Subscriber access provided by TEXAS A&M INTL UNIV
Article
Biocompatible Collagen Paramagnetic Scaffold For Controlled Drug Release Simona Bettini, Valentina Bonfrate, Zois Syrgiannis, Alessandro Sannino, Luca Salvatore, Marta Madaghiele, Ludovico Valli, and Gabriele Giancane Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.5b00829 • Publication Date (Web): 13 Aug 2015 Downloaded from http://pubs.acs.org on August 15, 2015
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Biomacromolecules is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 30
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Biomacromolecules
Biocompatible Collagen Paramagnetic Scaffold For Controlled Drug Release Simona Bettini§‡, Valentina Bonfrate||‡, Zois Syrgiannis˦, Alessandro Sannino||, Luca Salvatore||, Marta Madaghiele||, Ludovico Valli§, Gabriele Giancane¶* §
Department of Biological and Environmental Sciences and Technologies, DISTEBA, University
of Salento, Via per Arnesano, I-73100 Lecce ||
Department of Engineering for Innovation - Campus University Ecotekne, Via per Monteroni, I-
73100 Lecce, Italy ˦ Centre of Excellence for Nanostructured Materials (CENMAT), INSTM, unit of Trieste, Dipartimento di Scienze Chimiche e Farmaceutiche, Università di Trieste, via L. Giorgieri 1, 34127, Trieste (Italy). ¶
Department of Cultural Heritage, University of Salento, Via Birago 64, I-73100 Lecce, Italy
E-mail:
[email protected] Abstract A porous collagen-based hydrogel scaffold was prepared in presence of iron oxide nanoparticles (NPs) and was characterized by means of infrared spectroscopy and scanning electron microscopy. The hybrid scaffold was then loaded with fluorescein sodium salt as a model compound. The release of the hydrosoluble species was triggered and accurately controlled by the application of an external magnetic field, as monitored by fluorescence spectroscopy. The biocompatibility of the proposed matrix was also tested by the MTT assay performed on 3T3 cells. Cell viability was only slightly reduced when the cells were incubated in presence of the collagen-NPs hydrogel, compared to controls. The economicity of the chemical protocol used to obtain the paramagnetic scaffolds as well as their biocompatibility and the safety of the external trigger needed to induce the drug release, suggest the proposed collagen paramagnetic matrices for a number of applications including tissue engeneering and drug delivery.
Keywords Collagen, Iron Oxide Nanoparticles, Spectroscopy, Drug delivery
ACS Paragon Plus Environment
Biomacromolecules
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 2 of 30
Introduction For many years, porous materials able to deliver hydrosoluble drugs, cells or active compounds have been largely employed in biomedicine.1 Materials used as scaffold can be natural and synthetic polymers, metals, ceramic and glasses.2, 3, 4 A common feature for all these materials is, of course, a high biocompatibility, in addition to a porous microstructure, which is the crucial requisite for loading and releasing of chemical compounds.5, 6, 7, 8 Biological agents are commonly adsorbed onto the surface of scaffolds, used as “passive” matrices, according to different methods.
9, 10, 11
However, one of the major problems with this approach is
related to a difficult control of the release kinetics, with an almost instantaneous “burst” release when the matrix is placed in physiological fluids.12 In general, the release of active molecules from a given scaffold is regulated by their diffusion through the matrix and their solubility in the external environment. For this reason, chemical agents promoting crosslinking of the scaffold can be used to form a reticular structure of interconnected pores, which are able to retain the drugs and ensure their progressive release. 13, 14 Biocompatible hydrogels are particularly attractive for use as controlled drug delivery devices. The mesh size of hydrogels, which depends on the crosslinking reaction as well as the swelling degree (or water content), is a powerful means to tune the diffusion of molecules within the polymeric matrix.15,16 In particular, polyelectrolyte hydrogels are extremely sensitive to changes of physiologically relevant variables, such as pH and ionic strength, and the corresponding swelling/deswelling transitions might be exploited to target the release of loaded molecules to selected body sites (e.g. in oral delivery). In the last few years, increasing attention in biomedicine has been directed towards the synthesis of biocompatible hydrogels from natural polymers, e.g. chitosan 17, 18 and collagen 19, which also show the capability to be enzymatically resorbed in vivo. It is well known that hydrogels can be obtained by either chemical or physical crosslinking methods.20,
21, 22
However, the paramount drawback of
ACS Paragon Plus Environment
Page 3 of 30
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Biomacromolecules
physical hydrogels23, represented by their poor thermal stability and short residence time in vivo, makes chemical hydrogels more appealing for the sustained release of bioactive molecules. Collagen can be dispersed into aqueous solution and molded into various forms of scaffolds and/or drug delivery systems for a number of biomedical applications, including but not limited to: shields in ophthalmology,24 sponges for the treatment of burns/wounds,25 mini-pellets,26 gels, patches 27and nanoparticles for gene delivery.28 Among the most popular and most effective chemical cross-linkers adopted for collagen, there are glutaraldehyde
29
and formaldehyde
30
, that induce the formation of stable matrices with high
crosslinking density. Nevertheless, their well-known toxicity strongly limits their use for in vivo experiments and applications.31 In this work, magnetite nanoparticles (NPs) are proposed and used in a double role: NPs can offer interaction sites to protein fibers for the generation of collagen-based porous hydrogel matrices simultaneously providing an accurate sensitivity to an external trigger. The biocompatibility of iron oxide nanoparticles is widely reported in the literature 32, 33, 34 and such an important feature allows to overcome the problem related to the toxicity of several chemical crosslinking agents. Furthermore, the paramagnetic behavior of iron oxide nanoparticles permits to use an external stimulus, i.e. the application of a magnetic field, to induce and control the release of any loaded substance from the gel matrix. It is worth noting that the interaction among nanoparticles and collagen fibers can also guarantee the NPs stabilization, preventing their aggregation and oxidation.
Materials and Methods Chemicals. FeCl3, FeSO4.7H2O, NaOH, fluorescein sodium salt, formaldehyde (FA) were purchased from Sigma Aldrich® and used as received, without further purification. Type I collagen isolated from calfskin was kindly provided by TypeOne Srl, Lecce, Italy, and used as received.
ACS Paragon Plus Environment
Biomacromolecules
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 4 of 30
Fabrication of Porous Collagen Gel. Dry collagen flakes were dispersed in ultrapure water in order to prepare a 0.5% (w/v) suspension. The suspension was then homogenized under magnetic stirring for 6 hours at 10°C, to avoid the denaturation of the collagen fibres. The collagen slurry was cast in 100 mm Petri dishes, de-aerated under vacuum to remove entrapped air bubbles and freeze-dried according to previously reported methods in order to obtain microporous matrices. With the aim of improving their mechanical properties and chemical stability, all samples were then subjected to a dehydrothermal crosslinking treatment (DHT), for introducing covalent crosslinks among the polypeptide chains.35 Such a treatment was performed by heating the matrices in a vacuum oven (p
