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1.5 Polymer erosion of MSN matrix and drug release - pages 15-16
1.6 The aims and objectives of the present experiment – pages 16-17
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Abstract
The “Stöber method” or better known – the sol-gel process has been utilised to
produce porous inorganic, silica thin films since before the 20th century. The type
and quantity of precursors used in this process are of paramount importance to the
resultant microstructure. Characterisation of the structures using Fourier Transform
Infrared spectroscopy (FTIR) and Scanning Electron Microscopy confirmed the
progressive formation of a dense, crosslinked, porous silica network. The polymeric
framework enables the encompassing of different antibiotics, such as gentamycin
and daptomycin, and their elution profiles were analysed. Drug release could be
controlled depending on the microstructure formed by varying synthesis parameters.
The gradual increase in pathogenic nosocomial infections stemming from enhanced
resistance mechanisms of bacteria such as biofilm formation invokes the use of a
drug carrier, able to maintain effective antibiotic doses achieving more targeted drug
action and avoiding systemic administration.
1.0 Introduction
During the last few decades, the successful synthesis of nanoscale inorganic
materials, producing porous, well-defined structures and complex configurations
using the sol-gel technique continues to be a prevailing field of research worldwide.
Their multifunctional properties facilitate a range of use forms. Use cases include
optical, electrical, solar, semiconductor and sensor applications to medicine,
pharmacology and biotechnology.1,2,3 Their role as drug carriers for local antibiotic
delivery has gained specific attention in the last few years. Antibiotics have
inaugurated modern medicine, increasing life expectancy, and reducing mortality in
infancy. However, antibiotic-resistant bacteria are provoking a global health crisis as
they render conventional antibiotics less efficient. In turn, this has significantly
increased the cases of severe clinical infections. Therefore, there is a compelling
requirement to develop new formulations of promising antibacterial agents as
alternatives, driven by the recognition of the decreasing number of new antibiotic
drugs in the pipeline.4,5,6,7 This issue, in particular, makes Mesoporous Silica
Nanoparticles (MSNs) an ideal candidate as a drug carrier due to their ability to
provide a high loading capacity and maintain a controlled release of incorporated
compounds as thin films, increasing the efficacy of treatment. With this in mind,
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MSNs have entered the spotlight in this respect; by virtue of their unique
physicochemical properties, they are able to distribute effective antibiotic doses while
avoiding the high toxicity affiliated with systemic administration. 2,8,9,10 These delivery
systems can be utilised in circumstances such as orthopaedic trauma, periodontal
disease, or skin wound healing where the presence of numerous pathogens exists.
Since the early 2000s, MSNs have been explored in controlled drug delivery systems
and are already administered in biosensing and bioimaging applications; their
effectiveness is a result of their favourable, inherent characteristics. These include
substantial internal surface area and volume with size controllability, excellent
biocompatibility due to intrinsic nanoporosity with predetermined functionalisation
deriving directly from the synthesis process, thermal stability and low homolytic
activity.9,11
A more detailed explanation of the P.aeruginosa biofilm demonstrates that there are
typically gradients of oxygen and nutrients present within its structure. These gradi-
ents are associated with decreased bacterial metabolic activity at the low end, and
with the quorum-sensing nature of this bacteria, they are able to elevate doubling
times. The above-mentioned, predominantly dormant bacterial cells, account toward
the resilience of the biofilm; being particularly resistant to phagocytosis through eva-
sion of the host's immune defence mechanisms.
