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Results of NANOMED project.
Phase 2011
Objective 1. Study of the molecular and colloidal self association
and self organization process in aqueous solutions
Activities:
1.1. Comparative analysis of the models of homomolecular self association in
aqueous solutions of barley aleurone protein (BAP), collagen (COL) or
chitosan (CHI)
Molecular or colloidal self association consists in the spontaneous
association of molecules into stable molecular aggregates, structurally well
defined, and built by non covalent bonds. Self association is a process of
self organization, where molecules are associated into supramolecular
structures in aqueous solutions, at fluid interfaces such as liquid/air or
on solid supports, following the process of adsorption and formation of the
supramolecular assemblies in interaction with the solid support.
Two proteins, namely a globular protein, the stocking protein extracted and
purified from the aleurone cells of barley seeds (noted BAP protein), and a
fibrous protein, type I collagen (COL) from bovine Achilles tendon, as well
as a carbohydrate, chitosan (CHI) were investigated. These biomolecules are
widely spread in vivo, and present unique homomolecular self assembly
properties in solution, at the air/water interface or in thin films on solid
supports. Our research project developed new experimental strategies for the
preparation and characterization of homomolecular self association of
biomolecules in aqueous solutions, or by adsorption on solid support, as
well as of the biomolecules association with gold or silver nanoparticles,
in well controlled experimental conditions. Results are in substantial
agreement with the molecular structure of biomolecules and with the proposed
models for homomolecular self association.
1.2. Determination of the influence of operation parameters on the
processes of molecular and colloidal self assembling in aqueous solution of
BAP protein, collagen (COL) or chitosan (CHI)
In order to characterize the influence of experimental conditions on the BAP
protein, COL and CHI self assembly, we undertook studies on these
biomolecules in aqueous systems and on their adsorption on solid substrates
(mica, glass). We used different bulk techniques coupled with specific
interfacial techniques, like Langmuir-Blodgett Technique (LBT), that
provides a direct method to produce COL, CHI or BAP protein substrates
(scaffolds), well oriented at a chosen interfacial pressure, which can be
used in cell cultures. Their behavior proves to be strongly dependent on the
salt (NaCl) concentration in the water subphase and on working temperature.
The results have also shown that a large series of experimental variables
affect both the processes of molecular and colloidal self assembly in
aqueous solutions, and the structure of the layers of biomolecules adsorbed
on glass or mica. Such parameters are: pH value, ionic strength,
biomolecules concentration in the aqueous solution, the structure of
biomolecules and temperature. Various nanostructures are obtained by direct
adsorption on glass and mica, immersed in the aqueous phases of selected
biomolecules, at different concentrations of biomolecules and depending on
the adsorption time. The methods used to prepare the aqueous dispersions, as
well as the methods of preparation of thin films at the water/air interface
have an important effect on the obtained structures.
Phase 2012
Objective 2: Characterization of homomolecular interactions in
aqueous solutions of BAP protein, collagen or chitosan by spectroscopical,
optical, electrical methods and microscopy
Activities:
WP2.1-WP2.4, referring to ♦ physical and chemical characterization of
molecular interactions; ♦ determination of the self assembling
characteristics at the colloidal solution/air interface; ♦ determination of
characteristic parameters (size and shape of supramolecular aggregates) for
the molecular self association and ♦ self organization process in colloidal
solutions of BAP protein, collagen or chitosan; WP2.5-WP2.6 led to ♦
dissemination of partial results; ♦ good practice exchanges/ working visits.
The experimental research included the determination of shape and size for
the supramolecular aggregates prepared in aqueous biosystems, as well as of
the zeta-potential of the assemblies resulted by molecular or colloidal
self-association, using DLS, TEM and AFM imaging, as well as optical
measurements, NMR, FTIR and Raman spectroscopy. Thermodynamic properties of
the biosystems were investigated using differential scanning calorimetry
(DSC). Self-assembly characteristics for the BAP protein, COL and CHI at the
air/colloidal aqueous solution interface were determined by the Langmuir-Blodgett
technique (LBT) coupled with atomic force microscopy (AFM) and electron
microscopy (SEM and TEM).
