Federico Caneva Soumetz*, Mauro Giacomini*, Laura Pastonno*, James B. Phillips**, Robert A. Brown**, Carmelina Ruggiero*. *D.I.S.T, University of Genova, ...
2004 4th IEEE Conference on Nanotechnology
Drug Delivery for Nerve Tissue Regeneration Federico Caneva Soumetz*,Mauro Giacomini*, Laura Pastonno*, James B. Phillips**, Robert A. Brown**, Carmelina Ruggiero*
*D.I.S.T, University of Genova, Via Opera Pia, 13, 16145 Genova, Italy **University College London, Tissue Repair and Engineering Centre, United Kingdom. Abstract - The use of implantable biomedical devices for the repair of peripheral nervous system injuries shows great promises for a complete recovery of the physiological nerve function. The neutralhtion of pro-fibrotic factors like TGFp1 and the promotion of an oriented growth of the transected nenrites are of pivotal importance in uerve regeneration. This study focus on the investigation of the dillusion kinetics of a non specific antibody in nerve guidance channels based on the use of natural compounds. They have been found to be able to work as a depot of antibodies but the release kinetics resulted to be rather fast Index Terms - Drug delivery systems, fluorescence spectroscopy, implantable biomedical devices, nanotechnology, nervous system, repair.
I. INTRODUCTION It is well known how cells react specifically both to the nano-topographic and chemical features of their surrounding environment. To this regard, in order to elicit specific cellular responses and to improve the success of any tissue repair strategy, the use of appropriate biocompatible materials plays a pivotal role [ I]-[7]. As concem the nervous system, neurons exhibit a regenerative potential, but the recovery of function following a peripheral nervous system injury is negligible in absence of surgical reconnection. A major obstacle to recovery of function and regeneration of transected nerves is the scarring process with formation of a connective tissue barrier to neurite elongation [I]. To improve the repair of injured human nerves it is of fundamental importance to inhibit the activity of pro-scarrhg agents and to actively guide the extension of the neurites toward their targets. To this end the use of nanobioengineered materials which release therapeutic factors to direct regeneration shows great promises [Z,6, 81. The cytokine Transforming Growth Factor PI (TGFp l ) plays a pivotal role in the regulation of connective tissue deposition and scar formation and its local neutralisation by means of specific antibodies (Ab) bas already shown to improve the repair of nerve injuries [8]-
UOI. This short study, focuses on the investigation of the absorption and release kinetics of a non specific Ab by
0-7803-8536-5/04/$20.0002004 IEEE
medical devices based on biocompatible materials which can be used in vivo as controlled delivery systems and nerve guidance channels. More specifically, the devices under construction are made of an inner fibronectin (FN) fibre mat [ll, 121 surrounded by an outer HYAFF 11 tube (benzyl ester of the hyaluronic acid) [13]. FN is a protein present both in connective tissues and in blood plasma and is involved in many aspects of wound healing [14]. FN based biomaterials have been widely studied as templates to promote tissue repair by contact guidance [15]-[I71 and as controlled release systems [18, 191. HYAFF-11 is produced by esterification of Hyaluronic acid (HA), a polysaccharide ubiquitous in all soft tissues of higher organisms and at tissue interfaces and joints where it acts as a tissue lubricant and prevents adhesion. HA can be isolated from rooster combs or produced by biological fermentation and can be easily processed without loss of biocompatibility. At present, different HA based products are used to promote tissue regeneration and to reduce postoperative surgical adhesions, a critical factor in the repair of peripheral nerve injures [20]-[23]. 11. MATERIALS AND METHODS
In place of a specific anti-TGF-pl a non specific immunoglobulin has been used to characterise the diffusion dynamics. A Goat Anti-Mouse IgG labelled with R-Phycoerythrin was (Sigma Aldrich Italia) was used without further purification. Aprotinin from bovine lung (Sigma-Aldricb Italia,) was used at 300 I U h l in Phosphate Buffer Saline (PBS) to neutralise the activity of any contaminant serine proteases. In order to detect the fluorescent Ab in solution and in the thickness of the materials a Perkin Elmer LS90B Luminescence Spectrometer and a Leica confocal single photon microscopy were respectively used [24, 251. The uptake and the release rates of IgG have been characterised as a function of the device lengths. Consbucts 1 cm and 0.5 cm long have been used to
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characterise the release kinetic over a period of 4 months. The Ab absorption has been carried out by soaking the materials for 96 hours, in a solution of Ab at the concentration of 5 p d m l in PBWAprotinin. The release kinetic was followed by incubating the Ab embedded materials in PBSiAprotinin solutions. Aliquots of these solutions were sampled after 1, 2, 6, 14, 27 and 120 days and analysed by means of the Perkin Elmer fluorimeter. The concentrations of Ab have been calculated by means of appropriate interpolation functions obtained from the analysis of different standard solutions ( 5 , 3, 2, 1, 0.5, 0.25,0.1,0.05, 0.025, 0.01,0.005,0.001 pg/ml). 111. RESULTS The experimental results from the analysis of the solutions sampled over a period of 4 months, for tubes 1 cm and 0,5 cm long showed Ab storage of respectively 344.4 and 234.73 nanograms. Ab release from the devices was respectively 78.85 and 57.64 nanograms. The ratio of Ab release from 1 cm and 0.5 cm long devices was 1.37, which is comparable to the ratio for absorption dynamics (1.47). All the devices released only 23% of the withheld levels of Ab, most part of which was delivered in the fust 24 hours. In this period the 1 cm tubes showed a faster release kinetic, delivering 1.43 times more Ab than the 0.5 cm tubes. These differences decreased with time and disappeared after the sixth day. These results are currently being complemented by confocal microscopy analysis to investigate the distribution of the Ab through the thickness of the materials. As discussed below, these analyses show that absorption of Ab is confined to the surface of the FN mats.
N.DISCUSSION AND CONCLUSIONS Our previous results [26] suggest that hyaluronan based materials were not able to absorb the antibody. According to this, it bas been assumed that only the FN mats were responsible of the absorption of Ab even though the outer HYAFF 11 layers will have probably affected the diffusion dynamics. The obtained results support such hypothesis. The ‘long’ devices, in fact, did not absorb a double amount of antibody compared to the shorter ones, but less then what would have been expected on the basis of a linear relationship. The amount of Ab released in the first day from all the devices was higher than that found subsequently, suggesting that the Ab was mainly stratified only onto the surface of the Fn mats and not deeply inside the material.
In support of this Ab was released faster from tubes l c m long than for the shorter ones. The fact that the ratio of release for longer and shorter devices decreased towards 1 indicates a superficial absorption of the bulk of Ab. The experimental findings here support our previous idea that only traces of Ab were taken up by the HA tubes, whereas the majority appears to be localised in the surface of the FN core. The total amount of antibody released has been found to be rather low, but it is important to note that the low release rates could be due to the small amounts of Ab absorbed by the materials. Further experimental work will be necessary to clarify in which way the release dynamics changes as a function of the amount of Ab stored inside the device and their length. A mathematical model is in preparation to analyse the diffusion dynamics of Ab. It is expected to help surgeons to estimate the levels of drug needed inside the devices for in vivo treatment of nerve injures of various extension. ACKNOWLEDGEMEN7 The present paper has been supported by the European Union within the project “Tissue engineered nerve repair devices: development of European medical implantable devices and research training focus” (contract number: QLK3-CT-1999-00625). REFERENCES
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