Pharmaceutics 2019 , 11, x doi: FOR PEER REVIEW www.mdpi.comjournal pharmaceutics [621792]

Pharmaceutics 2019 , 11, x; doi: FOR PEER REVIEW www.mdpi.com/journal/ pharmaceutics
Article 1
Polymeric Mesoporous Silica Nanoparticles for 2
enhanced delivery of 5 -Fluorouracil in vitro 3
Thashini Moodley , Moganavelli Singh * 4
Nano-Gene and Drug Delivery Group , Discipline of Biochemistry, School of Life Sciences, University of 5
KwaZulu -Natal, Private Bag X54001, Durban 4000, KwaZulu -Natal, South Africa 6
*Correspondence: [anonimizat] 7
Received: date; Accepted: date; Published: dat e 8
Abstract: There is a need for the improvement of conventional cancer treatment strategies, by 9
incorporation of targeted and non -invasive procedures aimed to reduce side -effects, drug resistance 10
and recurrent metastases. The anti -cancer drug, 5 -fluorourac il (5-FU) is linked to a variety of induced – 11
systemic toxicities, due to its lack of specificity and potent administration regimens, necessitating the 12
development of delivery vehicles that can enhance its therapeutic potential, while minimising 13
associated s ide-effects. Polymeric mesoporous silica nanoparticles (MSNs) have gained popularity as 14
delivery vehicles, due to their high loading capacities, biocompatibility and good pharmacokinetics. 15
MSNs produced in this study were functionalised with the biocompati ble polymers, chitosan and 16
poly (ethylene )glycol to produce monodisperse NPs of 36 -65 nm, with a large pore surface area of 17
710.36 m²/g, large pore volume, diameter spanning 9.8 nm, and favourable zeta potential allowing 18
for stability and enhanced uptake of 5-FU. Significant drug loading (0.15 – 0.18 mg 5FU/mg msn), 19
controlled release profiles (15 – 65 %) over 72 hours, and cell specific cytotoxicity in cancer cells 20
(Caco -2, MCF -7 and HeLa) with reduced cell viability (≥50 %) over the non -cancer (HEK293) cell s, 21
were established. Overall, these 5FU -MSN formulations have shown to be safe and effective delivery 22
systems in vitro , with potential for in vivo applications. 23
Keywords: 5 -Fluorouracil; mesoporous nanoparticles; cancer; chitosan; poly(ethylene)glycol. 24
25
1. Introduction 26
5-Fluorouracil (5 -FU) is a potent anti -metabolite that was first patented in 1956 for application 27
in chemotherapy regimens 1. It is administered as a pro -drug and is rapidly converted to its 28
intermediates 5 -fluoro -2′- deoxyuridine -5′monophosphate and 5 -fluorouridine triphosphate, with 29
the former actively inhibiting thymidylate synthase and ultimately DNA replication and repair. The 30
latter has been linked to RNA, inhibiting replication and cellular functioning accordingl y [2-4]. The 31
ultimate bioavailability and induced -toxicity is linked to the unique properties of 5 -FU. 5 -FU is a low 32
molecular weight, negatively charged (130.08 g/mol; pKa ~ 8) compound which is quickly excreted 33
from the body (7 -20 % unchanged in urine w ithin 6 hours, up to 90% excreted within first hour), with 34
only a small percentage of the administered dose being metabolised, primarily in the liver. It has low 35
lipid solubility and has a dose -dependent half -life average between 10 – 20 minutes [1,2,5] . Th e 36
associated intravenous toxicities noted with 5 -FU includes neuropathy, depression of white blood 37
cells, cardiac toxicity and hepatic or renal associated toxicity [6-8]. 38
Alternative drug delivery strategies which reduce the administered dose and dosing i nterval, 39
and which can passively or actively target tumour tissue are attractive, and can be accomplished with 40
the use of nanoparticle (NP) based drug delivery systems [9]. These systems aim to provide targeting 41
potentials, sustained drug release profiles, biostability, and can administer a comparatively higher 42
intra -tumour drug concentration with reduced drug -induced toxicity and side -effects commonly 43
seen in vivo [10,11]. 44

