Depth Profiling of PLGA Copolymer in a Novel Biomedical Bilayer

Apr 17, 2013 - This method of measuring through the Z height to obtain the depth profile where the laser beam is incident normal to the sample surface...
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Depth Profiling of PLGA Copolymer in a Novel Biomedical Bilayer Using Confocal Raman Spectroscopy Colm McManamon,†,‡ Paul Delaney,†,‡ Claire Kavanagh,§ Jing Jing Wang,† Sozaraj Rasappa,†,‡ and Michael A. Morris*,†,‡ †

Centre for Research on Adaptive Nanostructures and Nanodevices (CRANN), Trinity College Dublin, Dublin 2, Ireland Department of Chemistry, Supercritical Fluid Centre and Materials Section, University College Cork, Cork, Ireland § DePuy Ireland, Loughbeg, Ringaskiddy, Cork, Ireland ‡

ABSTRACT: Confocal Raman spectroscopy was undertaken to identify separate layers of PLGA and gentamicin sulfate (GS) coatings on a titanium alloy substrate for a novel drug-delivery system. Additionally, it was found that it was possible to measure the layer thickness and uniformity of the PLGA accurately by detecting intensity and wavelength changes in the vibrational bands of the copolymer bonds. Further analysis of the materials was done using FIB, SEM/EDX, and profilometry; these techniques were used to confirm the findings of the Raman data. It was determined that the substrate was extremely rough and therefore the coating was not uniform in thickness but the materials were uniformly dispersed. Most importantly, two distinct GS and PLGA layers were present.

1. INTRODUCTION Much recent biomedical research has been focused on advancing prosthetic implants to give the patient increased mobility, reduced pain, and faster rehabilitation.1−3 According to the Voluntary Health Insurance Board Ireland (VHI), the number of total hip arthroplasty (THA) procedures in Ireland is expected to increase by 137% by 2030.4 One of the most important aspects in the successful implementation of biomaterials is the characterization of the external coating. An extensive knowledge of this is needed to predict the body’s reaction to the implant and avoid infection. Poly(lactic-coglycolic acid) (PLGA), since being approved by the FDA, has been used for a wide variety of medical applications such as treating cardiovascular disease, vaccines, tissue engineering, and drug delivery.5,6 The primary reason that it is commonly used in drug delivery is its ability to decompose in vivo through either hydrolysis of the ester linkages and/or enzymatic degradation and does not need to be removed after the release of the drug.7 Biomedical companies are continually trying to find ways to optimize drug delivery to fight bacterial infections. Infections from biomaterials is the second most common cause of implant failure, and the increase in their use has resulted in a rise in bacterial infections, often due to Staphylococcus spp..8 In the EU, concerns around the increased prevalence of surgical site infections (SSI) and, specifically, the incidence of methicillinresistant Staphylococcus aureus (MRSA) and Clostridium dif f icile led to the instigation of a program for the surveillance of SSI by the, then, Public Health Laboratory Service and Department of Health in 1997.9 As a result, surveillance of SSI in orthopedic © XXXX American Chemical Society

surgery became mandatory in April 2004. MRSA, in particular, is extremely dangerous because of the difficulty in treating the infection despite a 19% drop in cases, in Ireland, over the last 8 years.10 These devices are not strictly medical devices or pharmaceutical devices but rather a combination of both. The introduction of the aminoglycoside antibiotic, gentamicin sulfate (GS), into the body, with the implant, as a form of treatment has been proven to be very successful.11 Current methods use a copolymer/drug composite that is released into the system after surgery.12,13 The degradation of this matrix in vivo is predicted by analytical analysis including calorimetric and fluorometric techniques and chromatography14,15 This new product is composed of a separate drug and polymer bilayer and can significantly reduce the infection rates associated with prosthetic surgery and the enormous financial burden associated with surgical-related hip implant infections. It is therefore important to develop a quantitative method of characterization that is specific and sensitive to the material in question as well as reproducible and economically viable. Raman spectroscopy is a commonly used and valuable tool for diagnosing disease and studying biological tissue by analyzing inelastically scattered laser light to provide detailed information about vibrations of molecular bonds.16,17 Confocal Raman has been used to analyze PLGA in a variety of forms such as microspheres, nanoparticles, and thin films.6,18,19 Received: February 1, 2013 Revised: April 17, 2013

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identify layer positions, with the sulfate peak at 980 cm −1 corresponding to the GS layer and the peak at 1770 cm −1 corresponding to the ester groups in the PLGA layer. Measurements were taken at various Z positions in order to build up a chemical signature of the layers present.

