Concurrent delivery of dexamethasone and VEGF for localized inflammation control and angiogenesis
Introduction
Recent years have witnessed significant progress in the use of implantable bio-medical devices such as drug-eluting stents [1], pumps [2], artificial organs [3], biosensors, catheters, scaffolds, pacemakers [4], defibrillators, heart valves, artificial joints, vascular grafts, and prostheses as a part of treatment programs for a wide range of medical indications [5], [6]. Implantable biosensors [7] and devices [4] are particularly useful for the stabilization of chronic conditions such as cardiovascular diseases [4], [8] and diabetes [6], [9] since they can continuously sample and monitor blood metabolites and bio-chemical disease markers and thus help determine dosage, time of administration, and frequency of the appropriate medicinal agent [2], [10], [11].
Advances in design, engineering, and fabrication have generated miniaturized biosensors that are capable of performing multiple complex functions such as selectively measuring glucose levels in physiological fluids in vitro [10], [11], [12], [13]. However, accuracy and precision of the in vitro performance of sensors can rarely be replicated in vivo, especially over extended periods of time [10], [12], [14]. The longest in vivo residence time of a functionally active FDA-approved implantable glucose-monitoring biosensor is 3 days [15], [16]. Additionally, devices that do retain some functionality have been shown to suffer from high fluctuations and inaccuracy in the measurement of blood glucose levels in circulation, progressive loss of sensitivity, and occasional failures; thereby rendering them unsuitable for use in diabetes management in humans [16], [17], [18].
One of the reasons for the loss of in vivo performance of bio-medical devices is attributed to the changes in the immediate tissue environment that take place post-implantation. Implanted devices and drug delivery systems have been shown to typically trigger a cascade of immunogenic reactions in response to the trauma caused during implantation and result in acute inflammation characterized by a dense infiltration of inflammation-mediating cells at the device–tissue interface [19], [20], [21]. Acute inflammation constitutes a major part of the classic immune response to the presence of foreign objects in vivo and can lead to complications that can adversely impact or compromise the performance of implantable devices [22]. Continuous stimuli provoked by chronically implanted devices advance acute inflammation into the chronic phase that is marked by the accumulation of dense fibrotic tissue encapsulating the device and regression of blood vessel growth [20], [23], [24]. The entombment of the device may cause obstructed blood sampling, compromised function, and eventually result in complete rejection [18], [25]. Additionally, biofouling of sensor membranes and corrosion of sensor parts can limit selective transfer of sensor ligand to the appropriate detector surface and have been shown to compromise sensitivity and function [17], [18], [25].
To maintain sensor functionality over long term (weeks to months) it is vital to maintain a healthy, inflammation and fibrosis-free, and well-vascularized tissue environment surrounding the biosensor. In vivo residence time and functionality of implantable devices can be significantly improved using immunosuppressive anti-inflammatory drugs [26] and agents that promote angiogenesis around the sensor [17], [18], [25]. However, doses associated with oral delivery of corticosteroids required to generate these effects are significantly high to compensate for metabolism and may be unsuitable for patients with impaired renal or hepatic function. The possibility of therapeutic complications associated with toxicity and side-effects of these agents can also be elevated due to systemic exposure. Corticosteroid therapy has also been shown to exacerbate diabetes for which many implantable biosensors are currently being developed. Oral delivery of angiogenic growth factors such as VEGF and transforming growth factor-beta3 is significantly restrictive due to gastric degradation and instability, whereas parenteral delivery may require repetitive administration. In addition, due to a high volume of distribution, oral and parenteral i.v. bolus doses drugs levels at the local sites may not achieve the minimal effective concentration.
We have recently reported a composite delivery system that is fabricated by dispersing drug-loaded poly(lactic-co-glycolic acid) (PLGA) microspheres in a poly(vinyl alcohol) (PVA) hydrogel matrix [20]. These composites can be programmed to locally release corticosteroids such as dexamethasone at the implant site and can be used as an inert biocompatible external coating for biosensor probes and other implantable bio-medical devices [20], [27].