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The use of mesoporous silica nanoparticles (MSNs) in drug delivery has become incr easingly 45
desirable due to the large surface area [12] that can be selectively modified to ensure higher loading 46
of cargo into the mesoporous framework for sustained release profiles in disease models [13,14] . 47
Thus, in this chapter a polymeric coated MSN with a net positive charge was synthesised to 48
favourab ly load the chemotropic drug, 5 -FU. Polymers, chitosan (CHIT) and polyethylene glycol 49
(PEG), which at defined concentrations confer biodegradability [15], bioavailability [16,17] , 50
biocompatibility [18,19] , hemocompatibility [20,21] , increased circulating h alf-lives [22], and 51
improved cellular uptake rates in tumour tissue [23,24] , were incorporated into the delivery system. 52
The release profile of 5 -FU from MSN was investigated in vitro , with the release kinetics from 53
the polymeric MSN matrix assessed for i ts potential biological performance. These MSNs were 54
assessed for cytotoxicity and induced cell -death mechanisms in human cancer cell lines, with the 55
overall therapeutic efficiency of 5FU -MSN formulations being evaluated to ascertain the suitability 56
of MSN as a safe and efficient drug delivery vehicle, and an appropriate alternative to conventional 57
free drug administration formulations. 58
2. Materials and Methods 59
2.1. Materials 60
Tetraethyl orthosilicate (TEOS, Si(OCH2CH3)4), Triton X -100 (TX100), cetyl 61
trime thylammonium bromide (CTAB, 99%), polyethylene glycol 2000 (PEG 2000), chitosan (75 -85 % 62
deacetylated), sodium tripolyphosphate (TPP), Tweens 20, ammonia solution (28 -30%), sulphuric 63
acid, sodium carbonate (Na2CO3), 5 -fluorouracil (5 -FU), and deuterium oxide, were all purchased 64
from Sigma Aldrich (St Louis, USA). Eagle’s minimum essential medium (EMEM), Foetal Bovine 65
Serum (FBS), penicillin/streptomycin solution (10 000 U/mL), and trypsin−EDTA (0.25% trypsin, 0.1% 66
EDTA) were obtained from Lonza (Viviers, Belgium). Phosphate -buffered saline (PBS) tablets were 67
purchased from Calbiochem, Canada. The MTT salt (3 -(4,5-dimethylthiazol -2-yl)- 2,5- 68
diphenyltetrazolium bromide) and trichloroacetic acid (TCA) were purchased from Merck, 69
Darmstadt, Germany. The HeLa, Caco -2 and MCF -7 cells were originally purchased from the ATCC 70
(Manass as, USA), while the HEK293 cells were donated by the Anti -viral Gene Therapy Unit, 71
University of the Witwatersrand, South Africa. All sterile plasticware for tissue culture were obtained 72
from Corning Inc., Corning, NY, USA. All other reagents were of analy tical grade . 73
74
2.2. Synthesis of MSNs 75
MSNs were synthesised based on a sol -gel reaction adapted from literature [25,26] . Briefly, cetyl 76
trimethylammonium bromide (CTAB, 100 mg) was dissolved in 48 mL ddH2O and 350 µL of 2M 77
NaOH, and vigorously stirred in a round -bottom flask at 80 °C. Thereafter, 500 µL tetraethyl 78
orthosilicate (TEOS) was added, and the solution incubated for 2 hours. The nanoparticles (NPs) 79
were collected by centrifugation (4000 rpm, 30 minutes) and washed three times with ethanol, 80
followed by d eionised water. The CTAB surfactant was removed by overnight reflux in acidic 81
methanol (20 mL methanol, 1 mL 37 % hydrochloric acid) at 80 °C. The particles were then collected 82
by centrifugation, dried and calcinated at 70 ℃ for 24 hours , to remove any temp late reagents. 83
2.3. Chitosan Functionalisation 84
Approximately, 40 mL of acetic acid (10 % v/v) was added to 200 mg of dry, powdered MSNs 85
and 15 mg of chitosan (CHIT) [27,28] , and stirred at ambient temperature for 24 hours. The CHIT – 86
MSNs were then recovered from the solution by centrifugation, followed by washing with ethanol 87
and deionized water three times. The final CHIT -MSN product was dried at 60 ℃ for 24 hours. 88
89
2.4. Functionalisation with 2% and 5% PEG 90
Dilute acetic acid (30 mL, 2% v/v; pH 4.6) was added to separate beaker s containing 22.5 mg of 91
CHIT and 179 mg or 449 mg of PEG2000 , respectively , followed by the addition of 7.725 mg of TPP 92

