the scattering [Figs. 6(c) and 6(d)]. This is because of the different solubilities of thiazone and PG in water. Thiazone is unsoluable in water, yet PG can be mixed with water at any ratio. Meanwhile, the water content of fresh porcine muscle is up to 70%, whereas fresh porcine skin only contains 30%.⁵⁷ Hence, thiazone is easily extracted when thiazone-PG solution contacts with the free water in muscle. In addition, the dehydration effect of a high concentration of PG contributes to the discharge of water from tissue. Much thiazone crystal are extracted in muscle, whereas no crystals are observed on the skin. With all of the mentioned effects, thiazone is an excellent penetration enhancer for the OC treatment on skin, yet it is not suitable for muscle. Since thiazone mixed with OCAs can easily penetrate through skin and quickly have an effect on subcutaneous muscle tissue, our results demonstrate the inhibition effect of high concentrations of thiazone on the OC of muscle and provide such evidence for the use of thiazone as a penetration enhancer on OC of muscle.

5.

Conclusion

Previous studies show that thiazone can remarkably promote the efficiency of OC on skin and is widely used in the transdermal drug delivery. In this paper, we have studied the effect of OC on fresh in vitro porcine skin and muscle with the application of different concentrations of the thiazone PG solution. The results of skin experiments suggest that OCA, mixed with thiazone, can sharply improve the effect of OC on porcine skin and thiazone is a good penetration enhancer to skin. These results agree well with other research reports.^33,43,44,46 Interestingly, the effect of thiazone on OC of in vitro porcine muscle is absolutely the opposite. With the treatment of thiazone PG solution, crystals of thiazone in muscle are observed and decrease the OC efficiency by an increase in the scattering coefficient of the muscle. Moreover, high-concentration thiazone even inhibits the effect of OC. Hence, our experimental results of thiazone provide valuable evidence for the choices of penetration enhancer during OC studies on muscle. Considering the rapid development of the imaging technique and OCAs such as clarity,⁵⁸ scale,⁵⁹ see DB,⁶⁰ and so on, we expect to be able to expand this work by combining these OCAs with respect to skin, muscle, and neuroimaging. This work may remind researchers that the ancient methods, including the integrating sphere technique, can still play an important role in evaluating the OC efficiency of OCAs with the rapid demand for biological imaging area.