The supramolecular associates of BAP protein give rise to nanoparticles,
sometimes aggregated in clusters, tightly packed on the glass or mica
support. The apparent width of BAP protein nanoparticles is in the 70-80 nm
range. These results lead to the understanding of the molecular
structuration bioprocess, of BAP protein molecules into biological
nanoparticles, which takes place in the vacuoles where these protein
molecules are stocked in vivo. Similar results are obtained for CHI
molecules assembled in nanoparticles on solid substrate, with the diameter
of nanoparticles situated in the range of 80-90 nm, depending of
experimental conditions. Results indicate that layered scaffolds of BAP
protein or of CHI can be used for cells growth in vitro.
It is remarkable that the supramolecular structure of the COL protein
molecules has a high stability showing the fibrillar structure of COL self
assemblies in substantial agreement with self assembled models of COL
association and with molecular structure of COL molecules. The COL fibrils
and fibers are evidenced in the NANOMED project for the first time by using
LBT technique, TEM and AFM. The COL fibrillar supramolecular structure has
important biological effects as identified by using the collagen fibers as
scaffolds for osteroblasts in vitro. The COL fibrillar scaffolds can have
potential use in regenerative medicine.
Objective 3: Research and development of mixed polyfunctional
biosystems composed of BAP protein, collagen or chitosan, in presence of
gold or silver nanoparticles
Activities:
WP3.1-WP3.5 refer to ♦ preparation and characterization of noble metals
nanoparticles of controlled size and shape; ♦ spectral and structural
characterization of the mixed biosystems composed from biomolecules and gold
or silver nanoparticles; ♦ elaboration and experimental verification of
models for the molecular self assembly in aqueous solutions and in oriented
structures at interfaces ♦ new applications of Langmuir-Blodgett technique;
WP3.6-WP3.7 lead to ♦ successful materialization of good practice exchanges/
working visits; ♦ dissemination of partial results.
Experimental research included the development of new synthesis methods for
gold nanoparticles (GNPs) and silver nanoparticles (AgNPs) with the aim to
obtain stable monodisperse systems, with controlled particles size for GNPs
and for AgNPs, in order to satisfy the demands for biomedical applications.
We selected natural compounds as reducing agents in biogenic syntheses, e.g.
β-cyclodextrin (βCD), glucose or plants extracts, and determined the optimal
conditions to obtain GNPs or AgNPs with controlled shape and size, described
by UV-Vis spectroscopy and TEM and AFM images.
The mixed polyfunctional biosystems composed of BAP protein, collagen or
chitosan, in presence of gold or silver nanoparticles were also prepared,
which were stable for many weeks, as visualized by various said techniques,
including AFM. For GNPs and COL fibrils, a new fibrous biomaterial is
developed, which stimulates the development of osteoblasts cells in cell
cultures in vitro. For comparison, anesthetics, like procaine, tetracaine,
and dibucaine, as well as arginine and lipoic acid were also evaluated, both
in homomolecular self-assembly, and in mixed biosystems with GNPs or AgNPs.
We elaborated models for the self-assembly in aqueous phase and in oriented
structures at interfaces, which fully corresponded to the experimental data.
Phase 2013
Objective 4 (1st part): Research and development of mixed
polyfunctional systems composed of BAP protein, collagen or chitosan in
presence of anticancer compounds, anesthetics, or antioxidants and in
presence of noble metals nanoparticles (1st part),
Activities:
WP4.1-WP4.2 related to ♦ characterization of molecular interactions in
presence of anesthetic, antioxidants or anticancer compounds, and gold or
silver nanoparticles, by DLS, zeta-potential measurements, spectral
measurements, AFM, SEM or TEM; WP4.3 led to ♦ successful materialization of
good practice exchanges/ working visits; ♦ dissemination of partial results.
