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Abstract
In this paper, a drug loading and release device fabricated using nanopore thin film and layer-by-layer (LbL) nanoassembly is reported. The nanopore thin film is a layer of anodic aluminum oxide (AAO), consisting of honeycomb-shape nanopores. Using the LbL nanoassembly process, the drug, using gentamicin sulfate (GS) as the model, can be loaded into the nanopores and the stacked layers on the nanopore thin film surface. The drug release from the device is achieved by immersing it into flowing DI water. Both the loading and release processes can be monitored optically. The effect of the nanopore size/volume on drug loading and release has also been evaluated. Further, the neuron cells have been cultured and can grow normally on the nanopore thin film, verifying its bio-compatibility. The successful fabrication of nanopore thin film device on silicon membrane render it as a potential implantable controlled drug release device.
© 2017 Optical Society of America
1. Introduction
Ideally for many diseases, including various ocular diseases, some chronic diseases and coronary heart disease, the controlled dose of drugs can be delivered locally for a sustained treatment [1-2]. Toward this goal, a variety of nanostructured materials, devices and systems have been developed to fulfil this requirement [3–7]. Among them, the nanoporous structure such as the porous Si has been used as a controlled drug delivery device [2]. Another widely used technology for controlled drug release is to synthesize capsules for the nanoscale-sized drugs, which is achieved by coating the drugs with multiple layers of chemicals by a layer-by-layer (LbL) nanoassembly process [8-9]. LbL nanoassembly process is based on the electrostatic attraction between oppositely charged polyelectrolytes. It is a simple yet powerful way for fabricating micrometer diameter shells/capsules to encapsulate drugs. Usually the wall permeability of synthesized capsules can be readily modified either by the chemical structures of the wall compositions or by external stimuli [8–12], which include pH and light [10-11]. Specifically, by changing the pH of the solution around the chemically encapsulated drugs or using the light illumination to the capsules of drug, the nanopores inside the capsules can be dynamically changed or the wall of the capsules can be broken partially, facilitating the controlled drug release. Anodic aluminium oxide (AAO) nanopore, another nanoporous structure, is also an ideal drug delivery system with the following properties [13-14]. First, its nanopore size and volume can be easily modified [13]; second, various drug loading to the nanopore structure is easy to carry out with simple chemical modifications; and finally, AAO is biocompatible [14]. In addition, a unique property of AAO nanopore thin film is its ability to optically report on the loading or the release of a molecule or a drug within or from the porous nanostructure due to the change of the effective refractive index of the nanostructures [15]. In this paper, drug loading and release enabled by the AAO nanopore thin film and layer-by-layer (LbL) nanoassembly is reported. In addition, the effect of the nanopore size/volume on the release time has also been studied. Finally, the biocompatibility of the device and the successful fabrication of AAO nanopore thin film on silicone membrane have been demonstrated, indicating this type of device can be used as an implantable drug delivery device for localized drug treatment for an extended period of time.
2. Materials and methods
Sketch showing how to load the drug to and release drug from the AAO nanopore thin film-based device is illustrated in Fig. 1. Drug model gentamicin sulfate (GS) is stored in the nanopores and in the stacked between poly(acrylic acid) (PAA) and chitosan (CHI) layers. The chemicals PAA, GS, CHI, and phosphate-buffered saline (PBS) are all purchased from Sigma. Concentrations of PAA, GS, and CHI are prepared to be 2mg/ml, 5mg/ml, and 0.12mg/ml, diluted in PBS 6.5. The drug loading process is illustrated in Fig. 1(a). During this process, an LbL nanoassembly process [16] is carried out. PAA, GS, PAA, CHI are applied onto the Au-coated nanopore thin film device in sequence and repeatedly. Due to their opposite charges and attractive electrostatic force among them, layers of [PAA/GS/PAA/CHI] is formed. Briefly, after PAA is applied for 5 minutes on the oxygen-plasma treated Au-coated nanopore thin film surface with positive charges, a two-consecutive rinse step with PBS 6.5 is followed. The rinse ensures the less firmly bonded chemical PAA to be removed from the surface, allowing the next chemical layer GS deposition. Similarly, two consecutive rinse is carried out after the GS layer is formed before next layer of chemical is deposited. Following the same procedure, ten layers of [PAA/GS/PAA/CHI] combination are deposited in the whole loading process, resulting in totally forty layers of chemicals deposited onto the nanopore thin film surface. Figure 1(b-c) gives the optical micrographs of the surface of the nanopore thin film before and after the ten layers of [PAA/GS/PAA/CHI] combination have been deposited. Due to the nature of AAO nanopores [15, 17], the deposition of each chemical layer on the nanopore surface can be monitored by measuring the reflectance signals from the sample. The drug release process is illustrated in Fig. 1(d). The sample is immersed into flowing DI water, which is utilized as the release solution. The release procedure can also be optically monitored by measuring the reflectance signals from the sample.