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in 15 mL of deionized water. To this, was added 300 mg of MSN, and the mixture s stirred at room 93
temperature for 24 hours. The final product s (PEG_CHIT_MSN) were collected by centrifugation 94
(1000 rpm, 30 minutes), washed and dried at 60 ℃ for 24 hours. 95
96
2.5. Formation of 5 -Fluorouracil:MSN nanoconjugates 97
Approximately, 100 mg of the above f unctionalis ed MSNs (f -MSNs) were soaked in a 3 mg/ mL 98
saturated 5 -FU solution for 30 hours, with stirring to allow the drug to enter the mesopores and the 99
MSN framework. At 0, and 30 hours, 1 mL of drug solution was extracted, centrifuged, and the 100
supernatant analyse d by UV spectrophotometry at a wavelength of 266 nm, while the precipitate 101
was returned to the drug solution. The final 5 -FU-loaded MSNs were collected by centrifugation 102
and dried (60 ℃, 24 hours). The drug loading capacity was calculated using the followin g equation: 103
𝐿𝑜𝑎𝑑𝑖𝑛𝑔 𝑐𝑎𝑝𝑎𝑐𝑖𝑡𝑦 (𝑤𝑡%)=𝑀𝑎𝑠𝑠 𝑜𝑓 𝑑𝑟𝑢𝑔𝑠 𝑖𝑛 𝑓−𝑀𝑆𝑁𝑠
𝐼𝑛𝑖𝑡𝑖𝑎𝑙 𝑀𝑎𝑠𝑠 𝑜𝑓 𝑀𝑆𝑁𝑠 (1) 104
2.6. 5 -FU Release 105
Approximately, 5 mg of 5 -FU:MSN nanoconjugates were dispersed in 25 mL of PBS at pH 4.2 106
and 7.4, respectively, with stirring at 37 ℃ for 72 hours [29]. MSN suspensions (0.5 mL) were 107
regularly removed, centrifuged and analysed by UV spectrophotometry (266 nm), with addition of 108
0.5 mL of fresh PBS into the drug solution to maintain the volume. The experiments were conducted 109
in triplicate and the mean results reported. The drug release was calculated using the following 110
equation: 111
% 𝑅𝑡=𝐶𝑡.𝑉1+𝑉2.(𝐶𝑡−1+𝐶𝑡−2+⋯+𝐶0)
𝑊0.𝐿 × 100 % (2) 112
Where 𝐶𝑡 is the drug concentration at time interval t; 𝐶𝑡−1+𝐶𝑡−2 are drug concentrations prior to 113
time interval t (𝐶0=0); 𝑉1 is the total volume of the drug release bath (25 mL), and 𝑉2 is the 114
volume extracted for UV -vis analysis (0.5 mL). 𝑊1 is the initial weight of the 5 -FU-loaded MSNs 115
(0.005 g), and L is the drug loading capacity of the 5 -FU-MSNs (taken from equation 1). 116
117
2.7. Electron Microscopy 118
The size and morphology of all MSNs and their drug nanoconjugates were determined by 119
transmission electron microscope (TEM ; JEOL JEM 1010), at an accelerating voltage of 100 kV, and a 120
high -resolution transmission electron microscop y (HRTEM) (JEOL JEM 2100) at an accelerating 121
voltage of 100 kV. The samples for TEM and HRTEM imaging were prepared by dispersing ≈ 5 mg 122
MSN sample in 5 mL ethanol for 5 minutes in an ultra -sonic water bath. A carbon grid was then 123
dipped into the liquid sa mple and allowed to dry. Spherical shaped particles were individually 124
measured and shown in mean size distribution graphs . 125
The MSN surface was studied using a LEO 1450 SEM , employing SmartSEM software Version 126
5.03.06 . The powdered samples were placed onto the front of a double -sided carbon tape, and affixed 127
onto an aluminium stub. The samples were coated with gold through a BAL -TEC SCD 050 sputter 128
coater (Leica Microsystems, Australia . The scanning rate was 5 to 10 kilocounts per second, using and 129
accelerating voltage of 20 kV and the working distance of 5 – 10 mm. 130
131
2.8. Nitrogen adsorption and desorption 132
Nitrogen adsorption and desorption isotherms of the MSNs were obtained using a 133
Micrometrics Tri -Star I I 3030 version 1.03 instrument ( Micrometrics, USA ), operating at 77 K. The 134
total surface area was determined using the Brunauer -Emmet -Teller (BET) equation , and the pore 135
volume by the single point method. The pore size distribution was determined using the Barrett – 136
Joyner -Halenda (BJH) m odel and the desorption branch of the isotherm [30]. 137
138

