Therapeutic Micro-Nano Technology BioMEMs - Tejlal Desai & Sangeeta Bhatia

FIGURE 14.15. AFM scanning images of surfaces before and after surface modification. A) PMMA. B) Amine functionalized PMMA. C) Porous silicon. B) Lectin-modified porous silicon.

14.6.1. Welled Silicon Dioxide and PMMA Microdevices

These reservoirs were filled with picoto nanoliters of a polymeric solution using microinjectors (Figure 14.16). Water quickly evaporates from these reservoirs leaving behind the drug contained in polymer which acts as a timed-release plug. Using a specific type of polymer predetermines the time and rate of release of drug from the reservoir; for example, a hydrogel that swells in response to a specific pH, solvent or temperature or a polymer with a known dissolution rate. Different polymers with various dissolution rates can then be used in separate reservoirs to obtain controlled release of several compounds compounds.

14.6.2. Porous Silicon Microdevices

Capillary action was exploited in order to load the porous silicon microdevices. Homogeneous liquid formulations were prepared and slowly loaded onto the porous silicon particles using a micropipettor. The solution was then taken into the porous particles by

FIGURE 14.16. Time sequence of a microdevice reservoir filled by microinjection.

capillary action. The microdevices were then dried by applying vacuum pressure until any air trapped within the pores was released.

14.6.3. Caco-2 In Vitro Studies

The Caco-2 cell line, derived from human colorectal carcinoma cells, is one of the most useful in vitro models for the study of intestinal epithelial permeability and function as well as a model for drug delivery. The Caco-2 cell line is known to differentiate spontaneously into enterocytes to form polarized monolayers in culture [50]. When grown to confluency these cells highly resemble the small intestinal epithelium both, structurally and functionally. The microvilliated cells in the monolayer maintain tight intercellular junctions and expose only their apical, brush border membranes [51]. The cells also maintain transport systems, enzymes, and ion channels similar to those of the intestinal epithelium. The glycosylation pattern of the Caco-2 cells is characterized by high amounts of N-acetylglucosamine containing oligosaccharides followed by fucosyland mannosyland minor amounts of galactosamineand N-acetyl-galactosamine-residues [52].

14.6.4. Cell Culture Conditions

Caco-2 cells were grown in culture medium consisting of Eagle’s minimum essential media supplemented with Earle’s Balanced Salt Solution, 2.0 mM L-glutamine, 1.0 mM sodium pyruvate, 0.1 mM nonessential amino acids, 1.5 g/L sodium bicarbonate, 20% FBS and 20 μg/ml gentamycin sulfate antibiotic. Cells were maintained in a humidified 5% CO2/95% air atmosphere at 37◦C while renewing media every second day. Cells were subcultured by trypsinization with 0.25% trypsin-EDTA. For experiments, cells were seeded into either six-well plates or Costar Transwell inserts (0.4 μm pore size, and 4.7 cm2 surface

area). Before seeding, culture surfaces were coated with rat collagen in order to promote cell adhesion. Caco-2 monolayers grown between 14 and 30 days were used in all experiments.

14.6.5. Assessing Confluency and Tight Junction Formation

Cell confluency and tight junction formation, as a function of time, can be assessed using a number of techniques. All techniques rely on the measurement of selected substance from one tissue compartment to the opposing compartment. The most frequently used method is by measuring transepithelial electrical resistance. A square current pulse ( I) is passed across the epithelium and the resulting voltage response ( V) is measured. Using Ohm’s law, the conductance can be calculated ( I/ V). Here, cell monolayers exhibiting TEER values of at least 220 cm2 were used in transport studies. Although this method is rapid and simple to implement, it can often be inaccurate due to nonuniform current fields caused by the point source nature of the current passing electrodes and can increase infection of the culture system [53]. A simple, inexpensive spectroscopic method can instead be used to assess cell confluency, tight junction formation, and fluid flow [54]. Using this method, the flux of phenol red across a cell monolayer when grown on a transwell permeable support is observed. Phenol red is a pH indicator used in commercially available media. It is nontoxic to epithelial cells and is not metabolized or synthesized by the cells. Because phenol red is a pH indicator, its absorbance properties are a function of pH; however at a wavelength of 479 nm, its isosbestic point, it is not pH sensitive. The concentration of phenol red in a solution can be calculated by applying the Beer-Lambert Law using the extinction coefficient for phenol red (8450 M−1 cm−1). The flux of phenol red and the permeability of the monolayer can be calculated using the following equations:

Where J is the phenol red flux, A479b is the absorbance of the basolateral solution at 479 nm, Volb is the volume of the basolateral solution at the time of measurement, t is time, A is the surface area of the epithelium, EC is the extinction coefficient, and Pl is the light path. P denotes the permeability to phenol red and [PR] is the phenol red concentration in the apical compartment. Days 1–4 represent cell confluence, days 4–8 represents complete formation of tight junction and inhibition of cell proliferation, and days 8–21 represent a small but marginally significant further decrease in permeability (Figure 14.17).

14.6.6. Adhesion of Lectin-Modified Microdevices

In vitro studies were performed using the Caco-2 cell line to measure the bioadhesive properties of lectin-conjugated microdevices (Figure 14.18). The binding characteristics of microdevices modified with two types of lectin (tomato, known to agglutinate Caco-2 cells, and peanut, an unrelated lectin) were observed as a function of time. Although both lectin conjugates produce a higher degree of binding than the pristine microdevices, a marked difference still remains between the peanut and tomato lectin conjugates. The amount of binding associated with tomato lectin-modified microdevices is thought to be a direct result of the high amounts of N-acetylglucosamine-containing oligosaccharides which are

have contributed to this work: Christopher Bonner, Aamer Ahmed, and Michael Lubeley or the University of Illinois-Chicago; Ketul Popat and Simon Su of Boston University; colleagues from iMEDD, Inc.

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15

Nanoporous Implants for

Controlled Drug Delivery

Tejal A. Desai1, Sadhana Sharma2, Robbie J. Walczak3, Anthony Boiarski3, Michael Cohen3, John Shapiro2, Teri West3, Kristie Melnik3, Carlo Cosentino4, Piyush M. Sinha, and Mauro Ferrari5

1Department of Bioengineering and Physiology, University of California, San Francisco, CA 2Department of Physiology and Biophysics, University of Illinois at Chicago, Chicago, IL

3IMEDD Inc., Foster City, CA

4Department of Experimental and Clinical Medicine, University of Catanzaro “Magna Græcia”, Catanzaro, Italy

5Professor, Brown Institute of Molecular Medicine Chairman, Department of Biomedical Engineering, University of Texas Health Science Center, Houston, TX; Professor of Experimental Therapeutics, University of Texas M.D. Anderson Cancer Center, Houston, TX; Professor of Bioengineering, Rice University, Houston, TX; Professor of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, TX; President, the Texas Alliance for NanoHealth, Houston, TX

15.1. INTRODUCTION

15.1.1. Concept of Controlled Drug Delivery

Over the last three decades considerable advances have marked the field of drug delivery technology, resulting in many breakthroughs in clinical medicine. However, major unmet needs remain. Among these are broad categories of: 1) Continuous release of therapeutic agents over extended time periods and in accordance to a pre-determined temporal profile [38, 60]; 2) Local delivery at a constant rate to the tumor microenvironment, to overcome much of the systemic toxicity and improve anti-tumor efficacy; 3) Improved ease of administration, increasing patient compliance, while minimizing the needed intervention of healthcare personnel and decreasing the length of hospital stays [6, 46]. Success in addressing some or all of these challenges would potentially lead to improvements in efficacy and patient compliance, as well as minimization of side effects [66].