Fig. 1 (a) Load the drug (GS) to the AAO nanopore-thin film device using LbL nanoassembly process; (b-c) optical micrographs showing the surfaces of the nanopore thin film (b) before and (c) after LbL nanoassembly; (d) release the drug from the device by flowing DI water (not to scale).
The optical setup for monitoring the drug loading and release is shown in Fig. 2(a). It consists of a white light source, an optical fiber spectrometer, an optical fiber illuminating and collecting probe and a laptop computer. The reflection from the AAO nanopore thin film device (Fig. 2(b)) is the inference fringes (Fig. 2(c)) [15, 17], which are collected by the spectrometer. When the drug along with PAA and CHI is loaded to the nanopores and/or deposited on the nanopore thin film surface layer-by-layer, the effective refractive index of the device is changed. As a result, the reflectance optical interference fringes from the device shift [15, 17], which can be optically monitored. Similarly, when the drug (GS) is released from the device, namely the layers of GS/PAA/CHI is dissolved, again resulting in the optical signals’ change of the device.
Fig. 2 (a) Setup for optical monitoring of drug loading and release process for a nanopore thin film device; (b) incident light is reflected by the nanopore thin film device (1<n0≤n2, 1<n1≤n2, 1.35<n2<1.58 since the refractive indexes of PAA, CHI and GS are nPAA = 1.442,nCHI = 1.35, nGS = 1.58, respectively, n3 = 1.7, d = 50nm, t = 3µm); (c) reflected light from the AAO nanopore thin film device is interference fringes as the transducing signals.
3. Results and discussion
AAO nanopore thin film device for drug loading and release: The fabrication procedure is detailed in our previous work [18]. Anodization is carried out in 0.3M oxalic acid at 5.8 °C with a DC voltage of 30V for 1.5 hrs to fabricate AAO thin film. During the drug loading process, [PAA/GS/PAA/CHI] layer is repeatedly deposited ten times on the nanopore thin film. The measured reflectance optical signals for each [PAA/GS/PAA/CHI] layer is shown in Fig. 3(a). In Fig. 3(a), one of the peaks of optical signal from bare AAO nanopore thin film at wavelength of 609 nm is selected as the reference peak. In the first two layers of [PAA/GS/PAA/CHI] deposition, the peak shifts to 607.8 nm. Starting from the third layer of deposition, the peak has a redshift eventually to 612.3 nm after deposition of the tenth layer, respectively. Overall, the peak has a blueshift of 1.2 nm and then has a redshift of 4.5 nm, leading to a net 3.3 nm redshift. Figure 3(b) shows almost linear increase of wavelength (i.e., peak red-shift) from the third layer to tenth layer deposition. The relationship of refractive index, thickness and resonant wavelength of this device is [19]: 2(n3t + n2d-n1t-n0d) = mλ, where n3 is the effective refractive index of AAO, n2 is the effective refractive index of chemicals (composite of PAA, CHI and GS) on AAO surface, n1 is the effective refractive index of chemicals filled in nanopores, n0 is the effective refractive index of chemicals deposited above nanopores, t is the thickness of AAO structure and d is the chemicals thickness on surface of AAO (details in Fig. 2(b)). As seen from Fig. 3(b), the resonant wavelength peak shifts from 607.8 nm to 612.3 nm, suggesting that n2d increases linearly while other items remain unchanged in Eq. (1). This indicates that [PAA/GS/PAA/CHI] layer is deposited on the surface of AAO thin film uniformly and the thickness of chemical layers increases linearly, consistent with previous work of other researchers [2]. However, for the first two layer chemical deposition as shown in Fig. 3(b), it shows a linear decrease of wavelength (i.e., peak blueshift). In the process of first two layers, chemicals are not only deposited on the surface of AAO, they are also trapped into the nanopores of AAO. The nanopore would accommodate a certain amount of chemicals, chemicals deposited onto the surface is less than chemicals trapped into nanopore. This leads to a blueshift of the resonant wavelength. After first two layers deposition, nanopore is filled with chemicals and no more chemical can be trapped into nanopore. Therefore, chemicals can only be deposited on the surface of AAO due to the attraction force between positive and negative charges, which leads to a redshift.