GNPs and AgNPs represent some of the most attractive nanomaterials for nano-technology,
nanobiology , or nanomedicine, both as aqueous and colloidal solutions, and
as layers deposited on different solid surfaces. Our project developed
various innovative methods for the preparation of GNPs by reduction of
gold(III) compounds (particularly AuCl3 or HAuCl4) with chemicals, like
sodium citrate, Na3C6H5O7 or sodium borohydride, NaBH4, and “green”
reduction agents, like β-cyclodextrin (βCD), or diverse plant extracts.
Within our project, β-CD was used for the first time, in the state of the
art, both as reduction agent and as stabilizer, for the preparation of GNPs
and AgNPs. In addition, the use of β-CD can control the size and size
distribution of the particles, both for GNPs and AgNPs. GNPs or AgNPs were
subsequently functionalized (coated) with inclusion complexes of
cyclodextrins, for instance the inclusion complex of β-CD with lipoic acid,
or with anti cancer drugs (doxorubicin, cisplatin, 5-fluorouracil or taxol)
as potential carriers for cancer therapy.
AgNPs were fabricated also by the reduction with glucose, with hydrated
silica and L-asparagine as stabilizers. The AgNPs colloidal systems proved
to be very stable. These AgNPs systems showed a strong interaction with
three local anesthetics (procaine, dibucaine and tetracaine), as presented
for the first time in the state of the art, by our recent publication. The
colloidal systems were characterized by UV-Vis spectra, FTIR, DLS,
zeta-potential measurements, TEM and AFM. The results show that these AgNPs
can be used for biomedical applications, such as the analytical detection of
anesthetics in biological fluids.
At the same time we have tested the activity of functionalized GNPs with
different NANOMED biomolecules on diverse cell systems in vitro, in order to
discover their therapeutic effect. Further, GNPs functionalized with
collagen were utilized for the scaffolds fabrication and were also used in
cell cultures, for potential biomedical applications, such as for bones,
cartilage, ligaments, tendons and skin regeneration.
Phase 2014
Objective 4 (2nd part): Research and development of mixed
polyfunctional systems composed of BAP protein, collagen or chitosan in
presence of anticancer compounds, anesthetics, or antioxidants and in
presence of noble metals nanoparticles (2nd part).
Activities:
WP1.1-WP1.2 refer to ♦ study of the molecular interactions in polyfunctional
systems in presence of antioxidant compounds, anticancer compounds or
anesthetic and gold or silver nanoparticles, by spectral measurements, DLS,
zeta-potential measurements, LBT, AFM, SEM or TEM.
The NANOMED research project was realized by the research activities
included in the objective of this phase. The results of the scientific and
technical research over passed the frame of initially estimated results. In
the following we evidence the results of the phase and the achievements of
the research goals.
For medical applications it is important that the prepared AgNPs dispersions
do not contain toxic substances, and the particles size distribution is as
narrow as possible (preferable monodisperse systems). It is quite difficult
to obtain such systems and to stabilize them for large periods of time. The
NANOMED solution was to apply “green chemistry” methods in the syntheses of
AgNPs, using non toxic reagents, of natural origin, such as β-CD or glucose.
These green syntheses were optimized in this NANOMED phase.
The NANOMED project has additionally developed new mixed polyfunctional
biosystems with antimicrobial effect for several different pathogenic
species: Escherichia coli, Staphylococcus aureus, Staphylococcus spp.,
Bacillus cereus, Candida albicans, with potential medical applications.
These innovative materials contain different amounts of AgNPs and nano
hydroxyapatite (HAP), which is doped with 0,20% Zn, 0,25% Ag and 0,025% Au.
These polyfunctional nano materials have demonstrated an antimicrobial
effect, and were firstly synthesized, in the state of the art, by an
advanced nanotechnology recently published by us. Furthermore, innovative
composite materials were obtained by incorporating different amounts of HAP
and AgNPs into an organic matrix, for a retard release of Ag+ ions. The
composite materials were characterized from the structural point of view and
for antimicrobial properties, recently published by us. These results can
find medical applications in the realization of implants in bone tissue, to
prevent potential infections during surgical interventions.