Current Raman applications in the biomaterial sector range from understanding and analyzing the structure of polymer composites to understanding drug/excipient mixing in delivery systems.20,21 Here, we use Raman to examine the homogeneity of the mixing between the PLGA and the antimicrobial drug GS. This novel technique determines the coating thickness, which is paramount to understanding drug delivery, by detecting intensity changes in the vibrational bands of the copolymer bonds.

3. RESULTS AND DISCUSSION The profilometry spectra of the grit-blasted Ti substrate, shown in Figure 2, displays an extremely rough surface with a

2. EXPERIMENTAL SECTION 2.1. Methods. Titanium alloy (Ti-6Al-4 V) coupons, representative of the hip implant, with a diameter of 25 mm and a thickness of 3.5 mm were grit blasted with aluminum oxide prior to being coated with a bilayer of GS and PLGA (represented in Figure 1).

Figure 2. Profilometry spectra of 5 mm scans of the grit-blasted Ti substrate and the GS-, PLGA-, and GS-/PLGA-coated Ti substrates.

Figure 1. Schematic diagram of stem section showing the polymer/ drug bilayer.

maximum height difference of 40 μm over the measured area. When coated with GS, the softer material planarizes on top of the jagged Ti surface and reduces the height difference by 50%. The PLGA takes the shape of the material beneath it, and when coated on top of the Ti substrate, it appears extremely rough and displays a maximum height difference of 28 μm. However, when coated onto the GS surface it gives a much smoother appearance and reduces the maximum height difference to 12 μm. The nature of this coating over the surface may provide an advantage for the release of the underlying GS layer because it will enable the PLGA layer to break down uniformly. Figure 3 displays a series of Z-stack images on the surface coated with the fluorescein-loaded GS obtained with CLSM. The undulant nature of the material is consistent with the surface morphology

2.2. Characterization. Profilometry measurements were carried out using a Dektak 6 M fitted with a 25 μm radius tip and a point-topoint resolution of 1 Å. A load of 1 mg was used on Ti surfaces, and loads of 2 and 3 mg were used on GS- and PLGA-coated surfaces, respectively. All scans were performed at a horizontal distance of 5000 μm over a time period of 200 s. All confocal laser microscopy analysis was performed using an Olympus Fluoview FV1000 confocal microscope, and illumination was provided by a Kr/Ar laser (488 nm laser excitation). To investigate the surface morphology of the gentamicin layer, we treated it with flourescien prior to deposition on the metal substrate. The elemental composition and surface morphology of the coated coupons were determined with a high-resolution (30 μm), but because of the roughness of the material, it is not representative of the whole sample. Figure 7 shows the Raman spectra for the PLGA and GS bilayer at different Z heights (from the surface (0) to 12 μm into the sample). The peaks used to analyze the GS can be seen at 980 and 1500 cm−1, and these are due to the asymmetric stretching vibration of SO42− and the stretching vibration of N−H, respectively. The PLGA is represented by the ester D

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group peak at 1770 cm−1 and the asymmetrical stretching of the C−O−C band at 1046 cm−1.22 It is evident, from this graph, that the peak height increases as the laser moves deeper into the sample and decreases as it moves from the center of the material. The PLGA thickness was determined by analyzing these spectra. The peak area of each spectrum at 980 and 1770 cm−1 for GS and PLGA, respectively, was obtained (Figure 7). Another important observation obtained from the spectra is that there was no Raman shift of the data recorded for the peaks examined, which provides evidence that there is no composite mixing of the PLGA and GS. This is important for the controlled release of the drug, which is time dependent on the degradation of the PLGA. The data obtained for each scan performed was then fitted with a normal distribution curve, which is shown in Figure 8. The largest peak area represents the

