Key Engineering Materials Vol. 583 (2014) pp 145-149 © (2014) Trans Tech Publications, Switzerland doi:10.4028/www.scientific.net/KEM.583.145
Online: 2013-09-10
Development of Modified Viscoelastic Solution with Magnetic Nanoparticles – Potential Method for Targeted Treatment of Chondral Injuries Rodica Marinescu1,a, D.Laptoiu1, I.Antoniac2,a , C Petcu2 1
Colentina Clinical Hospital Bucharest, Romania; 2University Politehnica of Bucharest, Romania, b
[email protected]
Keywords: viscoelastic, magnetic nanoparticles, chondral injury.
Abstract Intra-articular treatments offer the advantage of achieving high active substance concentrations at the site of cartilage lesion. One problem with these drugs is that of low remanence. A method to prolong the survival time of substance inside the joint may improve the cartilage repair and patient symptoms. Targeted therapy aims to optimize the delivery of these drugs set in a solution of magnetic nanoparticles, by injection into the knee joint, in order to increase the residence time of active substances through the application of high magnetic field gradients in the joint area by using extracorporeal magnets. Synthesized magnetic nanoparticles, which were covered by layer-by-layer method in consecutive layers of protection, are stable and retain their character and magnetic-polymer composites features required to be used for their intended purpose. FTIR analysis demonstrated the presence of iron salts in the synthesized ferrofluid, and of the characteristic groups of polymer coating which demonstrated that the technique of embedding in polymer layers by layer-by-layer method is viable. INTRODUCTION Among chronic diseases, one of the most frequent and incapacitating is osteoarthritis. Several treatment protocols are available, with some conservative procedures included. New therapies emerge in order to preserve the joint surface and to improve patient physical performance and pain free life. The intra-articular treatments offer the advantage of achieving high active substance concentrations at the site of lesion. One problem with these drugs is that of low remanence - they don’t remain long enough inside the joints (as days versus weeks needed). A method to prolong the survival time of substance inside the joint may improve the cartilage repair and patient symptoms. Adding magnetic particles in such a substance may add magnetic properties and so, it may be potentially manipulated with the help magnets. In the last years, magnetic particles have been used in medicine either as diagnostic tool either as carriers in diseases treatment. [1,2] Magnetic drug targeting using iron oxide nanoparticles as carrier systems has been experimented for targeted therapy of joint diseases. Studies in human and animals revealed that intraarticular application of functionalized magnetic nanoparticles is possible, safe and that the use of extracorporeal magnets enhances their effect in situ [2,3]. Previous studies of biomedical applications show that these nanoparticles must have high magnetization values and size around 100 nm with overall narrow particle size distribution. [4] Biofunctionalization is obtained through special surface coating of the magnetic nanoparticles, which has to be not only biocompatible but also allow a stable delivery of the active substances in the designated area [5]; as we targeted articular cartilage our study started development of hyaluronic acid – Fe2O3 hybrid magnetic nanoparticles. MATERIALS AND METHODS The colloidal stability and behavior of nanoparticles in suspensions can be tailored during synthesis and it is related to the pH, additives in the solution as in cell media or body fluids, their viscosity and any other fluidic properties. Magnetic nanoparticles were prepared by Massart’s method (co-precipitation) from FeCl2 and FeCl3 in a concentrated aqueous alkaline solution All rights reserved. No part of contents of this paper may be reproduced or transmitted in any form or by any means without the written permission of Trans Tech Publications, www.ttp.net. (ID: 141.85.36.98, University Politehnica of Bucharest, Bucharest, Romania-14/08/15,13:26:26)