The NANOMED project has also developed the syntheses of GNPs through green
syntheses using individual reducing agents, like an antioxidant compound,
for instance resveratrol, which can ensure a good stability of colloidal
systems against aggregation and coagulation, allowing at the same time for
GNPs conjugation with biologically active molecules, for example with
doxorubicin, for their use in medical purposes, for cancer therapy and
diagnosis.
Phase 2015
Objective 5: ♦Research and
development of mixed polyfunctional systems composed of BAP protein,
collagen, chitosan or lipids in the presence of anticancer compounds,
anaesthetics, or antioxidants and in the presence of noble metals
nanoparticles.
Activities:
WP1.1-WP1.2 refer to ♦the study of the molecular interactions in
polyfunctional systems in the presence of gold nanoparticles (AuNP) or
silver nanoparticles (AgNP), obtained by green synthesis in alkaline aqueous
media by using β-CD, trans-resveratrol (Resv, as antioxidant component) and
doxorubicin (Dox, as anti-cancer drug) was investigated by spectral
measurements, AFM, TEM, SEM. ♦Langmuir-Blodgett (LB) monolayers made of L-α–dipalmitoyl
phosphatidylcholine (DPPC) and procaine (anaesthetic compound) were used for
mimicking cell membranes and were investigated by contact mode atomic force
microscopy (cm-AFM).
The structure and properties of different lipid phases induced by procaine
were evidenced for the first time by using topography, friction force, and
force modulation images. The results indicate that procaine interacts with
DPPC monolayers, stabilizes the lipid membrane interface and induces
condensed lipid domain formation, modulating membrane functional properties.
The procaine effect on lipid domains is also important for the emergence of
lateral membrane heterogeneity and can intervene in the generation process
of anaesthesia.
NANOMED project developed innovative polyfunctional biosystems
self-assembled in colloidal dispersions or at the air/water interfaces.
NANOMED project opens novel strategies to develop innovative vehicles to
transport anticancer drugs in vivo with various biomedical applications in
the treatment of cervical cancer.
The innovative vehicles are made in a pioneering work using AuNP,
synthesized by green chemistry with β-CD, and stabilized by β-CD and Resv.
The aqueous AuNP dispersions are rather homogeneous and very stable, ready
for biological and biomedical applications. The stability of Dox
nanocomplexes is high in phosphate buffer saline as estimated by UV–vis
spectra, TEM and AFM analysis. Effects of Resv-Dox mixtures, AuNP and
Dox-AuNP complexes on HeLa and CaSki cells, of cervical cancer, after 24 h
drug incubation, were assessed using MTT cell viability assay. AuNP
functionalized with Resv and Dox showed a strong anticancer effect on the
two cell lines Hela and CaSki. This is an inter- and multi-disciplinary work
developed by using low and safe doses of Dox, thus, reducing the side
effects of Dox in vivo, particularly its toxicity on heart. Our results
provide strong evidence that novel drug delivery vehicles developed on
Dox-AuNPs nanocomplexes and Resv could have wide applications in cancer
diagnosis and treatment.
Phase 2016
Objective 2016: ♦Research and
development of mixed polyfunctional systems composed of BAP protein,
collagen or chitosan in the presence of anticancer compounds, anesthetics,
or antioxidants and in the presence of noble metals nanoparticles.
Activities:
WP1.1 - WP1.5 refer to ♦ the study of the molecular interactions in
polyfunctional systems in the presence of gold nanoparticles (AuNP) or
silver nanoparticles (AgNP), obtained by green synthesis in alkaline aqueous
media by using trans-resveratrol (Resv, as antioxidant component) and
glucose as well as doxorubicin (Dox, as anti-cancer drug) and pure or
multi-substituted hydroxyapatite (HAP, as carrier), was investigated by
spectral measurements, UV-Vis, XRD, AFM, TEM, SEM. ♦ The anticancer activity
of AuNP-Resv-Dox nanocomplexes is evaluated by MTT test and apoptosis tests.
