Manuscript Details

Manuscript number COLCOM_2017_137

Title Microemulsion assisted sol-gel method as approach to load a model anticancer

drug inside silica nanoparticles for controlled release applications

Article type Full length article

Abstract
Silica nanoparticles are attractive carriers for drug delivery due to their improved safety and effectiveness in drug
delivery. Silica nanoparticles were synthesized by using microemulsion assisted sol-gel method and a model
anticancer drug 5-fluorouracil (5-FU) was added to the silica precursor before hydrolysis and condensation reactions
start. The obtained materials were characterized by means of Transmission Electron Microscopy (TEM) and Fourier
Transform Infrared Spectroscopy (FTIR). The drug encapsulation within silica nanoparticles causes a particle size
increase. However, the particle morphology is not affected. The drug release profile was obtained through high
performance liquid chromatography (HPLC). The encapsulation approach showed to be effective to sustain a
continuous and increasing release during the testing time (98 hours). Further studies were performed to evaluate the
cytotoxic effects of silica nanoparticles with loaded 5-FU on Chinese hamster ovary (CHO- K1) cells. The materials are
non-cytotoxic for all concentration tested (5-200 µg/mL)

Keywords anticancer drug; drug delivery; silica nanoparticles; sol-gel method;

encapsulation

Corresponding Author Claudia Garcia

Corresponding Author's Universidad Nacional de Colombia sede Medellín

Institution

Order of Authors Natalia Jaramillo, Carlos Paucar, Asuncion Fernandez, Carlos Garcia Negrete,
Claudia Garcia

Suggested reviewers Christine Vauthier, Ali Olad, Ali A. Alshatwi, Yizao Wan

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Dear Editor,

Please find enclosed the manuscript entitled “Microemulsion assisted sol-gel method as approach to load

a model anticancer drug inside silica nanoparticles for controlled release applications” Natalia Jaramillo,

Carlos Paucar, Asunción Fernández, Carlos García Negrete and Claudia García to be considered for

publication in “Colloid and Interface Science Communications”. This manuscript is original and has not

been considered for publication elsewhere.

The research performed is aimed to explore the feasibility of microemulsion assisted sol-gel method as
strategy to load a model anticancer drug (5-fluorouracil) inside silica nanoparticles for controlled release
applications. Compared to other strategies to load drugs on silica nanoparticles such as covalent binding
and electrostatic interactions, the presented methodology offers a complete entrapment of the
drug within siloxane matrix. This approach is also low-cost and promises higher drug protection
toward enzyme attacks or acidic degradation.

We provide herein experimental evidence about how after the loading approach is applied, 5-fluorouracil
is released to a physiological media in a continuous and sustained way. This is a important
contribution to the development of more suitable anticancer drug release systems in the future as
some anticancer drugs such (e.g., 5-fluorouracil) have short biological half life time. Moreover,
Cytotoxicity data obtained for silica nanoparticles also reveal a favorable in-vitro
biocompatibility.

Yours sincerely,

The authors
 Highlights

 Silica nanoparticles were sintetized by sol-gel assisted by microemulsion method.

 5-Fluorouracil was completely encapsulated inside the silica nanoparticles, providing drug
protection from the surrounding environment.
 Drug release from the silica nanoparticles in PBS occurred slowly depending directly of the
concentration.
Microemulsion assisted sol-gel method as

approach to load a model anticancer drug inside

silica nanoparticles for controlled release

applications

AUTHOR NAMES: Natalia Jaramillo, Carlos Paucar, Asunción Fernández, Carlos García

Negrete, Claudia García.

AUTHOR ADDRESS:

Natalia Jaramillo Universidad Nacional de Colombia sede Medellín. Ceramics and glass

group. Calle 59A # 63-20. Medellín, Colombia.

Carlos Paucar. Escuela de Química. Facultad de Ciencias. Universidad Nacional de

Colombia sede Medellín. Calle 59A # 63-20. Medellín, Colombia.

Asunción Fernández. Instituto de Ciencia de Materiales de Sevilla ICMSE. Universidad de

Sevilla. C/Américo Vespucio, 49 - 41092 Sevilla (España).

Carlos García Negrete. Facultad de Ciencias e Ingenierías. Universidad del Sinú. Cra 1w No

38-153 Barrio Juan XXIII Montería, Colombia.