Although administration of anti-inflammatory agents such as dexamethasone can minimize implantation-associated inflammation [20], their delivery can inhibit endogenous blood vessel growth [28], [29], [30]. Numerous studies have shown that dexamethasone administration can inhibit or down-regulate some of the key factors responsible for the formation of blood vessels such as endogenous VEGF and other angiogenic proteins in a variety of cell types that include chondrocytes [29], gastric mucosa cells [28], and renal cell carcinoma cells [30]. Thus, although inflammation can be arrested and the implanted biosensors are expected to be in a relatively less immunologically activated environment, the lack of vascular tissue can potentially limit functionality by significantly decreasing the available blood circulation surrounding the implant. Lack of vasculature at the implant site may retard healing the trauma caused during implantation or impact localized drug delivery and may be especially detrimental for sensor applications where the presence of well-vascularized tissue is a critical requirement. Therefore, it is essential to be able to administer both anti-inflammatory corticosteroids as well as growth factors in a localized environment at low doses so that both agents can function symbiotically without interference.
Localized delivery of growth factors using implantable drug delivery systems can be successfully used to achieve site-specific pharmacologic effects such as neo-angiogenesis using vascular endothelial growth factor (VEGF) [31], [32] and bone growth using bone morphogenic proteins [33]. Delivery of VEGF has been shown to not only stimulate new blood vessels but also improve circulation in the intended target area [34]. However, in vivo release of proteins may result in a more severe inflammatory response compared to that resulting from the implant alone, as a consequence of the body's response to the foreign protein and can further exacerbate the condition and thus aggravate the adverse effects. The use of combinations of anti-inflammatory agents and growth factors to simultaneously control inflammation and ensure neo-angiogenesis has not been previously reported. The present work reports the effect of concurrent delivery of dexamethasone and VEGF at the tissue/implant interface from PLGA microsphere–PVA hydrogel composites. The combined pharmacodynamic effects of inflammation and fibrosis control as well as stimulation of therapeutic neo-angiogenesis in a rat model are reported.
Section snippets
Materials and methods
PVA (average M.W., 30–70 kDa), methylene chloride, humic acid, dexamethasone, rat serum albumin (RSA), and phosphate-buffered saline (PBS), pH 7.4, were purchased from Sigma (St. Louis, MO). VEGF was purchased from Peprotech Inc. (Rocky Hill, NJ). PLGA (average M.W., 26 kDa) was a generous gift from Boehringer Ingelheim Corp. (Danbury, CT). PVA (99% hydrolyzed; M.W., 133 kDa) was purchased from Polysciences Inc. (Warrington, PA). [3H]Dexamethasone was purchased from Amersham Pharmacia Biotech
Results and discussion
Simultaneous site-specific parenteral elution of medicinal agents to modify the immediate tissue environment can be one of the best strategies to improve biocompatibility of implantable devices in general and can have a positive impact on the potential applications of biosensors in particular. In this work a combination of dexamethasone and VEGF was investigated for localized pharmacotherapeutic effects, using a PLGA microsphere/PVA hydrogel composite delivery system. The composite delivery
Conclusions
PLGA microsphere/PVA hydrogel composites released poorly water soluble small molecular weight compounds as well as protein therapeutics with low burst and approximately zero order release kinetics over the 4 week study period. Concurrent release of dexamethasone and VEGF effectively minimized inflammation, inhibited fibrosis and promoted neo-angiogenesis at the implant site at a fraction of the typical oral or parenteral i.v. bolus doses. This strategy of simultaneous delivery of VEGF in
Acknowledgements
This work was supported by the US Army Medical Research TMM grants (#DAMD178-02-10713, W81XWH-04-1-0779 and #W81XWH-05-1-0539). The authors would like to thank Denise Woodward, Department of Pathobiology, Upkar Bhardwaj and Craig Trusley, Department of Pharmaceutical Sciences, University of Connecticut for technical assistance.
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Current address: Analytical Research and Development Department, Amylin Pharmaceuticals Inc., San Diego, CA 92121, United States.