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2.9. Nanoparticle tracking analysis (NTA) 139
The hydrodynamic size and zeta potential of the MSNs were analysed using NTA ( NanoSight 140
NS500 , Malvern Instruments Ltd., Worcestershire, UK). The MSN preparations contained 100 μg/mL 141
MSN in deionized water. The particle size dist ribution based on the particle tracks in Brownian 142
motion within the laser scatter volume was calculated u sing the Stokes -Einstein equation . Zeta 143
potentials was calculated u sing the Smoluchowski approximation , based on Laser -Doppler 144
microelectrophoresis. All data collected are presented as the mode ± standard error , as calculated by 145
NTA software v3.0. 146
147
2.10. C ytotoxicity 148
The 4,5 – dimethylthiazol -2,5-diphenyltetrazolium bromide (MTT) [31], and the sulforhodamine 149
B (SRB ) assay s were used to assess the cytotoxicity of the MSNs in vitro . HEK293, Caco -2, MCF -7 and 150
HeLa cells were seeded at a density of 1 × 104 cells/well in 96 well plates and incubated at 37 ℃ in 5% 151
CO 2 for 24 hours. Cells were then treated with drug loaded MSNs of different concentrations (20, 50 152
and 100 μg/mL) in triplicate, and incubated for 24 a nd 48 hours. A positive control of untreated cells 153
was included. For the MTT assay, following incubation, the medium was replaced with 200 μL fresh 154
medium containing 20 μL of MTT solution (5 mg/ml in PBS), and incubated at 3 7℃ for 4 hours. The 155
MTT–medium m ixture was then removed, and 200 μL of DMSO was added for cell permeation and 156
solubilisation of the formazan crystals , and absorbance measured at 540 nm using a Mindray MR – 157
96A microplate reader (Vacutec, Hamburg, Germany). 158
For the SRB assay , 25 μL of cold TCA (50% w/v) was added directly to each well, and the plate 159
incubated at 4 ℃ for 1 hour, washed and air dried. Approximately, 50 μL of SRB solution (0.04% w/v) 160
was then added to each well and cells incubated at room temperature for 30 minutes. The plate w as 161
washed four times with 200 μL of acetic acid (1% v/v), air dried, and 100 μL of 10 mM Tris base 162
solution (pH 10.5) was added to each well, followed by agitation on an orbital shaker for 10 minutes 163
to solubilize the protein -bound dye. The absorbance was measured as previously, and growth 164
inhibition for both assays was calculated using the following formula e: 165
% 𝐶𝑒𝑙𝑙 𝐺𝑟𝑜𝑤𝑡 ℎ= 𝐴540 𝑛𝑚 𝑜𝑓 𝑡𝑟𝑒𝑎𝑡𝑒𝑑 𝑐𝑒𝑙𝑙𝑠
𝐴540 𝑐𝑜𝑛𝑡𝑟𝑜𝑙𝑙 𝑐𝑒𝑙𝑙𝑠 (𝑢𝑛𝑡𝑟𝑒𝑎𝑡𝑒𝑑 ) ×100 % (4) 166
% 𝐺𝑟𝑜𝑤𝑡 ℎ 𝐼𝑛ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛 =100 −% 𝐶𝑒𝑙𝑙 𝐺𝑟𝑜𝑤𝑡 ℎ (5) 167
168
2.11. Apoptosis 169
Cells were seeded into a 24 -well plate at a density of 1.5 × 105 cells/well and incubated for 24 170
hours to allow for attachment. Following incubatio n, the medium was replaced, and cells treated 171
with the drug nanoconjugates (50 μL/well) , at pre -determined IC 50 concentrations for 48 hours, in 172
triplicate. Untreated ce lls were used as control. T he medium was then removed, cells washed twice 173
with 200 μL of PBS, and 12 μL of acridine orange:ethidium bromide dye solution (AO: EB, 1:1 v/v 1 174
mg/mL) was added to each well for 5 minutes. The excess dye was then removed, and c ells washed 175
with 200 μL of PBS, and v iewed under an Olympus inverted fluores cence microsc ope U -RFLT50 176
(200x magnification), fitted with a CC12 fluorescent camera (Olympus Co., Tokyo, Japan). The 177
apoptotic indices were calculated according to the following equation : 178
𝐴𝑝𝑜𝑝𝑡𝑜𝑡𝑖𝑐 𝐼𝑛𝑑𝑒𝑥 = 𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝐴𝑝𝑜𝑝𝑡𝑜𝑡𝑖𝑐 𝐶𝑒𝑙𝑙𝑠
𝑇𝑜𝑡𝑎𝑙 𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝐶𝑜𝑢𝑛𝑡𝑒𝑑 𝐶𝑒𝑙𝑙𝑠 ×100 % (6) 179
180
181
2.12. Cell cycle Analysis 182
Cells were seeded and treated with the drug nanoconjugates as for the apoptosis assay. After 183
incubation , the cells were centrifuge d at 300 × g for 5 minutes. The pelleted cells were washed with 184

Pharmaceutics 2019 , 11, x FOR PEER REVIEW 5 of 18
PBS before resuspension in 200 μL ice cold ethanol (70% v/v). The cells were then fixed by incubation 185
at -20℃ overnight , thereafter centrifuged and washed with PBS. Finally, 200 μL of Muse® cel l cycle 186
reagent ( containing propidium iodide and RNase A) was added to each tube for 30 minutes at room 187
temperature in the dark. The samples were then analysed, and data generated using the Muse™ Cell 188
Cycle software module. 189
190
2.13. Statistical Analyses 191
All data were presented as mean ± SD (standard deviation). Statistical analyses were performed using 192
ANOVA (one -way analysis of variance), (GraphPad Prism version 6, GraphPad Software Inc., USA). 193
The Dunnett multiple comparison and Tukey honestly significa nt difference (HSD) tests were used 194
as post hoc test comparatives between groups and a pre -set control, and across groups, respectively. 195
P values less than 0.05 were regarded as significant. Dissolution kinetics parameters were evaluated 196
using Microsoft Ex cel 2018™ and excel Add -in DD Solver software. 197
198
3. Results 199
3.1. Synthesis and Chara cterisation 200
Monodisperse MSNs of 36 nm in size were synthesised , which after functi onalisation with CHIT 201
and 2% / 5% PE G increased to ~ 40 nm (Table 1). Upon 5 -FU loading, t he zeta potential of the MSN 202
particle stayed positive, but was reduced suggesting that the negatively -charged 5 -FU molecules may 203
have adhered to the free amine groups of the chitosan layer on the MSN surface. The hydrodynamic 204
size (NTA) was slightly larger, indicating some swelling in aqueous water may have occurred. The 205
loading of 5 -FU into the 5% PEG -CHIT -MSN formulation resulted in an almost polydisperse 206
distribution of MSNs. This was visually assessed and confirmed by TEM images (Figure 1 -2). 207
208
Table 1. TEM size, PDI, hydrodynamic size and zeta potential of all MSNs, and 5 -FU-loaded MSNs. 209
210
211
212
213
214
215
216
217
Figure 1. Selected TEM images; a) CHIT -MSNs (scale bar = 200 nm), and b) 5-FU loaded 5% PEG -CHIT – 218
MSNs (scale bar =500 nm) 219 Nanoparticle Mean Diameter
(TEM) (nm± SD) PDI
(SD/mean)2 Hydrodynamic size
(NTA)(nm ± SD) Zeta Potential
(mV)
MSN 36.09 ± 7.08 0.0385 188 ± 51.6 -9.8±1
CHIT -MSN 39.43 ± 7.22 0.0335 62.2 ± 16 32.4 ± 0.4
2%PEG -CHIT -MSN 40.75 ± 7.11 0.0422 12 ± 3.3 17.0 ± 16.5
5%PEG -CHIT -MSN 40.37 ± 7.70 0.0364 54.8 ± 2.1 7.4 ± 0.7
2%PEG -CHIT -MSN -5FU 48.32 ± 8.20 0.0287 54.8 ± 40.7 11.2 ± 7.0
5%PEG -CHIT -MSN -5FU 64.54 ± 25.11 0.1514 47.4 ± 5.5 3.4 ± 0.0

