Raman spectroscopy analyzes drug distribution for potential HIV prevention
When it comes to the human immunodeficiency virus (HIV), the old saying does not go far enough: An ounce of prevention is worth more than a pound of cure. Preventing the disease from being passed on avoids the need for lifesaving antiviral cocktails, an expensive and time-consuming treatment. It is a particularly problematic therapy in Africa, where millions infected with HIV face a daily dosing regimen for the rest of their lives.
Now Raman spectroscopy could help make a new prevention delivery system even better. In collaboration with the International Partnership for Microbicides, Queen’s University School of Pharmacy in Belfast, UK, is looking into microbicide TMC120 to prevent heterosexual transmission of the disease, which accounts for four out of five new infections worldwide. The university’s Innovative Molecular Materials Group, directed by Steven E.J. Bell of the School of Chemistry and Chemical Engineering, is participating in the research by performing materials analysis.
Sometimes models need tweaking, as can be seen in this plot of relative Raman signal intensity of silicon to the microbicide TMC120 in a vaginal ring. The solid line shows the actual readings from a core-type ring, which should have all of the microbicide in the middle, encased in an inert sheath. If the drug concentration were to fall off sharply, the readings would follow that of the model system. Because they do not, some fraction of the microbicide has penetrated the sheath, and that fact will play a part in drug delivery.
Raman spectroscopy was the natural choice for this materials characterization because it offers the combination of information needed, Bell noted. To be effective, the microbicide must be applied at the correct dose in the right place at the right time without fail, and Raman spectroscopy can help do this.
One of the most popular formulations of the drug, a gel, suffers from a problem common to other preventive measures for sexually transmitted diseases: It isn’t always used. The solution being developed at Queen’s University is a vaginal ring loaded with TMC120 that is worn internally for up to months at a time. The ring releases the microbicide at low doses, providing protection that does not require any further action. What is more, rings, unlike condoms, do not provoke religious objections. Finally, similar rings are being marketed for contraception and hormone replacement therapy and have garnered regulatory approval. Thus, they have something of a track record.
Rings that deliver the microbicide TMC120 might one day be used to prevent HIV. On the left is a Raman chemical image of the cross section of a core-type vaginal ring, whereas on the right is a matrix-type ring. These maps consist of approximately 20,000 spectra and pinpoint the microbicide distribution inside the ring, an important factor in drug delivery and effectiveness. Images reprinted with permission of the Journal of Pharmacy and Pharmacology.
TMC120 can be put into an elastomer ring in two distinct ways. The microbicide can be put into a central reservoir so that there is a therapeutic core surrounded by an inert sheath, or it can sit in a matrix of small pockets dispersed throughout the ring. In both cases, the TMC120 has to diffuse out to be effective.
It is known that the distribution of drugs influences their release, but characterizing the distribution of TMC120 within the rings is difficult. Typical chemical analysis answers questions only about bulk properties. Inspection of cross-sectioned rings via microscope provides few clues because the loaded and unloaded sections look alike, making the dividing line nearly invisible. “Visually, it’s difficult to see,” Bell said.
He and his group, therefore, turned to Raman spectroscopy. Because it captures data about chemical bonds, the method provides information about composition. It also can distinguish between chemically similar forms of drug molecules and therefore is one of the few techniques that can check polymorphism. With the right optics, Raman spectroscopy can focus on very small areas and with micron resolution.
For the investigation, the researchers used a Raman instrument from PerkinElmer Inc. of Waltham, Mass. The device had a motorized stage under software control with a 785-nm diode laser light source, which they focused to a 100-μm spot size on the sample. With this setup they analyzed 2-mm-thick cross sections, capturing 20,000 data points per map. It took about a second for each point.
A key enabling technology in the instrument was an echelle spectrometer combined with a cooled CCD detector. Andrew C. Dennis, Raman business manager at PerkinElmer in Belfast, noted that Raman instruments traditionally use a linear dispersive element that separates the incoming light into its constituents along a line. That technique forces a trade-off between spectral resolution and range because the detector has a maximum line length it can accommodate.
The instruments, consequently, deliver high spectral resolution over a narrow range or low spectral resolution over a wide range. If high resolution over a wide range is needed, as in this application, then getting a complete spectrum requires repeating data acquisition multiple times and stitching together the readings as the instrument steps through the entire range. As a result, it might take 10 to 20 seconds to accumulate data for every point.
In contrast, the echelle spectrometer disperses light in two dimensions, leading to something that Dennis admitted does not look anything at all like the output of a typical Raman device. “It’s a pattern of dots on our CCD detector,” he said. “We process that data to generate a perfectly normal Raman spectrum.”
The technique provides high resolution over a wide range. Because there is no scanning or stitching involved, data acquisition is much faster. This speed and the associated data- and image-processing software are what made the materials analysis project possible.
Using the instrument, the researchers examined the TMC120 distribution in core- and matrix-type elastomer rings, obtaining both a complete map and a series of line scans at timed intervals. By looking at the CH3 and CN stretching bands, they tracked characteristic signals from the elastomer and microbicide, respectively. The work was detailed in the February Journal of Pharmacy and Pharmacology. In the core type, they found that TMC120 actually extended up to a millimeter farther into the sheath than had been expected. “There wasn’t a simple sharp falloff between the inner heavily loaded core and the nonmedicated sheath,” Bell said.
Information of this type should help in ongoing research. As for Raman spectroscopy studies of the ring drug delivery system, those continue, with equipment changes that should improve the research. Already released enhancements to the software should make the analysis easier and more powerful. Future changes could cut the analysis time needed per point, and other possibilities for speeding up data acquisition include faster stage movement.
Contact: Steven Bell, Innovative Molecular Materials Group, School of Chemistry and Chemical Engineering, Queen’s University, Belfast, UK; e-mail: email@example.com; Andrew Dennis, Raman business manager, PerkinElmer, Belfast, UK; e-mail: firstname.lastname@example.org.
- raman spectroscopy
- That branch of spectroscopy concerned with Raman spectra and used to provide a means of studying pure rotational, pure vibrational and rotation-vibration energy changes in the ground level of molecules. Raman spectroscopy is dependent on the collision of incident light quanta with the molecule, inducing the molecule to undergo the change.
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