The remaining portion of the biofilm represents the metabolically active component,
found throughout its surface. Tolerance is achieved through modification of a two-
part regulatory system known as PmrA/PmrB (polymyxin resistance A/B) enabling
the chemical alteration of the lipopolysaccharide (LPS) layer within the outer mem-
brane.12 This is an adaptive resistance causing the addition of aminoarabinose to al-
ter the LPS and combat several cationic agents including protamine, lysine polymers
and human neutrophils.5,12
The concern of these biofilms is exacerbated by that fact that they present them-
selves in clinical settings through their adherence to biomaterial surfaces, damaged
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tissue, and medical devices. Consequently, this causes numerous nosocomial infec-
tions affiliated through the use of equipment such as urinary and venous catheters,
prosthetic heart valves and orthopaedic devices.4 However, if the multicellularity of
the biofilm is conquered, the immune system of the host may be able to overcome
the infection and restore the efficacy of antibiotics. This can be accomplished by em-
ploying early eradication strategies, potentially using combination therapy methods
as different portions of the biofilm are susceptible to different antibiotics. For exam-
ple, Colistin is active against the dormant central sector of P.aeruginosa biofilms,
with ciprofloxacin being effective against the metabolically active layer.5 This, there-
fore, invokes a strategy for the use of a sophisticated pharmaceutical carrier to be
used as a controllable, implantable material, able to maintain safe and effective anti-
biotic concentrations over a sustained period of time. 4,8,13
MSNs can be prepared in a variety of porosity and chemical compositions, thus alter-
ing drug release profiles. This is key in applications such as bone and soft tissue re-
pair. In these situations, when antimicrobial treatment is affiliated with the implanta-
tion of biomaterial, typical hydrogel-based dressings are employed in which the ki-
netics of drug release is too rapid. In bone repair applications, MSNs can promote
the healing of tissue through a gradual dissolution process accompanied by the exhi-
bition of a large surface area which can stimulate osteoblastic activity. This sequen-
tially increases hydroxyapatite (an essential component of bone and teeth) and in
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turn, encourages tissue growth.2,11 As well as the controllable size and porosity of
membranes, various studies have validated that silica nanoparticles express limited
cytotoxicity in the 10-200nm size range. Their destructive effect can be further mini-
mised through the grafting of sulfonate, thiol and amine moieties, improving biocom-
patibility; making them an ideal candidate as nanocarriers for drugs and nucleic ac-
ids.10
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and acylates can be used, but alkoxides are the most common precursor due to
higher lewis acidity, and lower electronegativity. Unlike colloid chemistry, the use of
an alkoxide can be more conveniently controlled through the regulation of hydrolysis
and condensation reactions of the alkoxide. 18 The alcohol is present to combat the
immiscibility of water and the alkoxide, acting as a mutual solvent. The hydrolysis re-
action proceeds by replacing the (-OR) groups of the alkoxide with (-OH) hydroxyl
groups; releasing the corresponding R-OH alcohol molecules. The complete hydroly-
sis of an appropriate alkoxide can be seen in the equation below.
Figure 3 – Hydrolysis equation of silica-
alkoxide reacting completely with water
As shown in the equation, the stoichiometric molar ratio of water: alkoxide is 4. The
successive condensation reaction involves the reaction of hydrolysed monomers
consisting of silanol groups (Si-OH) yielded from the hydrolysis reaction and their
conversion to siloxane bonds (Si-O-Si). This occurs through a polycondensation re-
action and is affected by conditions such as pH, and H2O: Si ratio.
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TEOS is unique in the fact that its hydrolysis rate is heavily influenced by pH through
combination with an acid. Alkoxisilanes typically react very slowly with water. For ex-
ample, a solution of TEOS in ethanol took over 1000 hours to react completely; the
same reaction was reduced to 92 hours with HCl occupied as a catalyst.3
TEOS, shown above, is sensitive to pH changes due to the central Silica atom in the
molecule providing a relatively neutral charge due to low electronegativity. Therefore,
a surface charge can be induced by virtue of an alkaline or acidic catalyst to en-
hance the subsequent hydrolysis and condensation reactions. As a result, the rate of
hydrolysis is a minimum at pH 7 - neutral pH, increasing at higher and lower pHs.
Opposingly, the condensation rate is a minimum at pH 2 due to the isoelectric point
of silica (~2.2) and a maximum rate at roughly pH 7.
Figure 5 – The water condensation reaction of silanol molecules to form siloxane bonds.