b

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220
221
222
223
224
225
226
Figure 2 . SEM images of 5% PEG CHIT MSN (a) scale bar = 200 nm; (b) scale bar = 20 nm. 227
The hexagonal shape of the MSNs before drug loading (Figure 2 b) resulting in swelling , and loss 228
of porosity (Figure 1b) are noted . The porosity, shape and size were evaluated using nitrogen 229
adsorption -desorption studies prior to drug loading. 230
According to IUPAC definitions, a type IV isotherm was obtained with well -defined steps for 231
capillary condensation and desorption in open and interstitial mesopores. Two hysteresis loops were 232
formed between at P/P 0 = 0.6 – 0.75 and P/P 0 = 0.87 – 0.9, characteristic of a mesoporous silica material 233
with narrowly distributed pores spaced at 3.5 nm. The pores were defined as cylindrical, with the 234
specific surface area and pore volume defined as 710.36 m²/g and 1.74 cm²/g. A summary of the 235
adsorption -desorption d ata is prov ided in Table 2 . 236
237
Table 2. Summary of Nitrogen adsorption -desorption data for synthesized MSN s 238
Surface Area Surface Area
BET Surface Area: 809.4447 m²/g
t-Plot Micropore Area: 162.2294 m²/g
t-Plot External Surface Area: 647.2153 m²/g
BJH Adsorption cumulative surface area of pores
between 17.000 Å and 3000.000 Å diameter:
680.013 m²/g
BJH Desorption cumulative surface area of pores
between 17.000 Å and 3000.000 Å diameter:
710.3616 m²/g
Pore Volume
Single point adsorption total pore volume of pores less than 1047.206 Å
diameter at P/Po = 0.981156675: 1.529764 cm³/g
t-Plot micropore volume: 0.066025 cm³/g
BJH Adsorption cumulative volume of pores between 17.000 Å and 3000.000 Å
diameter: 1.726356 cm³/g
BJH Desorption cumulative volume of pores between 17.000 Å and 3000.000 Å
diameter: 1.743321 cm³/g
Pore Size
Adsorption average pore width (4V/A by BET): 75.5957 Å
BJH Adsorption average pore diameter (4V/A): 101.548 Å
BJH Desorption average pore diameter (4V/A): 98.165 Å
Average Particle Size 74.125 Å
239
240

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3.2. Drug loading efficiency 241
The loading of 5-FU relies on the physio -adsorption of the drug to the mesopores of MSN, as 242
well as electrostatic interaction s between the positively charged moieties of the polymers. The 243
loading capac ities (Table 3) shows a moderately high loading of low weighted 5 -FU. Since the 244
population was polydisperse, the loading of 5 -FU into each MSN was individually regulated by the 245
stability of the MSN and its favourable interaction with the saturated drug sol ution. 246
247
Table 3. Loading capacity of polymeric MSNs with 5 -FU 248
5-FU Loaded MSNs
5%PEG -CHIT -MSN 2%PEG -CHIT -MSN
Loading Capacity (%) 18.02 15.02
Loading capacity (mg) 0,1802 0,1502
249
3.3. 5 -FU release and kinetics 250
The maximum accumulated release at pH 7.4, was much higher than that at pH 4.2 (Figure 3). 251
The release profile at pH 7.4, displayed an initial rapid release with 80% of the total drug released 252
before 20 hours. This may be equated to the rapid diffusion of 5 -FU molecules that were adsorbed to 253
the polymeric surface and at the pore entrances of the MSNs. Furthermore, at pH 7.4 there was a 254
higher amount of positively charged species in equilibrium with negatively charged species, and as 255
the positively charged species interacted with the PEG outer c oating, more 5 -FU were released. 256
257
258
Figure 3. Drug release profile of 5 -FU at pH 7.4 (solid lines) and pH 4.2 (long -dashed lines) for 2%PEG – 259
CHIT -MSN (orange and yellow) and 5% PEGCHITMSN (green and dark brown). 260
261
Considering all the release profiles, the concentration gradient between the PBS buffer and the 262
dry centre of the MSN may have caused a rapid diffusion of the drug into the bath medium. This is 263
followed by a slow release during 72 – hour period. The conventional drug release kinetic models 264
tested w ere zero order, first order [32], Higuchi [33], Hixson – Crowell [34], and Korsmeyer – 265
Peppas [35]. The contribution of diffusion and erosion to the release patterns seen was evaluated and 266
quantified using the Kopcha model [36](8). In this model , the constants A, representative of diffusion 267

