Improved design and characterization of PLGA/PLA-coated Chitosan based micro-implants for controlled release of hydrophilic drugs.

Repetitive intravitreal injections of Methotrexate (MTX), a hydrophilic chemotherapeutic drug, are currently used to treat selected vitreoretinal (VR) diseases, such as intraocular lymphoma. To avoid complications associated with the rapid release of MTX from the injections, a Polylactic acid (PLA) and Chitosan (CS)-based MTX micro-implant prototype was fabricated in an earlier study, which showed a sustained therapeutic release rate of 0.2-2.0 µg/day of MTX for a period ∼1 month in vitro and in vivo. In the current study, different combinations of Poly(lactic-co-glycolic) acid (PLGA)/PLA coatings were used for lipophilic surface modification of the CS-MTX micro-implant, such as PLGA 5050, PLGA 6535 and PLGA 7525 (PLA: PGA - 50:50, 65:35, 75:25, respectively; M.W: 54,400 - 103,000) and different PLA, such as PLA 100 and PLA 250 (MW: 102,000 and 257,000, respectively). This improved the duration of total MTX release from the coated CS-MTX micro-implants to ∼3-5 months. With an increase in PLA content in PLGA and molecular weight of PLA, a) the initial burst of MTX and the mean release rate of MTX can be reduced; and b) the swelling and biodegradation of the micro-implants can be delayed. The controlled drug release mechanism is caused by a combination of diffusion process and hydrolysis of the polymer coating, which can be modulated by a) PLA content in PLGA and b) molecular weight of PLA, as inferred from Korsmeyer Peppas model, Zero order, First order and Higuchi model fits. This improved micro-implant formulation has the potential to serve as a platform for controlled release of hydrophilic drugs to treat selected VR diseases.

Noninvasive Electroretinography Assessment of Intravitreal Sustained-Release Methotrexate Microimplants in Rabbit Eyes.

PURPOSE: The purpose of this study is to noninvasively evaluate the safety and toxicity of a chitosan (CS) and polylactic acid (PLA)-based sustained-release methotrexate (MTX) intravitreal microimplant in normal rabbit eyes using electroretinography (ERG). METHODS: PLA-coated CS-based microimplants containing 400 µg of MTX and placebo microimplants (without drug) were surgically implanted in the vitreous of the right and the left eyes, respectively, in each of the 8 New Zealand rabbits using minimally invasive technique. At each predetermined time points (days 5, 12, 19, and 33), ERG was conducted on 2 rabbits to evaluate the safety of the microimplants administered in each eye. ERG was carried out using 2 protocols, scotopic and photopic, on each eye prior to surgery (PS) and prior to euthanasia (PE) conditions. The safety of the microimplants was assessed using statistical analysis of the ERG data (B/A ratio analysis, oscillatory potential analysis, and Naka-Rushton analysis) and subsequently quantifying and comparing functional integrity of the retina between the PS and PE conditions of each eye. RESULTS: Statistical analysis of the ERG data showed no change in retinal functional integrity because of the PLA-coated CS-based MTX microimplant and the placebo microimplant. ERG analysis also revealed absence of any evident bioelectrical dysfunction caused by the microimplants. CONCLUSION: ERGs were performed to determine whether the microimplants containing MTX and the placebo microimplants were associated with any profound retinal bioelectrical dysfunction that might be attributable to toxicity not apparent on histological studies of such eyes. The results shown in this report indicate that there were no such evident adverse effects of the microimplants or contained drug.

Ultrasonographical assessment of implanted biodegradable device for long-term slow release of methotrexate into the vitreous.

Our group has developed a biodegradable drug delivery device (micro-implant) for long-term slow intraocular release of methotrexate (MTX) that can be implanted in the peripheral vitreous. The purpose of this study was to assess the position of the implanted devices and the status of the adjacent vitreous and peripheral retina over time using B-scan ocular ultrasonography (US). In each of the eight New Zealand rabbits used in this study, a chitosan (CS) and poly-lactic acid (PLA)-based micro-implant containing approximately 400 µg of MTX and a placebo micro-implant without MTX were inserted into the peripheral vitreous of the right and left eyes, respective, employing minimally invasive surgery. B-scan US imaging was performed on all of the rabbits immediately after implant insertion and on two rabbits at each of several pre-determined time points post-insertion (post-insertion days 5, 12, 19, and 33) to evaluate the position of the micro-implants and identify any evident morphological changes in the micro-implants and in the peripheral retina and vitreous during treatment. US imaging revealed stable positioning of the PLA-coated CS-based MTX micro-implant and the placebo micro-implant in the respective eyes throughout the study and lack of any changes in size, shape or sonoreflectivity of the micro-implants or abnormalities of the peripheral vitreous or retina in any of the study eyes. In summary, US did not show any evident morphological changes in the micro-implants, shifts in post-insertion position of the micro-implants, or identifiable changes in the micro-implants or peripheral vitreous and retina of the study eyes.

Biodegradable chitosan and polylactic acid-based intraocular micro-implant for sustained release of methotrexate into vitreous: analysis of pharmacokinetics and toxicity in rabbit eyes.