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Altering the relative rates of the hydrolysis and condensation reactions can yield
varying microstructures. Alkali catalysed reactions favour the condensation reaction
producing species that can agglomerate into fine particles with high solubility, more
porous structures. Whereas, acid catalysed, low pH reactions obtain dense,
branched polymer structures with strong oxide, low porosity, microporous networks
(<2nm pore size) due to low dissolution and reprecipitation of reactive monomers. In
general, the formation of particulates is favoured by high pH due to their increased
porosity; forming mesoporous particulates (2nm<pore size<50nm) at the cost of
gelation time. This will be discussed more in-depth in a later section.2,14,18,3
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effect on the reactions within the sol-gel chemistry. As Figure 3 illustrates, the
complete hydrolysis requites a stoichiometric molar ratio (r) of H 2O:TEOS of 4. For
this reason, an r value of <4 can lead to the formation of intermediate species
(examples below) indicating the result of partial/incomplete hydrolysis.3,20,18
The reactions above illustrate the occurrence of condensation reactions prior to the
completion of hydrolysis, causing an increased formation ratio of nonbridging
oxygen, resulting in a high content of oligomers. Subsequently, this decreases the
yield of (O-Si-O) siloxane bond formation forming, larger particles with undesirable,
reduced porosity. To further this point, the presence of these larger chain length
oligomers irregulate the homogeneity, with the structure showing low mesoporosity
and microporosity, reducing the desirability of the final material.20,14 An experiment
performed in 199820 detailed that, at an r-value of 1, the solution resulted in a non-
porous sol-gel.
Contrastingly, increasing the r-value will typically encourage the reaction shown in
figure 3, improving hydrolysis. However, this consequently reduces the analogous
concentration of the silicon alkoxide in the system. At higher r values, TEOS, being
the silica-containing precursor and the source of reactive primary monomers through
hydrolysis, is in lesser supply. Dilution of silicate in the system naturally reduces the
hydrolysis and condensation reactions occurring. At more extreme r values - R=55.6,
this complication has the potential to cause inhibition of particle growth through the
hydrolysis of the siloxane bonds (reverse of figure 5), decreasing the quantity of
organic species in the final material and nullifying the entire sol-gel process. When
[H2O] is increased the solution converts from a non-aqueous medium to an aqueous
solution, drastically retarding the reactions as water and TEOS become immiscible,
explaining a radical drop in the yield.
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Figure 8 – TEM analysis of silica particles at TEOS Figure 9 – TEM analysis at TEOS
concentration 0.13mol L-1 at fixed conditions.20 concentration 1.65mol L-1 at fixed conditions.20
Generally, a higher r-value (figure 8) is said to increase the yield of particles (per
mole of silica precursor used), forming smaller particles with narrower size
distribution but slower particle growth (caused by inhibition of reactions), with less
cross-linking in the sol-gel. Opposingly, lower r values (figure 9), produced more
mesoporous particles with broader size distribution while having an improved "total"
yield of particles through the existence of more silica precursor, with the added
benefit of a reduced gelation time. Yield and size distributions of varying TEOS
concentrations is illustrated below.14,2,20
10 11
Figures 10 and 11 – Effect of varying TEOS concentrations on particle size (diameter) and yield and size
distributions with fixed concentrations of H 2O (1.09mol L-1).20
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1.4.3 The effect of gelation time on microstructure of MSNs
The gelation of the sol-gel has been alluded to various times in this review of
Mesoporous Silica Nanoparticles. During the ageing (curation) and drying process,
the sol-gel undergoes shrinkage. This is caused by the loss of alcohol, water, and
other volatile constituents. This procedure can occur at room temperature and
generally takes a considerable amount of time. However, heat treatment can
accelerate this mechanism and increase the densification of the final material, as
well as a higher pH and low water:TEOS ratio. The gelation of the gel is interpreted
by the completion of the condensation - polymerisation reaction of silanol (Si-OH)
groups to siloxane bonds (O-Si-O). The development in this stage is critical for
aspects concerning purity, porosity, and homogeneity of the silica gel. Formation of