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and B, representative of erosion, were used to mathematically illustrate which of these two factors 268
affected release d more. According to literature, when A/B = 1, diffusion and erosion is equal. 269
However, when the A/B<1, erosion dominates over diffusion and conversely for A/B >1, the diffusion 270
is not affected by erosion. The best release model was selected based on the correlation coefficient 271
(R2) obtained, and release exponents that described the release patter ns observed were defined based 272
on the below equations: 273
Zero Order model: [37] 274
𝑀𝑡= 𝑀0+ 𝑘0𝑡 (3) 275
First Order model: [38] 276
𝑙𝑜𝑔𝑀𝑡= log𝑀0+ 𝑘1𝑡
2.303 (4) 277
Higuchi model: [33]This model assumes release from an insoluble matrix as a time -dependent 278
progression in which Fickian diffusion is supposed. 279
𝑀𝑡= 𝑘𝐻√𝑡 (5) 280
Hixson – Crowell model: [34] This cube root model describes release by dissolution and accounts for 281
changes in the surface area and diameter of the particle. 282
(𝑀𝑡− 𝑀∞)1/3= 𝑘𝐻𝐶.𝑡 (6) 283
Korsmeyer -Peppas model: [35,39] Follows release from a spherical polymeric system in which there 284
may be diffusion or erosion. 285
𝑀𝑡
𝑀∞= 𝑘𝐾𝑃 .𝑡𝑛 (7) 286
Kopcha model [40]: is used to define the amount of diffusion and erosion, and its effects on the release 287
rate. 288
𝑀𝑡= 𝐴 .√𝑡+𝐵𝑡 (8) 289
Where M 0, M t and 𝑀∞ represent the amount of drug dissolved at time zero, time t, and at 290
infinite time, respectively. The kinetic constants are represented by k and subscripted with their 291
model initial. 292
The release expon ent, n is derived from the Korsmeyer -Peppas model and wa s used to define 293
the releas e mechanism. When the n -value = 1, the release is zero order, if n = 0.43 the release is best 294
described as Fickian diffusion where there is no relevant deformation or stresse s during drug release. 295
When , 0.43 < n < 0.85, the release is through anomalous diffusion where there may be swelling or 296
stress during drug release and these structural changes may be due to temperature, activity or 297
structural dimension related fluctuations . If n > 0.85 there is Case II transport. 298
The kinetic modelling of 5 -FU release provides a detailed description of the mechanism of 299
release and integrity of the matrix during drug delivery in vitro . The release kinetics of 5 -FU at pH 300
7.4 could not be linea rized, however for the purpose of comparison are still listed in Tables 4 -6. 301
302
Table 4: Correlation co -efficient (R2) obtained from modelling 5 -FU-loaded 2% PEG -CHIT -MSNs through 303
release kinetic models at pH 7.4 and 4.2. 304
305
306 pH Zero -order First -order Higuchi’s Hixson -Crowell’s Korsmeyer -Peppas’s Kopcha’s
Correlation value (R2)
4.2 0.81 0.55 0.91 0.27 0.95 0.98
7.4 0.35 0.26 0.48 0.64 0.65 0.16