Claudia García. Escuela de Física. Universidad Nacional de Colombia sede Medellín. Calle

59A # 63-20. Medellín, Colombia. cpgarcia@unal.edu.co

Microemulsion assisted sol-gel method as approach

to load a model anticancer drug inside silica

nanoparticles for controlled release applications

ABSTRACT

Silica nanoparticles are attractive carriers for drug delivery due to their improved safety and

effectiveness in drug delivery. Silica nanoparticles were synthesized by using microemulsion

assisted sol-gel method and a model anticancer drug 5-fluorouracil (5-FU) was added to the

silica precursor before hydrolysis and condensation reactions start. The obtained materials were

characterized by means of Transmission Electron Microscopy (TEM) and Fourier Transform

Infrared Spectroscopy (FTIR). The drug encapsulation within silica nanoparticles causes a

particle size increase. However, the particle morphology is not affected. The drug release

profile was obtained through high performance liquid chromatography (HPLC). The

encapsulation approach showed to be effective to sustain a continuous and increasing release

during the testing time (98 hours). Further studies were performed to evaluate the cytotoxic

effects of silica nanoparticles with loaded 5-FU on Chinese hamster ovary (CHO- K1) cells.

The materials are non-cytotoxic for all concentration tested (5-200 µg/mL).

KEYWORDS: anticancer drug, drug delivery, silica nanoparticles, sol-gel method,

Encapsulation

1. INTRODUCTION

1
 Anticancer drug administration faced many challenges such as inadequate biological half-

time of drugs and side effects, between others. New technological tools are intended to direct

the drug to its site of action, maintaining their concentrations over time and over the necessary

therapeutic levels (Bae and Park, 2011). In this vein, encapsulation of pharmacologically active

ingredients in nanosized particles is a topic of increasing research interest (Moghimi, Hunter

and Murray, 2001; Müller, Jacobs and Kayser, 2001; Brigger, Dubernet and Couvreur, 2002;

Panyam and Labhasetwar, 2003; Sahoo et al., 2008; Jack Hu and Zhang, 2009; Kumary, Yadav

and Yadav, 2010). The nanosized encapsulation not only provides decreasing lethal side

effects, it also offers prolonged activity and protection toward enzyme attacks or acidic

degradation (Gehrke and Cussler, 1989; Xu et al., 2006; Shen et al., 2008; Parveen, Misra and

Sahoo, 2012). By encapsulating a drug into nanosized particles, the drug can be positioned in

specific areas of the body reducing their unspecific distribution and providing the required

dosage to the desired effect. The drug is not administered directly as it is included inside the

nanoparticles that release it in small amounts continuously.

Silica nanoparticles are a good choice for encapsulating drugs due to its high thermal and

chemical stability in aqueous suspensions, large surface area, and chemical inertness (Carturan

et al., 2004; Zhong et al., 2008; Z et al., 2011). Moreover, they have attractive physical and

chemical properties such as transparency and surface functionalization capability (Carturan et

al., 2004). Due to the electrostatic stabilization, the surface of the silica promotes the

dispersion of the nanoparticles in aqueous solution which makes it suitable for testing at a

biological level. Traditionally, there are two main approaches for loading organic molecules

(e.g., dyes) into silica nanoparticles that could be extended to the case of drug encapsulation.

The first one involves covalent binding of the molecule to the silica network. This concept

might sometimes increase considerably the difficulty of preparation steps (Auger et al., 2011).

Another approach is based on non-bonding process such as electrostatic interactions or

2
entrapment within siloxane matrix. This approach represents a promising way and more

attention should be paid to its investigation since it has low-cost and does not emphasize the

limitation of the functionalization (Auger et al., 2011). According to the traditional sol-gel

method, the incorporation of molecules into silica nanoparticles under non-covalent bonding is

poor and dependent of the absorption force between the molecule itself and the silica precursor

(Auger et al., 2011). However, a microemulsion process is a plausible route to avoid these

drawbacks because reverse micelles can control the quantity of incorporated molecules into

silica nanoparticles.