♦ Antimicrobial activity of AgNP or Ag+ ions in the absence and the presence
of different biomolecules, using carriers as hydroxyapatite based
biomaterials (e.g. HAP, HAP-Ag-Au-Zn or HAP-Zn-Sr) under the form of nano
powders or discs, is determined by Kirby-Bauer diffusion test against
Staphylococcus aureus. ♦ In addition, the antimicrobial activity of Cu2+
ions in presence of biomolecules, like 8-hydroxyquinoline (HQ) and
5-nitro-8-hydroxyquinoline (NHQ) or its derivatives, is evaluated against
various pathologic microorganisms, in comparison with Ag+ ions, using as
carrier hydroxyapatite, for Escherichia coli, Staphylococcus spp.,
Microcoocus spp., Bacillus spp., Candida spp., Prototheca spp.
Selective Bibliography:
http://www.chem.ubbcluj.ro/romana/ANEX/cf/pcas/index.htm
(l) Research original
articles:
1) Gh. Tomoaia, O.
Horovitz, A. Mocanu, A. Nita, A. Avram, C.P. Racz, O. Soritau, M. Cenariu,
and M. Tomoaia-Cotisel,
“Effects of doxorubicin mediated by gold
nanoparticles and resveratrol in two human cervical tumor cell lines”,
Colloids and Surfaces B: Biointerfaces, 135, 726-734
(2015).
IF: 4.152 RIS: 1.483
2) A. Mocanu, G. Furtos, S. Rapuntean, O. Horovitz, C.
Flore, C. Garbo, A. Danisteanu, Gh. Rapuntean,
C. Prejmerean, and M.
Tomoaia-Cotisel,
“Synthesis; characterization and
antimicrobial effects of composites based on multi-substituted
hydroxyapatite and silver nanoparticles”,
Applied Surface Science,
298, 225–235 (2014).
IF: 2.711 RIS: 1.488
3) G. Tomoaia, M. Tomoaia-Cotisel, L.B. Pop, A. Mocanu,
and A. Pop,
“Nanopowders of hydroxyapatite and its
substituted derivatives to be employed for medical purposes
and process for preparing the same”,
Romania, Patent nr.125817,
date 28 June 2013, BOPI (Buletinul oficial de proprietate industriala-
Sectiunea brevete de inventive), nr.
6, 2013, p. 123.
4) A. Danistean, M. Gorea, A. Avram, S. Rapuntean, Gh.
Tomoaia, A. Mocanu, C. Garbo,
O. Horovitz, and M.
Tomoaia-Cotisel,
“Antimicrobial activity of ceramic disks
loaded with silver ions and nitroxoline”,
Studia Univ. Babes-Bolyai,
Chemia, 61 (3), Tom I, 275-283 (2016).
IF: 0.148 RIS: 0.049
5) S. Rapuntean, A. Pop, V. Miclaus, C. Garbo, F.
Chirila, Gh. Rapuntean, N. Fit,
H. Farcău, and M.
Tomoaia-Cotisel,
“Research concerning in vitro sensitivity
of some microorganisms at hydroxyquinoline and cupric derivatives,
deposited onto hydroxyapatite”,
Bulletin UASVM, Veterinary
Medicine 72(2)/2016; pISSN 1843-5270; eISSN 1843-5378;
an international,
open access journal, listed by Thomson Reuters Master Journal in 2016.
6) M. Tomoaia-Cotisel, A. Tomoaia-Cotisel, Gh. Tomoaia,
and A. Mocanu,
“Innovative substituted hydroxylapatites
and collagen scaffolds for enhanced adhesion,
growth and proliferation of human
osteoblasts in vitro”,
J. Biotechnol.
Biomater., 6 (1), (Suppl), 2016 ; ISSN: 2155-952X. C1.049-016,
JBTBM an open access
journal.