The presence of GS before the apex of the peak (i.e., the surface of the GS) is due to a high degree of reflectance from the laser upon contact. Table 1 gives the thickness of the PLGA at the 10 Table 1. Undulating Surface Thickness of the PLGA on GSa material

actual PLGA thickness (μm)

gentamicin surface (μm)

site A site B site C site D site E site F site G site H site I site J

4.1 5.2 1.3 4.4 2.8 3.7 4.6 2.9 4.9 2.9

4.2 5.4 1.6 4.5 3.1 3.8 4.9 3.1 5.1 3.0

a

The GS peak represents the surface that corresponds to the end of the PLGA layer.

sites measured using the Raman Z-height scanning and gives a clear picture of the variation of surface thickness and lack of uniformity of the copolymer. All sites were chosen to give a comprehensive overview of the material. The large variation in uniformity is due to the roughness of the Ti substrate (shown in Figure 6) as well as the agglomeration of the copolymer upon deposition. It is also evident, at each site analyzed, that the GS layer begins where the PLGA layer ends (±300 nm). This shows that the PLGA adheres to the GS layer and does not display any significant interaction between the materials. This analysis technique allows for large-scale depth profiling of PLGA on extremely rough surfaces where mechanical attachment is the dominant adhesion mechanism.

Figure 8. Shown here is a Gaussian fit of the peak areas of both PLGA and GS at one site measured (200 μm). The center of the peak of the PLGA represents the center whereas the peak of the GS represents the surface of the material.

4. CONCLUSIONS Demonstrated here is a successful method, using confocal Raman spectroscopy, for studying the PLGA thickness and uniformity. This technique allowed detailed information about the chemical composition of the copolymer particles to be obtained. The Raman analysis was backed up with additional techniques including FIB, profilometry, and SEM/EDX. This method of PLGA analysis may be crucial in determining the optimum amount of the copolymer required for drug delivery on biomedical devices. In addition, this will aid and enable further studies directed at the chemical investigation of biomaterial characterization.

most intense signal of PLGA and thus represents the center of the material. This method of measuring through the Z height to obtain the depth profile where the laser beam is incident normal to the sample surface causes refraction. As the laser moves deeper into the sample, it becomes impossible to obtain pure spectra of that point in the material. Therefore, a refractive index (RI) value of 1.4 was used in determining the PLGA coating thicknesses. The RI value is estimated from product literature values for the RI of polylactic acid (1.35−1.45, Natureworks LLC) and for the monomers (lactic acid = 1.42, glycolic acid = 1.41, Sigma-Aldrich Corp.).23 Thus, the following equation was used n1 ×2=Z n2 (1)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

where n1 is the Z height at the center of the PLGA layer and n2 is the refractive index (1.4) of PLGA. The Raman laser was unable to penetrate the GS layer because of the light being dispersed upon contact; therefore, the most intense peak of GS is obtained on the surface of the material (Figure 8). This gives further evidence as to the thickness of the PLGA layer because the GS layer begins when the PLGA layer ends, which is proven by FIB images (Figure 6) and the lack of composite mixing in the Raman shift (Figure 7).

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS

We acknowledge the assistance of Cathal McAuley and Dermot Daly of the advanced microscopy laboratory (AML) in CRANN. This research is independent of DePuy Ireland and does not necessarily reflect the views of the company, and no official approval should be assumed. E

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(20) Andrew, J. J.; Browne, M. A.; Clark, I. E.; Hancewicz, T. M.; Millichope, A. J. Raman imaging of emulsion systems. Appl. Spectrosc. 1998, 52, 790−796. (21) Morris, H. R.; Munroe, B.; Ryntz, R. A.; Treado, P. J. Fluorescence and Raman chemical imaging of thermoplastic olefin (TPO) adhesion promotion. Langmuir 1998, 14, 2426−2434. (22) Belu, A.; Mahoney, C.; Wormuth, K. Chemical imaging of drug eluting coatings: combining surface analysis and confocal Raman microscopy. J. Controlled Release 2008, 126, 111−121. (23) Clarke, F. C.; Jamieson, M. J.; Clark, D. A.; Hammond, S. V.; Jee, R. D.; Moffat, A. C. Chemical image fusion. The synergy of FTNIR and Raman mapping microscopy to enable a more complete visualization of pharmaceutical formulations. Anal. Chem. 2001, 73, 2213−2220.

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dx.doi.org/10.1021/la400402a | Langmuir XXXX, XXX, XXX−XXX