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(NH4OH 25%) as a mixture of iron salts in molar ratio FeCl2: FeCl3 of 1:2,7. Two solutions were prepared from 2M HCl FeCl2 * 4H2O and FeCl3 * 6H2O with water, were mixed, after the dissolution of solids in a three necks flask. The mixture was stirred mechanically under nitrogen pad, and after mixing the required amount of NH4OH was added and left at 25% slow stirring, 300 rpm at room temperature for one hour. Modified magnetic particles were collected, washed twice and redispersed in water. In the next stage, magnetic nanoparticles were coated by TMOH (a hydrophilic complex - tetramethylammonium hydroxide), in a layer by layer method. After washing, the magnetic particles were added to a TMOH (1M) solution, which was mixed until homogeneous and then was left overnight in order to obtain the peptization phenomenon. The solution thus obtained was kept at room temperature for next use. The purpose of this study was to synthesize a compound that once placed in the joints under the action of an external magnetic field, would give cartilage elasticity and suppleness in the desired areas, potentially acting as a targeted "lubricant". For this reason, we embeded the synthesized particles in a final layer (third) of hyaluronic acid compound which is known to have these features. Coverage of hyaluronic acid on the nanoparticles was achieved by layer-over-layer sonication technique (layer-by-layer) using two different concentrated solutions of hyaluronic acid, in order to study the effect of hyaluronic acid concentration on the final properties of the biopolymer composites - magnetic nanoparticles. At this time a stable colloidal dispersion was obtained. Ferrous chloride (II) (FeCl2 ∙ 4H2O) (Merck), ferric chloride (III) (FeCl3 ∙ 6H2O) (Merck) and ammonium hydroxide 25% aqueous NH4OH was used as such. Tetramethylammonium hydroxide (TMOH) (Fluka) and inulin (Dahlia tubers, Fluka) were also used as such. For the characterization of nanoparticles, Zeta Potential measurements, FTIR and TEM methods were used. All Zeta potential measurements were made on an instrument of dynamic light scattering, Zetasizer Nano ZS (Malvern Instruments Ltd, Worcestershire, UK). Particle size distribution was measured on a device the dynamic light scattering (DLS) Zetasizer Nano ZS. Refractive index of the material was fixed at 1.89. For polymer surface characterization were drawn ATR FT-IR spectra using a 37 Tensor tool (Bruker Optik GmbH, Rudolf-Plank-Str. 27, Ettlingen Germany). A further study by transmission electron microscopy (TEM) helped us to determine the average size and distribution of nanoparticles. RESULTS Preliminary particle size measurements, the dispersion of dimensional solution and the degree of aggregation of nanoparticles was made using dynamic diffusion of light (DLS). The results are shown in figure 1 and table 1. Initial magnetic particles are in the form of particle size around 100 nm. Agglomerates are deposited very quickly in the bottom container, which led us to stabilize the magnetic particles by covering with a layer of TMOH (peptization), leading to destruction of agglomerates.
Figure 1: Distribution of the particles size (black – numeric; red – volumetric) by dynamic light scattering (DLS) of the suspension (a)) before and after TMOH coating (b).
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Table 1 Medium size and Zeta potential value of the synthesized particles Nr. crt. Sample Composition DLS (nm) Zeta Potential (mV) 1 magnetic particle (PM) 109,4 -44,6 2 PM + TMOH 43,67 -42,4 3 PM + TMOH + HA conc. 106,8 -85,5 According to the results presented in table 1, the magnetic nanoparticles formed stable composites after adding TMOH layer. Particle size results were 43 nm, but Zeta potential evidence has not changed. The relative uniform distribution around 106 nm sizes allows potential intraarticular use without initial concern for the articular remanence – studies have defined the pore size for the cartilage dense collagen matrix around 60 nm. We can see that there is a difference between the size distribution of nanoparticles with and without coating. Dimensional dispersion is narrower than that of nanoparticles without surface coating; the nanoparticles significantly improved their stability in suspension. The FTIR results (figure 2) shows characteristic bands for ferroferic nanoparticles. Wavelength band at 630 cm-1, is present in the spectra of all synthesized samples and demonstrates the presence of maghemite (γ-Fe2O3). Also hematite (α-Fe2O3) is present in the analyzed ferrofluids, as demonstrated by the band from 537 cm-1.