Fig. 3 (a) Measured optical signals after each [PAA/GS/PAA/CHI] deposition cycle: clear optical signal peak shift is observed; (b) the shift of wavelength peak, relative to 609 nm, for each cycle of deposition.
Drug release process is facilitated by flowing DI water through the devices. As shown in Fig. 4(a), for the first 2 minutes, the peak has a blueshift of 1nm. Because of change with solution from PBS 6.5 to DI water, chemical ionization changes. The bonding force due to charge attraction becomes weak and deposited chemicals on AAO nanopore surface are dissolved, leading to drug release, resulting in a wavelength blueshift. In the next 8 minutes, the peak has a very small blueshift, approximately 0.5 nm. Peak shift has been observed for next 120 minutes, 600 minutes, 1440 minutes, and 2880 minutes. During this period of time, the peak has a redshift rather than a blueshift, which suggests the chemicals/drug stored inside the nanopores started to be released into the DI water. The much longer release time for them indicates that it is relatively difficult for the chemicals/drug inside the nanopores to be released into the DI water, in comparison with the chemicals/drug stored in the layers deposited on the AAO nanopore surface. The wavelength peak shift during the whole release process is summarized in Fig. 4(b).
Fig. 4 (a) Measured optical signals from the device after chemical/drug releasing through DI water at 0, 2, 120, 600, 1440, 2880 minutes, resulting in clear peak shift; (b) and the corresponding shifts at different release time.
Nanopore size/volume effect on drug loading and release: In order to evaluate the nanopore size/volume effect on the drug loading and release, AAO nanopore thin film devices with two different sizes (~10nm and ~50nm) have been fabricated. SEM images of the nanopore thin film devices are shown in Fig. 5(a). The device with enlarged pore size is supposed to provide larger volume to accommodate more drugs, and thus achieve longer release time.
Fig. 5 (a) SEM images for AAO devices with nanopore size of ~10 nm and ~50 nm, respectively; (b) measured shift of optical signals for the devices during the LbL loading process; (c) the measured peak shifts after chemical/drug releasing through DI water at different release time.
As shown in Fig. 5(c)-5(d), the measured optical signals of these two devices show the similar trend in both drug loading process and release process. During the loading process, the optical peak has blueshift in the first two layers and then redshift in the next eight layers. On the other hand, during the release process, the optical peak has blueshift in the first two minutes and then redshift until the releasing procedure is completed. But, for the Device2 with larger nanopore size (~50 nm), its optical peak shifts during the loading process and release process of the Device2 are significantly larger than those of the Device1 of smaller nanopore size (~10 nm). The larger shift indicates that more chemicals can be stored in the device, and thus larger dose of drug can be delivered for the same period of time (Fig. 5(d)). Hence, both the amount and the duration of the drug delivery can be modified by using AAO nanopore thin film with different nanopore size/volume to accommodate different amount of drugs. In these experiments, the time used for monitoring the drug release from these device is only from 0 to 2800 minutes. As can be seen in Fig. 5(d), even after 2800-minute release, the shift of the optical signals for both Device1 and Device2 still do not tend to become saturated, indicating that some drugs in both devices still remain to be released for an extended period of time.
