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Multifunctional Hybrid Silica Nanoparticles for Nanomedicine

Bioconjugation with DNA for Gene Therapy

Extended Abstract

Rui Manuel de Azevedo Pedrosa de Brito Colaço

Dissertação para obtenção do Grau de Mestre em Engenharia de Materiais

Orientadoras: Profª. Drª. Maria Clara Henriques Batista Gonçalves

Profª. Drª. Maria Bárbara dos Anjos Figueira Martins

Maio de 2011

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Abstract

Nanomedicine is an emerging new field combining nanotechnology and medicine. Silica

nanoparticles are chemical and biologically inert, optically transparent and can be doped with imaging

agents and/or functionalised to promote its bioconjugation with different therapeutic molecules. Silica

nanoparticles can be engineered to improve diagnosis, treatment and follow-up of diseases. A

combination of diagnosis devices and therapeutics (theragnosis) would be beneficial for patients.

In this work, amino-, methyl-, vinyl- and phenyl-functionalised silica nanoparticles as drug

carriers or non-viral vectors for gene delivery were prepared via different modified Stöber sol-gel

processes. Core-shell structures, using superparamagnetic iron oxide or silica nanoparticles as core,

coated with organically modified silica (ORMOSIL) shell were synthesised, as well as plain ORMOSIL

nanoparticles fabricated directly using sodium silicate solution as nucleating agent.

Dynamic light scattering, transmission electron microscopy and Fourier transformed infrared

spectroscopy were used to characterise the hybrid nanospheres. Synthesis parameters were studied

and fabrication methods have been optimised. Monodisperse spherical nanoparticles with desired size

and functionality have been obtained. Nanoparticle-DNA complexes were successfully obtained at

different nanoparticle / pDNA ratios and confirmed by agarose gel electrophoresis and ethidium

bromide exclusion test.

Keywords: ORMOSIL, nanoparticles, core-shell, bioconjugation, DNA, gene delivery

1. Introduction

Nanotechnology reaches medicine in a bottom-up approach, allowing in many cases parallel-

processing mechanisms at a single cell level. Drug delivery and gene therapy are regarded as

promising applications of nanotechnology in medicine, by making use of nanoparticle (NP) carriers.

The most accepted definition of NP is that their diameter should be 100 nm or smaller. However, in

drug delivery the particle size is selected according to the specificity of the therapy [1]. NPs have been

regarded as an attractive means for medical purposes due to their unique characteristics, such as

their large surface to mass ratio, their quantum properties and their ability to adsorb and carry other

compounds. Their surface is capable of being functionalised, which means it is able to bind, adsorb

and carry many other compounds such as drugs, probes and/or proteins. The composition of the

engineered NPs may vary from materials of biological or synthetic origin, resulting in different possible

interactions with the target cells [1]. Nanomedicine, by means of using NPs, has various possible

applications, such as clinical diagnosis (imaging and fluorescence), separation, drug delivery and

gene therapy. Clinical diagnosis has come to rely heavily on detection and monitoring of individual

chemical interactions of smaller and less abundant targets, such as single cells, mRNA, DNA,

proteins, and peptides. Such targets can only be detected by probes of the same size scale. The

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integration of biology and nanotechnology was made necessary by the demand for gene profiling and

high-throughtput drug and disease screening without complex instrumentation or processing steps [2].

Gene therapy can be achieved by either viral or nonviral vectors. Viral vectors are attractive in

terms of the scientific strategy of exploiting natural mechanisms [3]. The safety of this vector for gene

therapy is of great concern. There is a risk of excessive immune response (adenovirus) as well as

insertional mutagenesis (retroviruses) when viruses are used as transfection vectors. Although

attractive, they suffer from inherent problems in processing, scale-up, immunogenicity and reversion of

an engineered virus [4]. Some of the advantages of nanocarriers over viral vectors are the possibility

of producing them at a large scale and its low immunogenicity. Recent advances in vector technology

have yielded molecules and techniques with transfection efficiencies similar to those of viruses. For

them to be effective as gene delivery systems, it is essential that the negatively charged plasmid DNA

is condensed into a nanoparticulate structure. Depending on the specific application, their size can

vary between 50 and 200 nm [3]. For instance, organically modified silica (ORMOSIL) NPs are of

great use in DNA delivery. The delivery by this mean does not cause the tissue damage or

immunological side effects that have been observed frequently with viral-mediated gene delivery [5].

ORMOSIL is a highly versatile material; the ease of surface functionalising and incorporation of a

variety of biomolecules, combined with its noncytotoxicity and intrinsic biocompatibility, makes

ORMOSIL a benign material, ideal for drug and gene delivery.