Figure 2. FTIR results: a) magnetic particles stabilized with TMOH; b) magnetic particles stabilized with TMOH coated with HA FTIR analysis of tetramethylammonium hydroxide coated ferrofluid demonstrates the presence of the group-OH stretching vibration, which generates a symmetric and asymmetric broadband 3330 cm-1. Intense band at 1637 cm-1 can be assigned to symmetric stretching vibration of-OH ofCOOH groups, and the characteristic deformation vibration of C-N grouping of TMOH. Vibration at 1489 cm-1 is on the asymmetric stretching, C = O of-COOH group. During the analysis of the samples coated with hyaluronic acid, we observe a slight modification of the specific bands for hydroxyl-OH from 3330 cm-1 to 3340 cm-1 which means that there has been coverage with hyaluronic acid. TEM images indicates that the nanoparticles without surface treatment (Figure 3a) are very busy and form aggregates, while nanoparticles functionalized are spherical and have relatively uniform size (Figure 3b). Regarding the dimension of the nanoparticles, we observe that the nanoparticles size after synthesis have an average size of about 40 ± 2 nm and for the nanoparticles coated with hyaluronate the average size is around 100 nm.
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a)
b)
Figure 3: TEM Image of the magnetic nanoparticles (a) and after functionalization by coprecipitation (b) 100 nm scale DISCUSSIONS and CONCLUSIONS Targeted therapy aims to optimize the delivery of these drugs set in a solution of magnetic nanoparticles, by injection into the knee joint, in order to increase the residence time of active substances through the application of high magnetic field gradients in the joint area by using extracorporeal magnets. Synthesized magnetic nanoparticles, which were covered by layer-by-layer method in consecutive layers of protection, are stable and retain their character and magneticpolymer composites features required to be used for their intended purpose. FTIR analysis demonstrated the presence of iron salts in the synthesized ferrofluid, and of the characteristic groups of polymer coating which demonstrated that the technique of embedding in polymer layers by layer-by-layer method is viable. Anyway, our future studies related to the biocompatibility and the magnetic properties will be made in order to confirm the clinical potential for our developed materials. For the long evolutions of degenerative joint diseases, it is still need for a targeted delivery system, capable to reduce the side effects and to maintain a controlled quantity of active substance in the damaged area. BIBLIOGRAPHY [1] Sarma AV, Powell GL, LaBerge M. Phospholipid composition of articular cartilage boundary lubricant. J Orthop Res. 2001 Jul; 19(4):671-6. [2] Hofmann-Amtenbrink M, Hofmann H, Montet X. Superparamagnetic nanoparticles - a tool for early diagnostics. Swiss Med Wkly. 2010 Sep 17; 140:w13081. doi: 10.4414/smw.2010.13081. Review. [3] Rothenfluh DA, Bermudez H, O'Neil CP, Hubbell JA Biofunctional polymer nanoparticles for intra-articular targeting and retention in cartilage. Nat Mater. 2008 Mar; 7(3):248-54. Epub 2008 Feb 3. [4] Ajay Kumar Gupta, Mona Gupta Synthesis and surface engineering of iron oxide nanoparticles for biomedical applications Biomaterials Volume 26, Issue 18, June 2005, Pages 3995-4021. [5] Ajay Kumar Gupta, Mona Gupta Cytotoxicity suppression and cellular uptake enhancement of surface modified magnetic nanoparticles Biomaterials Volume 26, Issue 13, May 2005, Pages 1565-1573. [6] Butoescu N, Seemayer CA, Foti M, Jordan O, Doelker E. Dexamethasone-containing PLGA superparamagnetic microparticles as carriers for the local treatment of arthritis.Biomaterials. 2009 Mar; 30(9):1772-80. Epub 2009 Jan 8.
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Development of Modified Viscoelastic Solution with Magnetic Nanoparticles – Potential Method for Targeted Treatment of Chondral Injuries 10.4028/www.scientific.net/KEM.583.145