the polymeric species experiences a relaxation phase inducing high stress within its
structure. Deformation and cracks in the framework can arise, leading to random
porosity if stress it too substantial. Non-hydrolysing organic groups can be employed
to reduce this stress and allow for structural relaxation.18,2,16
As gelation time advances, naturally, the size of the silica monolith increases from
the attachment of organic species to the siloxane framework. As mentioned prior,
heat treatment is performed to speed up the gelation process. Heating to relatively
high temperatures (100-500°C) forces the removal/bonding of silanol (Si-OH) via
polycondensation. Thermogravimetric analysis performed by Pal and Kundu, 2009,21
confirmed a weight loss of approximately 15% up to 150°C and a further, gradual
loss of 15% up to 850°C. Supporting FTIR analysis (figure 12) shows the
corresponding decrease in transmission intensity of the 953cm-1 peak, referring to
the stretching mode of Si-OH, confirming its removal during heat treatment. In
addition, as the heat treatment temperature increased, as did the intensity of the
asymmetric stretching vibration at 1080cm-1 associated with siloxane bonds. In
conjunction, it is possible to assume from the table below, successive increases in
temperature also effectuated the development of a densely crosslinked structure
established through a viscous sintering mechanism. Gradual reduction in surface
area and unwavering presence and expansion in siloxane bond transmittance
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corresponding to increased thermal treatment indicates the forcible densification of
the structure. Surface area becomes null at 1000°C after reaching the fracture
energy of the siloxane bonds, in which the porous gels are converted to glass
microspheres. Although the surface area is reduced during heat treatment, the
porosity of the microstructure produced is significantly improved.3,21,20
Figure 12 – FTIR spectra of silica sol-gel heat treated to (a) 60°C, (b) 500°C, (c)
700°C, (d) 900°C and 1000°C.21
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1.4.5 The use of coating techniques for targeted drug delivery systems
MSNs have been employed as thin films to control antibiotic release and maintain
concentrations over an extended period, exceeding the minimum bacterial
eradication concentration without an initial "burst release". MSNs provide a high
loading capacity of embedded antibiotics with the potential incorporation of several
drugs in the biomaterial scaffold with different physiochemical properties.8 The
synthesis process can lead to functionalisation based on the particular application of
the nanomaterial, making these constructs extremely versatile. One strategy of
target and trigger responsive drug delivery is the coating of MSNs with the layer-by-
layer technique (LbL). LbL enables the alteration of properties such as surface
charge density, allowing the carrier system to be fabricated with stimuli-responsive
components such as pH, ionic strength and temperature.2
The LbL mechanism (shown above) involves the encapsulation of a substrate (MSN)
built up through multilayers provoked by the electrostatic attraction of oppositely
charged components. Characteristics such as morphology, stability and thickness of
films can be improved with this procedure. Polyelectrolyte coatings such as PSS
(negatively charged) and PAH (positively charged) can be utilised to form a bilayer
retarding the release kinetics of incorporated drugs. Analysis of these coatings with
embedded drugs has proven them to prevent an initial burst release by increasing
the extent of diffusion resistance. This is highly promising for future applications of
this technique as PAH and PSS are cost-effective and economical regarding the
release kinetics of nanoparticles. Alteration of this parameter and the others
mentioned in section 1.4, allows for the controlled and targeted release of
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compounds from thin, mesoporous films.22 Tailored release systems help mitigate
side-effects, avoiding systemic drug concentrations and prolonging drug action.
The degradation of the silica sol-gel matrix is a variable of the amount of drug
released. The release rate of an incorporated drug within MSNs is dependant of the
erosion of the matrix and diffusion of the drug typically following zero-order kinetics.
The physicochemical factors of the MSN govern drug dissolution; therefore, all the
previous sections determine the release factors of the particular microstructure. The
diffusion rate is controlled by changes in processing pH and type of catalyst, the
water:alkoxide ratio and whether or not a LbL treatment is utilised. Degradation of
the matrix describes the hydrolysis of (Si-O-Si) siloxane bonds within the body.