7) Maria Tomoaia-Cotisel (Author and coordinator),
“Multifunctional nanostructures formed of
gold or silver nanoparticles and different
biomolecules with medical applications”,
Cluj University Press,
Cluj-Napoca, 2016, pp. 322; ISBN 978 606 37 0017 0;
Monography: e-Book,
http://www.editura.ubbcluj.ro/bd/ebooks/pdf/1976.pdf
(II) Research original communications:
8) M. Tomoaia-Cotisel, A. Tomoaia-Cotisel, Gh. Tomoaia,
and A. Mocanu,
“Innovative substituted hydroxylapatites
and collagen scaffolds for enhanced adhesion,
growth and proliferation of human
osteoblasts in vitro”,
Oral invited presentation
by M. Tomoaia-Cotisel at Biomaterials 2016 Conference and expo on
biomaterials,
London University, UK.,
March 14-16, 2016;
published in J. Biotechnol.
Biomater., Volume 6, Issue 1 (Suppl), 2016 ;
ISSN: 2155-952X.
C1.049-016, JBTBM an open access journal.
http://dx.doi.org/10.4172/2155-952X.C1.049.
http://biomaterials.conferenceseries.com/pdfs/biomaterials-2016-scientific-program.pdf
9) M. Tomoaia-Cotisel,
“Frontier biomedical research: from
multi-functional bio-interfaces to
biomaterials and tissue engineering”,
Oral presentation, Sesiunea
Ştiinţifică de Primăvară 2016, a
Academiei Oamenilor de Ştiinţă din România,
Bucuresti, May 27-28, 2016.
http://www.aosr.ro/wp-content/uploads/2015/04/2016-05-25-PROGRAM-si-VOLUM-REZUMATE-SESPRIM2016.pdf
10) M. Tomoaia-Cotisel,
“Frontier biomedical research: from
multi-functional bio-interfaces to nano biomaterials and tissue
engineering”,
Oral invited presentation,
at International Conference On Nanomedicine And Nanobiotechnology
(ICONAN 2016), Paris,
Sept. 28-30, 2016.
http://programme.exordo.com/iconan2016/
11) Workshop with international participation, on the
theme:
“Gold and silver nanoparticles with medical
applicability”,
within the project
PCE - NANOMED - no. 257, from the National Plan II, organized by the Centre
of Physical Chemistry from the Faculty of Chemistry and Chemical
Engineering, Babes-Bolyai University from Cluj-Napoca (UBB), April 23, 2016.
The meeting was attended by Romanian scientific experts, along with foreign
experts in the field of the workshop, from 5 European research units, from 4
countries: Switzerland, France, Latvia and Romania, partners in
international Consortium of our research project “Multifunctional injectable
composites for the treatment of osteoporosis”, NANOFOROSTEO, ERANET project
no. 4-006, from the European Platform of Nanomedicine. Thus the workshop was
held in the framework of the second scientific meeting of the international
consortium, which took place in Cluj-Napoca, between April 22 and 27, 2016.
The workshop has been included in UBB RESEARCH: Weekly News about Results
and Impact, published at UBB, on April 30, 2016.
(III) PhD Theses:
12) R.D. Pasca,
“Lipid nanostructures in the presence of
some biomolecules soluble in water or various metal
nanoparticles of gold or silver”,
PhD Thesis, Babes-Bolyai
Universitaty from Cluj-Napoca, 2013.
13) A. Nita,
"Gold or silver nanoparticles,
functionalized with biomolecules. Biomedical applications”,
PhD Thesis, in
progress, Babes-Bolyai Universitaty from Cluj-Napoca.
14) A. Avram,
“Research and development of nanostructured
systems of biologic and biomedical interest”,
PhD Thesis, in
progress, Babes-Bolyai Universitaty from Cluj-Napoca.
15) Sz. Santa,
“ Research and development of micro- and
nanostructured systems made from various biomolecules”,
PhD Thesis, in progress,
Babes-Bolyai Universitaty from Cluj-Napoca.
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