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307
Table 5 . Correlation co -efficient (R2) obtained from modelling 5 -FU loaded 5 % PEG -CHIT -MSNs through 308
release ki netic models at pH 7.4 and 4.2. 309
310
The drug release kinetics for both PEG -CHIT -MSNs at pH 4.2, fitted into Higuchi’s mode, 311
indicating that release occurred by diffusion. According to the Korsmeyer -Peppas fitting, the release 312
profiles followed quasi -Fickian diffusion and correspondingly, a Kopcha’s model fitting displayed 313
high A/B values and small B values, indicating release mechanisms were predominately diffusion 314
based, with little erosion. 315
316
Table 6: Korsmeyer -Peppas model’s release exponent factor and the corresponding Kopcha’s release model 317
fitting results. 318
319
3.4. Cytotoxicity in vitro 320
The cytotoxicity in the HEK293 cell was almost negligible, suggesting that the MSN 321
nanocomplexes were well tolerated in this cell line (Figure 4). The Caco -2 cell viability decreased with 322
the smallest dosage of both polymeric coated MSNs after a 48 -hour duration (Figure 5 ). In the MCF – 323
7 and HeLa cell lines, a larger dose of 5 -FU loaded MSN was needed to elicit a significant response 324
after the 48 -hour treatment period (Figures 6 -7). The minimal concentration (Table 7) needed to 325
inhibit 50% of the cell proliferation was then applied to further tes ting to elucidate and confirm the 326
mechanism of cell death. 327
328
329 pH Zero -order First -order Higuchi’s Hixson -Crowell’s Korsmeyer -Peppas’s Kopcha’s
Correlation value (R2)
4.2 0.72 0.44 0.85 0.72 0.91 0.97
7.4 0.69 0.29 0.81 0.51 0.75 0.70
pH 4.2
Multidrug formulation Korsmeyer – Peppas Model Kopcha Model
n – value A B A/B
2%PEG -CHIT -MSN 0.39 2.97 0.13 22.85
5%PEG -CHIT -MSN 0.32 3.76 0.27 13.93
pH 7.4
Multidrug formulation Korsmeyer – Peppas Model Kopcha Model
n – value A B A/B
2%PEG -CHIT -MSN 0.74 5.16 2.02 2.55
5%PEG -CHIT -MSN 0.35 6.87 0.16 42.94

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Figure 4: MTT and SRB cell viability of f -MSNs and 5 -FU-loaded MSNs administered at various 330
concentrations (20, 50 and 100 μg/ mL) in HEK293 cells . Data is represented as means ± SD (n=3). *p < 0.05, 331
**p < 0.01 were considered statistically significant. 332
333
Figure 5: MTT and SRB cytotoxicity assay of f -MSNs and 5 -FU-loaded MSNs administered at various 334
concentrations (20, 50 and 100 μg/ mL) in Caco -2 cells .Data is represented as means ± SD (n=3). *p < 0.05, 335
**p < 0.01 were considered statistically significant. 336
337
338
339
340
341
342
343
344
345

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346
Figure 6. MTT and SRB assay of f -MSNs and 5 -FU-loaded MSNs administered at various concentrations 347
(20, 50 and 100 μg/ mL) in MCF -7 cells . Data is represented as means ± SD (n=3). *p < 0.05, **p < 0.01 were 348
considered statistically significant. 349
350
351
352
Figure 7: MTT and SRB cytotoxicity assay of f -MSNs and 5 -FU-loaded MSNs administered at various 353
concentrations (20, 50 and 100 μg/ mL) in Caco -2 cells .Data is represented as means ± SD (n=3). *p < 0.05, 354
**p < 0.01 were considered statistically significant. 355
356
Table 7. IC50 values of 5 -FU-MSN treated tested cell lines (Figures 4 – 7). 357
2%Peg -CHIT -MSN 5 -FU 5%Peg -CHIT -MSN 5 -FU
HEK293 – –
MCF -7 100 μg/ml(48h) 50 μg/ml(48h)
Caco -2 20 μg/ml(48h) 20 μg/ml(48h)
HeLa 100 μg/ml(48h) 50 μg/ml(48h)
358
359

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3.5. Apoptosis 360
Characteristic apoptotic events such as membrane blebbing, formation of apoptotic bodies, 361
chromatin condensation, irregular cell shapes can be observed in selected fluorescent images (Figure 362
8). The HEK293 cell s scored low apoptotic indices (Figure 9), with cells appearing morphologically 363
unchanged, emitting a green fluorescence with all treatments (Figure 8a). Caco -2 cells were extremely 364
5-FU s ensitive, with most cells undergoing chromatin condensation (Figure 8 b). MCF -7 cells and 365
HeLa cells showed significant morphological changes characteristic of apoptosis (Figure 8c). Treated 366
cells produced moderately high apoptotic indices (Figure 9), with the 5% PEG -CHIT -MSN producing 367
more of a cytostatic effect in the cancer cells. 368
369
370
371
372
373
374
375
376
377
378
379
Figure 8. Fluorescent micrographs of dual acridine orange /ethidium bromide stained cells showing 380
induced morphological changes in a) HEK293, b) Caco -2 cells treated 5 -FU loaded 2% PEG -CHIT -MSN c) 381
MCF -7 cell treated with 5 -FU loaded 2 %PEG -CHIT -MSN, and d) HeLa cell treated with 5% PEG -CHIT – 382
MSN (20× magnification). 383
384
385
386
387
388
389
390
391
392
Figure 9. Calculated apoptotic indices for each cell line. 393
394
3.6. Cell cycle analysis 395
These studies corroborated results from the apoptosis assay. The HEK293 showed no significant 396
changes in cell cycle distribut ions between the defined phases (Figure 10a) . For the Caco -2 cells, the 397
percentage distribution of cells between the cell cycle phases decreased, and the percentage of cell 398
debris increased, indicating that cells had undergone apoptosis and were fr agmented . Furthermore, 399
there was a decrease in cells in the G 0/G1 phase , and an increase in the G 2/M phase , which are key 400