Erosion can be classified into two categories: bulk (homogenous), or surface
(heterogenous). Within the bulk erosion process, material is lost from the entire
volume of the matrix and is therefore determined by the surface to volume ratio of
the matrix. This is dependent on the total amount of material; erosion typically
decreases as material decreases.
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clinical environments such as coating biomedical devices. Their efficacy was
analysed and by means of microbiological assays and ensuing release profiles were
quantified by the employment of LC-MC and HPLC. Due to some restraints on
laboratory time, not all results are conclusive and will be marked as (future work).
Results will therefore be assumed and replaced with relevant reviews of similar,
primary literature experiments.
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2.3 High Performance Liquid Chromatography-Mass Spectrometry (LC-MS) –
Detection of Gentamycin
Gentamycin was quantified via (LC-MS) using a Phenomenex Luca C18 column
(150mm x 1mm) coupled to a Finnigan LCQ ion mass trap mass spectrometer in
which separation conditions of the mass spectrometer were employed for the
detection of the gentamycin fragment ion. The mass spectrometer was operated with
an electron spray ionisation (ESI) source in positive ion mode with a source voltage
of 4.5kV, sheath gas flow 80 (arbitrary units). A total ion count was performed which
exhibited the presence of the protonated gentamycin with the molecular ion of m/z
478. Further confirmation was provided by operating the mass analyser in triple
quadrupole, applying the separation conditions for the detection of the gentamycin
fragment ion at m/z 322 with a 50% relative collision enerygy. Once detection was
confirmed, 10 standards were produced in the range of 0-100μg/ml. Each sample
was tested using an auto injector with a 5μL/min injection volume. The mobile phase
consisted of 40% Methanol + 0.1% Formic Acid and 60% water + 0.1% Formic Acid
with a flow rate of 50μL/min. A 5s needle wash containing 75% MeOH was used to
reduce contamination between analytes and a calibration curve was produced.
Analysis of the sol-gel was procured at various time points (0.5,1,3,9,24,36,48 and
72hrs after mixing) of gel curation as well as the precursors prior to mixing.
Evaluation was performed by means of coating a thin layer of sol-gel onto the KBr
disk and implanted into an FTIR machine for analysis.
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A similar adaptation of the method from Kortesuo,and Ahola, et al, 2002 and can
be employed to characterise the nanoparticles within the sol-gel. In which,
samples of sol-gel at differing time-points (same at FTIR) of gel curation were
dispersed in water for 5 seconds to ensure a homogenous dispersion of
particles. A 50 or 100mm lens was employed in the measurements and three
repeat measurements were made to ensure accuracy. The morphology and
size distribution was presented on account of the scanning electron
microscope – (SEM-EDX Stereoscan 360, Cambridge Instruments, UK).
Stock solutions of daptomycin and gentamycin would have been added to curated
sol-gel by means of direct impregnation “soaking” in the volumetric ratio of 0.05:0.95
(equivalent to antibiotic loading used in bone cement) at different gelation time
points. Loading efficiency would have been determined by HPLC and LC-MS for
daptomycin and gentamycin, respectively.2
Release profiles would have been studied in media containing Simulated Body Fluid
(SBF) at a physiological pH of 7.4. Differing volumes of SBF (1, 2 and 5mL) would
have been used to assess the impact of the concentration gradient as a factor of
drug release through diffusion as well as assessing the impact of different gelation
times on the subsequent release profiles. Liquid supernatant (0.5mL) would be
extracted at various time points and the concentrations of daptomycin and
gentamycin would be quantified by their respective methods (section 2.2 and 2.3) to
determine drug elution. The medium would have been refilled with SBF to maintain a
constant volume.2
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3.0 Results and Discussion
An initial difficulty arose from the analysis of daptomycin by HPLC. Commencing with
a mobile phase of 30% acetonitrile + 0.1% formic acid and 70% H 2O + 0.1% formic
acid lead to the improper integration of the daptomycin peak due to the considerably
acidic nature of the mobile phase, which can be seen in figure 18.