5-FU is a highly effective anticancer drug which, in combination with others, has been widely

used in the clinical treatment of many solid tumors such as breast and colorectal, and others

(Luo et al., 2016). Usually, the drugs work by damaging the RNA or DNA that tells to a cell

how to copy itself in division. Unfortunately, the biological half-life of 5-FU is rather short,

lying in the range of 10-20 min (Azhar and Olad, 2014; Ji et al., 2015). In order to maintain

the concentration level over sufficient time, the drug is often administered repeatedly which

could induce serious side effects (Azhar and Olad, 2014; Ji et al., 2015). Considering the

possibility to contribute to the development of more suitable anticancer drug release systems

in the future, in this work, we explore the feasibility of microemulsion assisted sol-gel method

as strategy to load a model anticancer drug (5-FU) inside silica nanoparticles. Specifically, 5-

FU is added to the silica precursor before hydrolysis and condensation reactions start and, the

as-prepared silica nanoparticles with loaded 5-FU is then studied by using different

characterization techniques. Details are revealed on how the drug is encapsulated and released

to a physiological media. Cytotoxicity data for silica nanoparticles are also presented.

2. MATERIALS AND METHODS

2.1 Synthesis of silica nanoparticles and drug encapsulation

3
 The synthesis of silica nanoparticles was carried out using the sol-gel method assisted by

reverse micelle microemulsion, using tetraethylorthosilicate (TEOS, Merck) as silica

precursor, Triton X-100 (Sigma-Aldrich) as surfactant, Methanol (Merck, 98%) as co-

surfactant and cyclohexane (Carlo Erba, 99.5%) as oil phase. The preparation of silica

nanoparticles initially involves the formation of a water in oil (w/O) microemulsion (Figure 1).

The molar ratio Triton x-100/Ciclohexano/Metanol used was 1/7.6/24.7.

The next step in the encapsulation procedure was the addition of 5-Fluorouracil (5-FU) to

the previously formed w/O microemulsion (Figure 1). Right after the drug is incorporated, the

silica precursor (TEOS) was added and hydrolysis and condensation reactions leading to drug

encapsulated silica particles were performed for two hours. This step is critical for nanoparticle

shape and sizes so that some molar ratios such as water to TEOS (h), methanol to triton X-100

(ρ) and water to triton X-100 (R) must be controlled carefully in the colloidal solution. Briefly,

by adjusting h, ρ and R to 59.1, 4.5 and 9.2 respectively, spherical silica particles in the

nanometer range are suitably obtained. Finally, the microemulsion was broken with ethanol,

washed and centrifuged in order to remove traces of surfactant.

Figure 1. Scheme of the encapsulation procedure of 5-fluorouracil in silica nanoparticles.

4
 The characterization of silica particles loaded with 5- FU was performed by using FTIR and

TEM techniques. Controlled experiments were carried out to obtain the corresponding drug

release profiles. Tests were conducted in phosphate buffer solution (PBS) thermostated at 37 °

C ± 0.1. The nanoparticles were initially dispersed in PBS (pH = 7.4) to 0.49 mg/mL (in terms

of the drug content). During 96 hours, small aliquots were taken from the PBS media each 24

hours and the samples were analyzed by using HPLC analysis.

2.2 Cell culture and treatment

CHO-K1 cells were propagated in RPMI 1640 medium (GIBCO) supplemented with 5% FBS

(GIBCO). Cells were cultured in T25 culture flasks (BD Falcon) and incubated at 37 ° C in a

humid atmosphere (> 95%) and 5% CO2 (standard conditions). After the CHO-K1 cells grew

to the expected concentration, they were harvested by trypsinizing the cell with 0.025%

trypsin/EDTA to exponential growth between 36 and 48 hours post seeding and incubated at

37°C for 5 minutes to obtain complete cell detachment. The cell suspension was centrifuged at

4000 rpm for 10 min, cell pellets were re-suspended with complete medium. Cell suspensions

were treated with different concentration of nanoparticles (5, 10, 15, 20, 25, 50, 75, 100, 200

μg/mL) and added separately into the wells of 96-well plate. Finally, the treated cells were

incubated for 48 h with 5% CO2 at 37°C for citotoxicity evaluation.

2.3 Citotoxicity

Citotoxic effects of silica nanoparticles loaded with 5-FU were assessed using MTT assay.

For each exposure concentration described in the section above, four replicates were examined

twice per plate. Four hours before compliance the setting time of 48 hours, 20 µL of MTT

reagent (5mg/ mL) was added maintaining incubation conditions described in the section

above. At the end of the incubation period, 100µL of acidified isopropanol (0.1% HCl fuming,

5
10% Triton X 100) was added to each well and culture plates were left under orbital shaking

and darkness for 4 hours to dissolve the formazan crystals. To eliminate background

absorption, the plates were centrifuged at 2700 rpm for 5 min and the supernatant of each well

was transferred to a new plate. Absorbance values at 540 nm and 570 nm were measured using

a spectrophotometer (Multiskan - Spectrum thermos Scientific).

