OCT-based Drug Diffusion Biosensor and Early Diagnostics

Nondestructive depth-resolved imaging of molecular diffusion in tissues can assist in development of novel therapeutic agents and drug delivery systems as well as provide a novel tool for early diagnosis of various epithelial disorders.

Successful management of many diseases (e.g. cancer, diabetic retinopathy, glaucoma and different vascular disorders) requires long-term treatment with drugs. In contrast to the traditional oral route, topical drug delivery (TDD) through epithelial tissues is currently recognized as a preferred route for drug administration. However, topical delivery of therapeutic agents to target tissues in effective concentrations remains difficult problem due to low permeability of epithelial tissues and drug washout. Significant research effort is currently devoted to development of therapeutically effective topical formulations such as drug delivery systems, gels, creams, ointments, lotions, as well as application of various permeation enhancers. Successful development of these formulations requires understanding of dynamics of molecular distribution in epithelial tissues for better manipulation and optimization the therapeutic processes and outcomes. However, currently available techniques for quantification of molecular diffusion processes in tissues are either invasive and requires isolation of tissues or have limited resolution and sensitivity. Therefore, development of a noninvasive biosensor capable of real-time and depth-resolved monitoring and quantification of molecular transport through epithelial tissues (and different layers of the tissues) would warrant development of novel therapeutic agents and assess pharmacokinetic properties of TDD systems. Additionally, difference in diffusion rates of macromolecules in healthy and diseased tissues could potentially be used for development of novel early diagnostic methods.

Here we briefly describe recent progress made on developing of a molecular diffusion biosensor based on Optical Coherence Tomography (OCT) technique. Diffusion of several macromolecules and drugs was monitored and quantified in cornea and sclera of the eye, skin, brain, and vascular tissues both in vitro and in vivo. Molecular diffusion inside tissue to interstitial space leads to alteration of its morphological and optical properties. The increase of tissue molecular concentration will, e.g., raise the refractive index of ISF that will decrease the scattering coefficient. Since OCT can provide depth-resolved analysis of tissue optical properties with high resolution, changes in the in-depth distribution of tissue scattering coefficient and/or refractive index are reflected in changes in the OCT signal amplitude and the slope.

An example of typical results obtained from molecular diffusion studies in epithelial tissues is shown in Figure 1. The propagation of glucose molecules (20% concentration) inside rabbit sclera changed the local scattering coefficient, which was reflected in change of OCT signal slope. The increase in local in-depth glucose concentration resulted in the decrease of the OCT signal slope and vice versa during the diffusion processes. The permeability coefficients for several molecules and drugs in sclera and cornea of rabbit eyes were noninvasively measured and are summarized in Table 1.

FIGURE 1: a) Typical OCT signal slope as a function of time recorded from sclera (in whole eyeball) during glucose diffusion experiment. The arrow indicates time of added agent. b) Typical OCT images of rabbit sclera recorded before topical application of glucose and after it diffused through.
Rabbit cornea Rabbit sclera

Agent

Permeability ± SD (cm/sec)

Permeability ± SD (cm/sec)
Water

(1.68 ± 0.54) ×10-5 (n=8)

(1.33 ± 0.28)×10-5 (n=5)

Metronidazole (1.59 ± 0.43) ×10-5 (n=5) (1.31 ± 0.29)×10-5 (n=4)
Ciprofloxacin (1.85 ± 0.27) ×10-5 (n=7) (1.41 ± 0.38)×10-5 (n=3)
Dexamethasone (2.42 ± 1.03) ×10-5 (n=7)  
Mannitol 20% (1.46 ± 0.08) ×10-5  (n=4) (6.18 ± 1.08)×10-6 (n=5)
Glucose 20% (1.78 ± 0.23) ×10-5 (n=6) (8.64 ± 1.12)×10-6 (n=14)
TABLE 1: Summary of permeability coefficients of different drugs measured in cornea and sclera of rabbit eye.

OCT has some unique properties such as high resolution and ability for truly non-destructive depth-resolved imaging. These allowed studying the molecular diffusion not only as a function of time but also as a function of depth. With the OCT, we monitored the diffusion process of different molecules at various depths away from the surface of tissues. Figure 2a shows typical OCT signal measured at depths 105, 158, 225, and 273 µm away from the surface of rabbit sclera during mannitol diffusion experiment. The arrows on each of the OCT signals depict the time the drug action reached that particular depth (manifested as sharp decrease of the OCT signal). Figure 2b shows typical permeability coefficients of glucose measured at different depths in a sclera. This figure shows that the glucose diffusion rate inside sclera is nonlinear. This nonlinearity is due to molecular diffusion through at least two layers: epithelium (low diffusion) and stroma (faster diffusion). Different inclusions of these two processes at different time intervals and difference of collagen organization in tissue layers are the source for such nonlinearity.

FIGURE 2: a) OCT signal as a function of time recorded at different depths during a mannitol diffusion experiment in sclera. Arrows indicate the mannitol’s front reaching different depth; b) glucose permeability coefficients measured at different depths in a sclera.

Optical turbidity of most tissues could prevent the ability of OCT to be fully engaged in various diagnostic and therapeutic procedures. Transformation of a turbid medium to a transparent one with no or minimal alteration of its composition has been subject of number of studies. An example of depth-resolved assessment of optical clearing of 40% glucose in rabbit sclera is shown in Figure 3: the permeability coefficient in the upper 80-100 µm (which constitutes a layer that includes the episclera) and the following 100 µm region (which is thought to be in the stroma or the second layer) was found to be (6.01 ± 0.37) × 10-6 cm/sec and (2.84 ± 0.68) × 10-5 cm/sec, respectively. These regions also showed a difference in the clearing: roughly, the first 100 µm cleared around 10% while the deeper 100 µm region cleared about 17-22%.

FIGURE 3: Optical clearing and permeability coeffificient at different depth in a rabbit sclera upon topical application of 40% glucose solution.

Advances in clinical diagnostic methods are often governed by innovations in imaging, measurement, and diagnostic technologies. Early diagnostics is unquestioningly is the key for successful treatment and therapy of many devastating diseases. In our pilot study we quantified permeability of several low and high molecular weight compounds in normal and diseased animal and human arteries. The results show dramatic change for the molecular permeability rate in healthy versus diseased samples (Fig 4). Moreover, structural OCT imaging of arteriosclerotic lesions at early stages was not able effectively differentiate between normal and diseased areas whereas functional method demonstrated superior contrast and sensitivity. Therefore, application of this novel method could make possible early diagnosis of, e.g., vascular abnormalities and bring us one step closer to understanding cardiovascular diseases.

FIGURE 4: Glucose permeability coefficients measured in normal and diseased aorta samples.