PURPOSE: The purpose of this study was to evaluate the pharmacokinetics and toxicity of a chitosan (CS) and polylactic acid (PLA) based methotrexate (MTX) intravitreal micro-implant in an animal model using rabbit eyes. METHODS: CS- and PLA-based micro-implants containing 400 µg of MTX were fabricated using lyophilization and dip-coating techniques. The micro-implants were surgically implanted in the vitreous of eight New Zealand rabbits employing minimally invasive technique. The PLA-coated CS-MTX micro-implant was inserted in the right eye and the placebo micro-implant in the left eye of each rabbit. Two rabbits were euthanized at each pre-determined time point post-implantation (days 5, 12, 19, and 33) for pharmacokinetics and histopathology evaluation. RESULTS: A therapeutic concentration of MTX (0.1-1.0 µM) in the vitreous was detected in the rabbit eyes studied for 33 days. The MTX release from the coated micro-implants followed a first order kinetics (R (2) ~ 0.88), implying that MTX release depends on the concentration of MTX in the micro-implant. Histopathological analysis of the enucleated eyes failed to show any signs of infection or tissue toxicity in any of the specimens. CONCLUSION: The PLA-coated CS-MTX micro-implants were able to deliver therapeutic release of MTX for a period of more than 1 month without detectable toxicity in a rabbit model. The micro-implants can be further investigated as a prospective alternative to current treatment protocols of repeated intravitreal MTX injections in intraocular disorders such as primary intraocular lymphoma, and selected cases of non-microbial intraocular inflammation.

Enhanced capture of magnetic microbeads using combination of reduced magnetic field strength and sequentially switched electroosmotic flow--a numerical study.

Magnetophoretic immunoassay is a widely used technique in lab-on-chip systems for detection and isolation of target cells, pathogens, and biomolecules. In this method, target pathogens (antigens) bind to specific antibodies coated on magnetic microbeads (mMBs) which are then separated using an external magnetic field for further analysis. Better capture of mMB is important for improving the sensitivity and performance of magnetophoretic assay. The objective of this study was to develop a numerical model of magnetophoretic separation in electroosmotic flow (EOF) using magnetic field generated by a miniaturized magnet and to evaluate the capture efficiency (CE) of the mMBs. A finite-volume solver was used to compute the trajectory of mMBs under the coupled effects of EOF and external magnetic field. The effect of steady and time varying (switching) electric fields (150-450 V/cm) on the CE was studied under reduced magnetic field strength. During switching, the electric potential at the inlet and outlet of the microchannel was reversed or switched, causing reversal in flow direction. The CE was a function of the momentum of the mMB in EOF and the applied magnetic field strength. By switching the electric field, CE increased from 75% (for steady electric field) to 95% for lower electric fields (150-200 V/cm) and from 35% to 47.5% for higher electric fields (400-450 V/cm). The CE was lower at higher EOF electric fields because the momentum of the mMB overcame the external magnetic force. Switching allowed improved CE due to the reversal and decrease in EOF velocity and increase in mMB residence time under the reduced magnetic field strength. These improvements in CE, particularly at higher electric fields, made sequential switching of EOF an efficient separation technique of mMBs for use in high throughput magnetophoretic immunoassay devices. The reduced size of the magnet, along with the efficient mMB separation technique of switching can lead to the development of portable device for detection of target cells, pathogens, and biomolecules.

Influence of variable native arterial diameter and vasculature status on coronary diagnostic parameters.

In current practice, diagnostic parameters, such as fractional flow reserve (FFR) and coronary flow reserve (CFR), are used to determine the severity of a coronary artery stenosis. FFR is defined as the ratio of hyperemic pressures distal (p(rh)) and proximal (p(ah)) to a stenosis. CFR is the ratio of flow at hyperemic and basal condition. Another diagnostic parameter suggested by our group is the pressure drop coefficient (CDP). CDP is defined as the ratio of the pressure drop across the stenosis to the upstream dynamic pressure. These parameters are evaluated by invasively measuring flow (CFR), pressure (FFR), or both (CDP) in a diseased artery using guidewire tipped with a sensor. Pathologic state of artery is indicated by lower CFR (<2). Similarly, FFR lower than 0.75 leads to clinical intervention. Cutoff for CDP is under investigation. Diameter and vascular condition influence both flow and pressure drop, and thus, their effect on FFR and CDP was studied. In vitro experiment coupled with pressure-flow relationships from human clinical data was used to simulate pathophysiologic conditions in two representative arterial diameters, 2.5 mm (N1) and 3 mm (N2). With a 0.014 in. (0.35 mm) guidewire inserted, diagnostic parameters were evaluated for mild (∼64% area stenosis (AS)), intermediate (∼80% AS), and severe (∼90% AS) stenosis for both N1 and N2 arteries, and between two conditions, with and without myocardial infarction (MI). Arterial diameter did not influence FFR for clinically relevant cases of mild and intermediate stenosis (difference < 5%). Stenosis severity was underestimated due to higher FFR (mild: ∼9%, intermediate: ∼ 20%, severe: ∼ 30%) for MI condition because of lower pressure drops, and this may affect clinical decision making. CDP varied with diameter (mild: ∼20%, intermediate: ∼24%, severe: by 2.5 times), and vascular condition (mild: ∼35%, intermediate: ∼14%, severe: ∼ 9%). However, nonoverlapping range of CDP allowed better delineation of stenosis severities irrespective of diameter and vascular condition.