3. RESULTS AND DISCUSSION

Figure 2 shows a TEM image for silica nanoparticles with encapsulated 5-fluorouracil. These

particles have typical sizes of 90 nm which are considerably greater than those presented by

silica nanoparticles without drug (data not shown). By encapsulating a drug within silica

nanoparticles, the number of chemical interactions increases inside particles due to the

introduction of new functional groups, which leads to increase in size. However, the

morphology of the nanoparticles would not be affected because reverse micelles are

responsible for controlling this parameter. Figure 3 shows FTIR spectra for 5-FU alone (a) and

silica particles with 5-FU encapsulated therein (b). The observed bands were ascribed to

specific vibrational modes as summarized in Table 1 for 5-FU or Table 2 for silica particles

with 5-FU encapsulated therein.

6
 Figure 2. TEM image of silica nanoparticles loaded with 5- FU.

Based in results showed above, absorption bands assigned to vibrations of C=O, C-F and

C-N bonds in 5-FU spectrum disappear when the drug is encapsulated within silica particles.

The drug encapsulation is possible by polymerizing the silica precursor. Although the drug is

within silica nanoparticles, unencapsulated drug could remain residual in the colloidal

dispersion. However, the absence of characteristic features of 5- FU in Figure 3b, suggests that

the drug must be completely within silica nanoparticles. The amount of drug inside

nanoparticles is determined as difference between drug added initially in the synthesis and that

found in the supernatant. This quantity is known as encapsulation efficiency and it is

determined by Equation 1 (Moghimi et al., 2001):

%𝐸𝐸 = (𝑊𝑖𝑛𝑖𝑡𝑖𝑎𝑙 𝑑𝑟𝑢𝑔 ‒ 𝑊𝑓𝑟𝑒𝑒 𝑑𝑟𝑢𝑔) 𝑥 100 Eq. 1

7
Figure 3. FTIR spectra of a) 5- Fluorouracil alone, b) 5 - Fluorouracil encapsulated in silica
nanoparticles.
Table 1. Assigned vibrations in FTIR for 5-FU (Zwierzchowska, 1997; Lewandowski,
Kalinowska and Lewandowsk, 2005).

Frequency Assigned vibrations

(cm-1)

1718 Carbonyl groups C=O

885 and 1731 Movements of oxygen in the carbonyl groups C=O (high intensity) and
C=O carbonyl groups (low intensity)
1631 C5C6 link that is close to halogen
1240 Flexion movements C5-F

8
 748 Link vibrations C5F = C6H
1346 Pyrimidine vibrations
1502 and 1423 Vibrational modes of the links C-N

Table 2. Assigned vibrations in FTIR for silica particles loaded with 5-FU (Lewandowski,
Kalinowska and Lewandowsk, 2005).

Frequency Assigned vibrations

(cm-1)

630 Vibrations of Si-O

800 Flexion of Si-O
920 Silane groups and stretching Si-O-Si (Serra et al., 2003)
1100 Asymmetric stretching of Si-O (Serra et al., 2003)
1509 to 1656 Deformation (Scissoring and flutter) of H alcohol
2819 to 3033 Presence of alcohol and stretching of the hydroxyl groups Si –OH
(Lewandowski, Kalinowska and Lewandowsk, 2005)

5-FU is a chemotherapy drug usually used in chemotherapy. It is required a long-acting so

that it is necessary that its release from silica nanoparticles occurs gradually to guarantee its

continuous therapeutic effect. As shown in Figure 4 the release profile for 5-FU from silica

nanoparticles as measured by HPLC over time exhibited a continuous sustained behavior

which can be described as follow. After first 24 hours, 0.00524 mg of FU/ mL were released

to the 0,012
physiological

media.
0,010
Concentration (mg/ml)

0,008

0,006

0,004

0,002

0,000
0 24 48 72 96
Time (hours)
9
Figure 4. In vitro release profile of 5-FU encapsulated in silica nanoparticles as measured by
HPLC.

Figure 5. Cytotoxicity percentage of different concentrations of silica nanoparticles on CHO-

K1 cells.
The interaction between silica particles and the fluid media continues over time leading to the

release of 0.00669 and 0.00785 mg of FU/ mL after 48 and 72 hours respectively. Finally,

0.010 mg of FU/ mL were released in a controlled way. This continuous and sustained release

behavior can be attributed to the specifically slow dissolution rate of silica particles in the

physiological fluid which act, in this case, as a control mechanism for drug release to the

surrounding media.