Here some simple yet efficient methods for synthesising ORMOSIL NPs with controlled

diameter, size dispersion and chemical properties are illustrated. Plain and core-shell ORMOSIL NPs

were synthesised (by bottom-up methods) with different functional groups:

1. Core-Shell ORMOSIL NPs;

1.1. Synthesis of superparamagnetic iron oxide core (IONPs) (~ 8 nm) by a precipitation-

reduction method [15];

1.2. Synthesis of silica core (~ 170 nm) by the classic Stöber method [14];

1.3. Functionalising silica and iron oxide cores: organically modified silica (ORMOSIL)

shell using a sol-gel method [13, 12].

2. ORMOSIL NPs fabricated directly from the hybrid sol-gel precursors, using sodium silicate

solution (SSS) as seeds [6];

Dynamic light scattering (DLS) was used to measure the hydrodynamic diameter of the NPs.

Particle morphology features were directly observed by transmission electron microscopy (TEM).

Fourier transformed infrared spectroscopy (FTIR) used to confirm the presence of the organic groups

in the structure of the ORMOSIL NPs.

DNA bioconjugation to ORMOSIL NPs has been accomplished by direct incubation of the

desired plasmid with the NPs. To prove the formation of NP-pDNA complexes and to determine the

NP-pDNA saturation ratio, agarose gel electrophoresis and ethidium bromide exclusion assay were

performed.

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2. Materials

Ferric chloride hexahydrate (FeCl3.6H2O) (97%, Sigm-Aldrich), sodium sulfite (Na2SO3)

(technical grade (TG), Merk) and hydrochloric acid (HCl) (37%, analar Normapur) were used as

received. Tetraethyl orthosilicate (TEOS) (99+%, Merck), 3-aminopropyl triethoxysilane (APTES)

(98+%, Sigma-Aldrich), methyl triethoxysilane (90%, Sigma-Aldrich), phenyl trimethoxysilane (PTMS),

N1-[3-(trimethoxysilyl)-propyl]diethylenetriamine (DETA) (TG, Aldrich), vinyl triethoxysilane (VTES)

(97%, Aldrich), ammonia (25%, José Manuel dos Santos, LDA.) and aqueous sodium silicate solution

(Na2O.SiO2, 27 wt.% SiO2, Sigma-Aldrich) were also made use of. Ethanol absolute (Panreac) and

methanol chromasolv (Sigma-Aldrich) were of analytical reagent quality. Plasmid DNA (pDNA)

expressing a “humanised” secreted Gaussia Luciferase as reporter gene (pCMV-GLuc, 5,7 Kbp

(pGLuc), New Englands Biolabs, USA) was used to transform E.coli and cell culture expansion was

performed on triptic soy broth (Biokar, France) at 37ºC with agitation. The cells were isolated by

centrifugation, the cell pellet was washed with pre-chilled phosphate buffer saline at pH 7,4 (10mM

PBS, Invitrogen, UK). The purification of the plasmid was performed according the procedure of Maxi

Quialfilter Kit (Quiagen, Germany).

3. Characterisation of the NPs

Nanoparticles were characterised with respect to hydrodynamic diameter and size distribution

by the polydispersity index (PI), using the Zetasizer Nano ZS (Malvern Instruments, UK). Particle size

measurements were performed at 25 °C in triplicate, and samples dilution was performed in filtered

distilled water. TEM micrographs were obtained using a Hitachi H-8100, with an applied tension of 200

kV. To analyse the samples, a droplet of the suspension was placed on the copper grid and dried at

room temperature. FTIR spectra of the ORMOSIL NPs were obtained using a Nicolet 5700 in

transmission mode in the medium infrared range. The ORMOSIL NPs were finely ground and mixed

with potassium bromide, and then pressed into a disc.

4. Core-Shell Structures

With the objective of synthesising core-shell organically modified silica (ORMOSIL) NPs for

biomedical applications, two types of core were produced: IONPs were synthesised with a reduction-

precipitation method as described by Qu and Yang (1999)[15], and NP monodispersion was achieved,

with diameters of around 7,8 ± 2,7 nm (TEM). Silica NPs were synthesised using a modified Stöber

method [14], resulting in monodisperse NPs with 170,6 ± 26,4 nm (TEM).

For growing different ORMOSIL shells, a process was adapted from Ge and Yin (2008) [12].

Phenyl, vinyl, methyl and amino functional groups were incorporated in different NPs.

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Amino-, methyl-, vinyl- and phenyl-functionalised ORMOSIL shells were grown successfully

around the IONP cores, creating IONP (core) / ORMOSIL (shell) nanostructures with shell thickness of

around 5 nm (TEM). FTIR analysis confirmed the presence of the organic groups, and TEM

micrographs allowed characterisation of size and morphology; some NP aggregation was observed.