Evaluation of biocompatibility of AAO nanopore thin film: In order to prove the biocompatibility of the devices based on AAO nanopore thin film as potential implantable devices, the neuron cells have been cultured on the AAO nanopore thin film. Briefly, immortalized rat mesencephalic cells (1RB3AN27, N27 for short) are grown in RPMI medium supplemented with 10% fetal bovine serum, 1% L -glutamine, penicillin (100 U/ml), and streptomycin (100 U/ml), maintained at 37°C in a humidified atmosphere of 5% CO2. Before we seed cells from the flask to AAO nanopore thin film, trypsin is used to detach the cells from the bottom of flask. After 2 or 3 minutes, we pipette normal medium with 10% fetal bovine serum to the flask. Then the cells are seeded to AAO nanopore thin film. An optical image of neuron cells (N27) cultured on AAO nanopore thin film is shown in Fig. 6. Clearly the cells can grow (spread and divide) normally during 3-day culture, verifying the good biocompatibility of the AAO nanopore thin film.
Fig. 6 Optical images showing the neuron cells’ (N27 cells pointed by read arrows) growth (spread and divide) on AAO thin film device for 3 days (left to right: day1 to day3, one cell is divided in to two cells, then four cells).
Transferring of AAO nanopore thin film to silicone membrane: Even though the fabricated AAO nanopore thin film on the glass substrate can be peeled off, but it is quite crispy and easy to become curly and thus damaged (Fig. 7(a)), in order to make implantable or wearable AAO nanopore thin film device, the nanopore thin film need to be transferred to biocompatible substrate from the glass substrate. To this end, a process has been developed to transfer the AAO nanopore thin film to the silicone substrate (Fig. 7(b)). The transferring process can be briefly summarized as the following: After the AAO nanopore thin film fabricated on glass substrate, the liquid adhesive PDMS-silicone membrane is applied uniformly on the nanopore thin film. After curing PDMS-silicone at 65 °C for 2 hours, the nanopore thin film along with the PDMS-silicone membrane can be gently peeled off the glass substrate. To verify the nanopore thin film is not damaged, the reflected optical signal from the thin film on silicone has been measured. As shown in Fig. 7(c), the optical interference fringes can be observed, similar to that of AAO nanopore thin film on glass substrate [15, 17]. While for the PDMS-silicone membrane without AAO nanopore thin film, no inference fringes can be observed. Further, it has been found no damages are observed after the nanopore thin film on PDMS-silicone membrane has been cyclically bent.
Fig. 7 (a) photos showing the nanopore thin films peeled off glass substrate are damaged and/or become curly; (b) photos showing nanopore thin film transferred on PDMS-silicone; (c) measured optical signals indicate the nanopore thin film perfectly transferred to PDMS-silicone membrane.
4. Conclusions
In this paper, a drug loading and release device based on AAO nanopore thin film and LbL nanoassembly is reported. Both drug loading and release processes can be monitored optically for this type of device. The nanopore size effect on the drug loading and release has been evaluated. The drug release time can be readily up to three days by using only 10 layers LbL nanoassembly to load the drugs to the AAO nanopore thin film devices with nanopore size from ~10 nm to ~50 nm. It is anticipated that the drug release time and the released dose can be readily further increased by simply adding LbL nanoassembly steps and/or increasing the nanopore size and density. The bio-compatibility of AAO nanopore thin film has been demonstrated by culturing neuron cells on its surface. Further, AAO nanopore thin film has been successfully transferred to PDMS-silicone membrane, indicating the AAO nanopore thin film based device can be used as an implantable device for drug delivery.
Funding
National Science Foundation (NSF) (0845370, 1461841).
Acknowledgments
The authors thank the technical supports from staffs at Microelectronics Research Center (MRC) at Iowa State University.
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