Method development lead to the adjustment of the mobile phase through the
introduction of a phosphate buffer (PBS), replacing H2O + O.1% formic acid, allowing
for the complete integration. A 500ml 0.2M phosphate buffer solution was prepared
by adding 13.1345g of monosodium phosphate and 1.2925g of disodium phosphate
to 500ml of water. A pH probe was used to confirm and adjust the pH to 5.5.
With the change in the mobile phase, the daptomycin peak was enhanced; this can
be seen in figure 19 (above). However, the peak in figure 19 shows the introduction
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of "fronting" which may indicate the requirement of a column change. This was
consistent in each analysis, and although introducing error, it was opted to remain
unchanged Under the improved mobile phase conditions, figure 20 illustrates an
average retention time, taking daptomycin approximately 8 minutes to elude through
the C18 column producing a calibration curve at various concentrations being shown
in figure 21.
Figure 20 – HPLC data of daptomycin containing average elution time and peak area
Page | 21
3.2 Quantification of Gentamycin – LC-MS
Contrastingly, the standard curve for gentamycin was rather straightforward with
peak area increasing linearly with the concentration of standards prodcued. This can
be seen in figure 22 below.
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Figure 23 – FTIR spectra of curated sol through smearing of sol on the surface of the KBr
disc
Figure 23 – FTIR spectra of curated sol through smearing of sol on the surface of the KBr
disc
The spectra depicted in figure 23 was a result of implantation of dried sol-gel powder
integrated within the KBr disc in its formation. This was achieved by transferring 3-
4μg of viscous sol-gel (1% in weight of KBr disc) to a mortar and pestle and allowing
it to dry into a powder. Subsequently, the addition and grounding of 300-400μg of
KBr incorporated the sol-gel into a disc effectively. Alternatively, figure 23 relied on
the coating of pre-prepared KBr discs with sol-gel that were left in a desiccator until
required. The different spectra vastly differ in transmittance due to the relative
amounts of sol-gel used in each method but coincide in the fact that they demon-
strate similar peaks.
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Inadequate experimentation time dismissed the ability for sufficient method develop-
ment and abolished the possibility of obtaining results at various timepoints. Never-
theless, the spectra obtained does manage to show critical information on the sol-gel
process occurring.
The analysis of these spectra shows characteristic bands relating to numerous vibra-
tions within the solid network. The broad band ranging from 3470-3400cm-1 corre-
sponds to the overlapping of O-H stretching caused by hydrogen-bonded water mol-
ecules and surface silanols SiO-H''''H2O. The stretching vibrations of the silanol
groups (Si-OH) appear of 960cm-1 indicating incompletion of condensation reac-
tions. Whereas the intense, silicon-oxygen bonds (siloxane bonds: O-Si-O) appear
predominantly in the range of 1000-1200cm-1 denoting its asymmetric stretching,
with its symmetric vibrations arising at 800cm-1 and bending mode at 469-476cm-1.
The existence of the siloxane bonds proves the occurrence of the figure 5, polycon-
densation reaction forming a dense microstructure.23 The intensity of the siloxane
bond peaks within the spectra, therefore, represent the successfulness of the poly-
condensation reaction, whereas, unreacted silanol groups prove the incompletion of
the reaction. As section 1.4.4 mentions, heat treatment of silica microspheres is
known to reduce gelation time through assisting the condensation reaction of silanols
to siloxane bonds. As a consequence of this, if heat treatment were to be utilised in
this experiment, the FTIR spectra would show a further decrease (compared to room
temperature gelation) in peak intensity of silanols and, in turn, would increase the in-
tensity of the siloxane bond peaks.