Their superparamagnetic properties give them possibility of remote-control targeting.

Figure 1 – TEM micrographs: (a) iron oxide NPs; (b) iron oxide NPs (core) / amino-functionalised ORMOSIL (shell) NPs.

Amino-functionalised silica was also grown on silica NPs, creating silica (core) / ORMOSIL

(shell) nanostructures, with shell thickness of around 100 nm (TEM). It was also verified that these CS

NPs swell when suspended in aqueous medium, due to their highly porous structure and hydrophilic

character. Such porosity could allow future loading with drug agents.

Figure 2 – TEM micrographs: (a) Silica core; (b) silica (core) / amino-functionalised ORMOSIL (shell).

CS NPs carry the potential to be used in the future for biomedical applications. Hence, the

fabricated CS NPs constitute a scaffold with great potential for creating multi-functional NPs to be

used in the field of theragnosis, since their specific functional groups display the potential to be

conjugated with different biomolecules.

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Table 1 – ORMOSIL core-shell NPs synthesised: core; shell functionality; diameter measured by TEM (TEMD); shell thickness (ST); hydrodynamic diameter (HD) and polydispersion index (PdI).

TEM DLS Core Shell

Functionality TEMD (nm) ST (nm) HD (nm) PdI

methyl 18,0 ± 3,0 ~ 5 125,4 ± 64,4 0,915 ± 0,205

phenyl 15,9 ± 0,9 ~ 4 130,6 ± 1,4 0,236 ± 0,000

vinyl 19,9 ± 3,0 ~ 6 162,0 ± 9,9 0,395 ± 0,034IONP

amino 17,7 ± 1,5 ~ 5 412,1 ± 37,6 0,399 ± 0,088

Silica amino 380,9 ± 45,0 ~ 105 436,4 ± 0,3 0,344 ± 0,046

5. Plain ORMOSIL NPs

Although a wide range of bulk ORMOSIL materials has been prepared by sol-gel route, and

silica NPs are currently synthesised by the classical Stöber method, the use of sol-gel process as a

route to organically-functionalise silica NPs has been limited until quite recently. Arhireeva and Hay

(2003) [6] modified a protocol established by Buining et al. (1996) [10] where sodium silicate solution

(SSS) was used as seed for silica growth. The presence of organic components bonded to a siloxane

or to silica backbone, due to both electronic and steric factors, plays a role in sol-gel condensation

kinetics, running difficult the ORMOSIL NP synthesis by the classical Stöber method without the

presence of any seeds [11]. Methyl- and amino-functionalised NPs were synthesised using SSS as

seeds, according to a modification of the work presented by Arkhireeva and Hay (2003) [6]. The effect

of the reaction parameters, such as the ratio of hybrid silica precursor to inorganic silica precursor, the

amount of SSS used, the total amount of precursor introduced, the nature of the solvent used, and

different precursor mixtures, on the diameter, size dispersion and morphology of the resulting NPs has

been studied.

Figure 3 – Plain ORMOSIL NPs: (a) methyl-functionalised; (b) amino-vinyl-functionalised.

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Characterisation by DLS, TEM and FTIR was performed, thus confirming the presence of the

organic functional groups in the plain ORMOSIL NPs. The fabrication process was studied: Plain

ORMOSIL NPs size was observed to increase with APTES/TEOS ratio: to optimise the number of

surface amino groups, a balance between the amino surface density (increase with APTES/TEOS

ratio) and NP size (decrease with TEOS/APTES ratio) should be established. The amount of seeds

(SSS) was also subject of study. By increasing the number of nucleation sites, more and smaller NPs

were expected. Further studies are required to assess the effect of SSS in the plain ORMOSIL NP

characteristics. By decreasing sol-gel precursor concentrations (keeping constant TEOS:APTES

ratio), a decrease in NP size and a narrow NP size distribution was observed. Using methanol instead

of ethanol was advantageous to achieve smaller and more monodisperse plain ORMOSIL NPs,

probably due to a faster hydrolysis rate. Monodisperse plain ORMOSIL NPs with sizes between 100

and 200 nm were fabricated, with different functional organic groups for future bioconjugation. By

combining different ORMOSIL precursors, multifunctional NPs were achieved. This is promising for

bioconjugation with different specific biomolecules. The combination of VTES, TEOS, APTES and

DETA, allows synthesising rougher (and probably more porous) monodisperse plain NPs. Such

rugosity can be useful for increasing loading with drugs for theragnosis in nanomedicine.