10
Figure 5 shows the results of MTT assay as graphic of cytotoxicity (%) vs concentration of

silica particles in the culture media. The cytotoxicity tends to increase with the rise in

concentration. However, the cytotoxicity corresponding to the maximum concentration tested

(200 µg/mL) is 22.9%. This value is widely below the limit established by the standard ISO

10993-5 (Wallin and Arscott, 1998) to define a material as cytotoxic, indicating that SiO2

nanoparticles can be considered as non-cytotoxic at the conditions tested for CHO-K1 cells.

Moreover, considering that the concentration of carrier materials used in drug and gene

delivery is usually below 100 µg/mL, the cytotoxicity data obtained for silica nanoparticles

reveal a favorable in-vitro biocompatibility for drug delivery applications.

CONCLUSIONS

Microemulsion assisted sol-gel method has been proven herein to represent a suitable route

by which to load a model anticancer drug inside silica nanoparticles, affording the capability

to release the drug (5-FU) to a physiological media in a continuous and sustained way. The

mechanism for the observed controlled release behavior is attributed to the specifically slow

dissolution rate of silica nanoparticles in the physiological fluid. Cytotoxicity data obtained for

silica nanoparticles also reveal a favorable in-vitro biocompatibility for drug delivery

applications. Further studies need to be performed in order to test the applicability of the

loading approach to others drugs different to 5-FU, preferably if they exhibit short biological

half-times.

AUTHOR INFORMATION

Corresponding Author

Claudia García. Escuela de Física. Universidad Nacional de Colombia sede Medellín. Calle

59A # 63-20. Medellín, Colombia. cpgarcia@unal.edu.co. Tel: +57-4-4309842.

11
Author Contributions

The manuscript was written through contributions of all authors. All authors have given

approval to the final version of the manuscript.

Funding Sources

This work was funded by the Administrative Department of Science, Technology and

Innovation —Colciencias— Young Researchers and Innovators "Virginia Gutiérrez de Pineda"

in 2009 and 2011. The Mayor of Medellin with the "Link Worlds" program. Materials Science

Institute of Seville and the Technological Institute of Construction AIDICO (Spain) and the

Research Medellin headquarters of the National University of Colombia in the 2012 draft

DIME 15519.

ACKNOWLEDGMENT

This work was funded by the Administrative Department of Science, Technology and

Innovation —Colciencias— Young Researchers and Innovators "Virginia Gutiérrez de Pineda"

in 2009 and 2011. The Mayor of Medellin with the "Link Worlds" program. University of

Seville and the Technological Institute of Construction AIDICO (Spain) and the Research

Medellin headquarters of the National University of Colombia in the 2012 draft DIME 15519.

ABBREVIATIONS

XRD X-ray Diffraction, FTIR Fourier Transform Infrared Spectroscopy, TEM Transmission

Electron Microscopy, 5-FU 5 Fluorouracil, PBS phosphate buffer, HPLC high performance

liquid chromatography, TEOS Tetraethylorthosilicate.

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15
Table 2. Assigned vibrations in FTIR for silica particles loaded with 5-FU (Lewandowski, Kalinowska
and Lewandowsk, 2005).

Frequency Assigned vibrations

(cm-1)

630 Vibrations of Si-O

800 Flexion of Si-O

920 Silane groups and stretching Si-O-Si (Serra et al., 2003)

1100 Asymmetric stretching of Si-O (Serra et al., 2003)

1509 to 1656 Deformation (Scissoring and flutter) of H alcohol

2819 to 3033 Presence of alcohol and stretching of the hydroxyl groups Si –OH
(Lewandowski, Kalinowska and Lewandowsk, 2005)
Table 1. Assigned vibrations in FTIR for 5-FU (Zwierzchowska, 1997; Lewandowski,
Kalinowska and Lewandowsk, 2005).

Frequency Assigned vibrations

(cm-1)

1718 Carbonyl groups C=O

885 and 1731 Movements of oxygen in the carbonyl groups C=O (high intensity) and
C=O carbonyl groups (low intensity)
1631 C5C6 link that is close to halogen
1240 Flexion movements C5-F
748 Link vibrations C5F = C6H
1346 Pyrimidine vibrations
1502 and 1423 Vibrational modes of the links C-N