Figure 24 – Time evolution of integrated areas for Figure 25 – Time evolution of integrated areas for
Page | 24
1200 and 1147cm-1 t(0) being the initial mixing of 1160 and 812cm-1 t(0) being the initial mixing of
precursors and water.24 precursors and water.24
The figures above (24, 25) provides evidence of a typical sol-gel reaction. The reac-
tion within a sol-gel after mixing precursors is expected to proceed as follows; the
primary, hydrolysis reaction of TEOS or silicate precursor used occurs. This de-
creases the IR bands associated with the presence of the heavy silica phase
(C2H5OH) at roughly 1168 and 812cm-1. As the hydrolysis of TEOS continues a
subsequent increase in silanol bond intensity should occur. This allows the progres-
sion of the condensation reaction of the silanol groups (960cm-1), forming siloxane
bonds (1000-1200cm-1). The FTIR analysis should, therefore, exhibit a reduced
gradual reduced peak intensity at 960cm-1 with an incremental increase at 1000-
1200cm-1 as gelation transpires.21,23,24
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Scrutiny of the SEM micrographs taken
from Kortesuo,and Ahola, et al, 2002, of
silica microparticles validates that vary-
ing synthesis properties such as pH and
r-values can noticeably transform the
structure of the microparticles. With the
low pH of the experiment conducted in
this review, it is safe to assume the par-
ticles synthesised through this particular
process would possess similar attributes
to both A and C. The reasonable wa-
ter:alkoxide ratio would impose slight ag-
glomeration of particles, and the low pH
of 1.72 would result in visible pores
throughout the surface while having a
narrower size distribution. The rough-
ness displayed on the surface of the mi-
croparticles caused by low pH catalysis
is a result of the formation of highly
branched polymers as the hydrolysis re-
action of the silica alkoxide is favoured.
Porosity is generally lower in low pH con-
ditions but is compensated by an in-
creased total surface area.
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3.5 Drug loading and release profiles of antibiotic-incorporated MSNs – (future
work)
The drug loading of MSNs can be maximised by optimising the microporous struc-
ture of MSNs through adjustments in pH and r-value. Tamanna, Bulitta and Yu,
2015,2 were able to load an impressive 219μg of gentamycin per mg of MSN under
optimal conditions.The present study aims to control the release of both gentamycin
and daptomycin from MSNs.
Drug loading would have been implemented through direct impregnation of the anti-
biotic, dispersing a stock solution of the drug within a sol-gel suspension.
Although unplanned in this particular experiment, the use of a layer-by-layer (LbL)
technique (detailed in section 1.4.5) is extremely desirable in targeted drug delivery,
it allows for the easy and cost-effective functionalisation of nanomaterials allowing
for the alteration of stimuli-responsive properties. The diffusion of drug through a
concentration gradient and the dissolution and degradation of the matrix are critical
components of its release. The concentration gradient effect would have been exam-
ined through release studies performed in varied volumes of SBF (1, 2, and 5ml) at
pH 7.4 (section 2.7). Variation in pH is known to have a drastic effect on the loading
capacity of the MSNs. Drug loading capacity is escalated at higher pHs. This stems
from the difference in microporous networks obtained from alkaline catalysed sol-gel
reactions as they constitute increased porosity, and solubility over their acid cata-
lysed counterparts. For this reason, the MSNs produced in this experiment (with pH
1.7) would most likely have a somewhat low drug loading capacity. This is inter-
preted exceptionally in figure 35 below.