Summing up, the process was studied and optimised. ORMOSIL NPs can be synthesised with

the controlled size, size distribution, surface morphology and surface chemistry, which can be chosen

to fulfil the requirements of biomedical applications.

Table 2 – Optimised plain ORMOSIL NPs: functionality; precursors used for the synthesis; diameter measured by TEM; hydrodynamic diameter (HD) and polydispersion index (PdI).

* Synthesised using methanol as co-solvent

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6. Bioconjugation with DNA

DNA loading of amino-functionalised ORMOSIL NPs was accomplished by direct incubation of

the desired plasmid with the NPs, with varying weight ratios (NP/pDNA), for 30 min at room

temperature. The conjugation is done by electrostatic interaction between the cationic amino groups of

the ORMOSIL NPs and the anionic phosphate groups of the plasmid. The binding of pDNA with NPs

was determined by 1% agarose (low melting point) gel electrophoresis. A series of different NPs to

pDNA weight ratios was loaded (20 μl of the sample containing 0,2 μg of pDNA). A 1:6 dilution of

loading dye was added to each and electrophoresis was carried out at a constant voltage of 100 V for

1 h in TBE buffer (4.45 mM Tris–base, 1 mM sodium EDTA, 4.45 mM boric acid, pH 8,3) containing

0,5 μg/ml ethidium bromide. The pDNA bands were then visualised under a UV transilluminator at a

wavelength of 365 nm. DNA condensation was measured by quenching of ethidium bromide (EtBr)

fluorescence. Briefly, quadruplicates of 0,2 μg of pDNA were complexed with increasing amounts of

NPs in 96-well plates in 10 mM PBS buffer at pH 7,4. After 10 min incubation time, 20 μl EtBr solution

(0.1 mg/ml) were added. The fluorescence was measured on a fluorescence plate reader (Tecan

Infinite M200, Austria) at excitation wavelength of 518 nm and at emission wavelength emission of 605

nm.

Figure 4 – Optimised plain amino-functionalized ORMOSIL NPs and respective agarose gel electrophoresis test after bioconjugation with plasmid-DNA.

Incubating the amino-functionalised ORMOSIL NPs (Plain and CS) with plasmid-DNA and

running agarose gel electrophoresis tests confirmed their capability of successfully forming NP/pDNA

complexes. Plain ORMOSIL NPs synthesised using sodium silicate solution as seeds were proven

efficient, despite the percentage of ORMOSIL precursor used in their synthesis, the chemical nature of

the solvent used (ethanol or methanol) and their final NP size and size dispersion. Amino-

functionalised CS structures also showed affinity to DNA, both with iron oxide and silica cores. Non-

functionalised NPs did not attach DNA, thus confirming the amino groups are essential for creating

NP/pDNA complexes. The amino-functionalised NPs fabricated for this work have potential for future

applications of gene delivery and therapy, which will be confirmed in future in vitro and in vivo tests.

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7. Conclusions

In this work, hybrid silica NPs were synthesised by several sol-gel procedures, with controlled

dimensions, size dispersion, surface chemistry and roughness, among other properties. Core-shell

structures were fabricated, using either amorphous silica NPs (~ 170 nm) or superparamagnetic iron

oxide NPs (~ 8 nm) as core, coated with an ORMOSIL layer with the desired functional organic

groups: amino, methyl, vinyl or phenyl. Plain ORMOSIL NPs were also synthesised using aqueous

sodium silicate solution as a seed for growth, functionalised with methyl, amino, vinyl groups alone or

combined. Synthesis parameters were studied, such as the amount of catalyser (ammonia), the

chemical nature of the solvent (ethanol or methanol), the amount of precursors introduced, how they

were introduced and the proportion between them, or the quantity of nucleation sites. Processes were

optimised to achieve the desired final properties. Core-shell (superparamagnetic and non-

superparamagnetic) and plain ORMOSIL NPs functionalised with amino groups were complexed with

plasmid-DNA, revealing their potential for using in gene therapy.

Because of the characteristics of the NPs synthesised, they are considered to be a good

scaffold for multifunctional applications in the area of nanomedicine. Their intrinsic mesoporosity could

allow them to be loaded with one or more types of drugs, for therapy by drug delivery, or even

complexed with a fluorophore, for real time imaging and diagnosis. Their superparamagnetic

properties could permit this drug delivery to be remotely controlled by the application of a magnetic

field in the diseased tissue. By synthesising ORMOSIL NPs with the appropriate surface functional

groups, such NPs could be conjugated with several types of biomolecules, such as specific linkers,

fluorescent labels, antibodies or DNA, giving them simultaneous diagnostic and therapeutic

(theragnosis) capabilities.

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