Figure 36 – Gentamycin release from uncoated, single coated, and dual coated
using PSS and PAH polyelectrolyte layering at physiological pH of 7.4.25
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through eradication of highly protected biofilm bacteria. The most common patho-
gens associated with bone implant surgeries and biomedical devices are Staphylo-
coccus aureus (gram-positive) and Pseudomonas aeruginosa (gram-negative) both
acquiring the ability to self-produce an extracellular matrix (biofilm).26
Daptomycin, a cyclic lipopeptide exhibits a dose-dependent killing activity against
gram-positive bacteria. With it being a lipopeptide, it binds readily to MSNs due to
the inherent lipophilicity of the MSN structure. Whereas gentamycin, an aminoglyco-
side, attains rapid bacterial killing effectiveness on both gram-positive and gram-neg-
ative bacteria. The possession of valuable chemical stability aids in the solubility of
the antibiotic in aqueous media. The five amino groups found within its structure can
promptly undergo protonation to facilitate drug attachment to the MSN matrix through
electrostatic attraction.2
The rapid formation and gelation of silica microporous matrices can be an essential
aspect of their use within clinical applications. Factors explained in sections 1.4 and
throughout section 3 equivalate to highly concentrated acidic silica sols demonstrat-
ing the most reduced gelation time. A highly acidic-catalysed sol produced in con-
junction with a surfactant was able to have a minimal gelation time of 9-10 minutes.21
Gelation time can be further reduced by heating above room temperature. Extreme
temperatures of 500 degrees may not be able to be performed on-site at hospitals
and clinics, but heating of the curating sol to equitable temperatures such as 60°C is
known to improve gelation time noticeably.
Other considerations include the addition of the flow rate of ammonia during the syn-
thesis of the sol-gel. The presence of ammonia increases the amounts of aqueous
media in the solution. NH3 subsequently increases the rate of hydrolysis of TEOS in
addition to the heightened rate of condensation of the hydrolysed monomers, in-
creasing silica size. Trends of particle size, narrow size distribution and yield (up to
95%) are accompanied through the use of ammonia flow which is demonstrated in
the figures below.20
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Figure 37 – The effect of ammonia concentration on (a) yield and particle
size, (b) size distributions of sol-gel prepared at fixed concentrations of TEOS
(0.08mol-1) and H2O (1.09mol-1).20
4.0 Conclusion
Matrix formation was dependant on the hydrolysis and condensation reactions of the
silica precursor; acidic catalysts favouring hydrolysis, forming branched polymer
structures with low porosity whereas with alkaline-catalysed reactions being
preferential to the condensation reaction forming contrastingly high solubility, porous
structures.
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The water: alkoxide ratio also played a critical role in synthesis, and thus, the
microstructure. Higher ratios diluted the silicate in the system, forming smaller
particles with narrower size distribution (SD) due to slower growth through reduction
of hydrolysis and condensation reactions which was almost completely inhibited at
extreme values. However, with lower ratios produced more porous particles with a
broader SD and reduced gelation time. Properties such as porosity, size distribution,
density can, therefore, be easily functionalised through adjustments in synthesis to
adapt to a particular application.
Characterisation of particles was carried out by SEM and FTIR detailing how the
successive parameters affect the microstructure and drug release rates and of
biofilm-effective antibiotics, gentamycin and daptomycin. The release profiles are
dependent on the diffusion of the drug in conjunction with polymer degradation of the
MSN matrix. Drug loading was quantified by HPLC for daptomycin and LC-MS for
gentamycin and reached a maximum, closer to physiological pH and a minimum
within acidic conditions. Release profiles could be better controlled through the
implementation of layering techniques, introducing a bilayer around the MSN-drug
complex and reducing the rate of diffusion. The LbL technique was found to prevent
an initial burst release and instead, maintained an effective antibiotic concentration,
improving drug efficacy. Gelation of the sol was also determined to be a critical
process as it defines the continuation of the polycondensation reaction of alkoxides
to siloxane bonds. This process could be effectively monitored through FTIR, as it
provides information on the rates of the hydrolysis and condensation reactions.
Gelation time was reduced by highly acidic precursors, and the application of thermal
treatment further accelerated the process assisting in the densification of
microstructure through increased conversion to siloxane bonds through
condensation.
Overall, MSNs show considerable promise as a drug carrier through their relatively
easy synthesis and functionalisation enables the adoption of specific drug profiles
(increasing bioavailability) with relatively low cost. The unintrusive nature of the
chemicals formed through erosion of the matrix within the body, accomplishing
exceptional biocompatibility and bioactivity.
Page | 31
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