Figure 5.12. Fluorescent micrographs of dual acridine orange /ethidium bromide sta ined cells
showing induced morphological changes in a) HEK293, b) Caco -2 cells treated 5 -FU loaded 2% PEG –
CHIT -MSN c) MCF -7 cell treated with 5 -FU loaded 2 %PEG -CHIT -MSN
a b
c
d

Pharmaceutics 2019 , 11, x FOR PEER REVIEW 13 of 18
checkpoint s for DNA damage. T hus, cells that had been arrested in G 0/G1 may have selectively 401
undergone apoptosis , and c ells in the G2/M phase may either undergo repair mechanisms or mitotic 402
catastrophe s. Coupled with the AO/EB images (Figure 10 b), it seems that the cells were more likely 403
to have undergone mitotic catastrophe. The apoptosis results for the MCF -7 and HeLa cells were 404
also c onfirmed with a slight shift in normal cell phase distributions (Figure 10 ). HeLa cells increase d 405
in G 0/G1 phase, whilst M CF-7 cells saw a shift of cells in S and G 1/M phase (Figure 10). This was 406
possibly due to early arrest of the S phase, a s 5-FU mitigates its effect on the key replicative enzyme, 407
thymidylate synthase. 408
409
410
Figure 10 . Cell cycle distribution in a) HEK293, b) Caco -2, c) MCF -7, and d) HeLa cells. 411
412
4. Discussion 413
5-FU is a widely used chemotropic drug with a variety of commercial forms available, including 414
oral [37,41] , topical [42] and intravenous administration [6], and often in combination with other 415
scheduled chemotropic drugs [43]. The wide arr ay of toxicities experienced by patients is dependent 416
on the metabolism of this pro -drug, and the individual patient’s unique epigenetic and genetic 417
profile, coupled with their physical traits and external environmental stimuli [44–49]. Thus, the 418
ultimate pharmacokinetic fate of 5 -FU cannot be predicted prior to treatment. Hence, as in most 419
chemotherapeutic therapies, strategies involve a trial and error base, in which patients undergo 420
prolonged administration of repetitive dosages of potent anti -neoplastic drugs [50]. 421
Hence, there is a need for improved delivery strategies and more biocompatible “packaging” of 422
potent cytotoxic drugs , with a variety of engineered materials currently being evaluate d for clinical 423
and commercial applications as drug delivery vehicles [51–53]. MSNs is one such delivery vehicle, 424
with extremely malleable properties that can be selectively optimis ed for a defined purpose. 425
The MSN in this study was coated with a polyelectrolyte layer [20,54–56], which has been 426
described in literature as a selective coating for many recent drug delivery vehicles, as they allow for 427
selective controlled -release properties. The loading of 5 -FU was moderate, with this low molecular 428
weight particle adsorbing to both the surface and inner pore channels of the MSN. Thus, when 429
immersed in solution, 5 -FU was rapidly released followed by a slow and gradual release for 72 hours. 430
The 2% PEG -CHIT -MSN released the most 5 -FU at pH 7.4, whilst both the 2% and 5% PEG -CHIT – 431

Pharmaceutics 2019 , 11, x FOR PEER REVIEW 14 of 18
MSNs released similar total percentages of 5 -FU at pH 4.2, indicating shared kinetic mechanisms 432
influencing release patterns in acidic conditions. 433
The release kinetics at pH 7.4 were non -linear, suggesting rele ase was subject to saturation of 434
one of the pharmacokinetic measures [57]. The models utilised in this study only accounted for the 435
rate of diffusion, and whether there was erosion or diffusion. However, a more suitable model could 436
be a multi -faceted one that includes the effects of solute diffusion co -efficients, electrostatic 437
interaction between 5 -FU and the polymer/drug matrix, and the heterogenous structure of drug 438
delivery systems [57–59]. The synthesized MSNs, especially the 5% PEG -CHIT – MSN displayed 439
polydispersity , while presenting with a heterogenous sized population. Th us, the release mechanism 440
could not be linearized for the heterogenous distributed samples, especially at pH 7.4, where there 441
was increased electrostatic interaction between the polymer surroundings and the 5 -FU encased in 442
the MSN [60,61] . The release of 5 -FU may thus be subject to pH -gradients, with burst -release 443
occurring at a more neutr al pH, than at acidic pH conditions [62]. 444
Burst release kinetics would be associated with the MSN’s geometry, surface characteristics, the 445
heterogenous distribution of drug in the MSN matrix, the intrinsic dissolution rate of 5 -FU, the innate 446
heterogeneity of the porous matrix and the pore densities [57,62,63] . Thus, a more conclusive model 447
that allows for the as sessment of burst release together with slow -controlled release, would be ideal 448
to elucidate the behaviour of these drug -loaded MSNs [58]. 449
The cytotoxic, apoptotic and cell cycle activities of the MSNs were evaluated in four human 450
cancer cell lines to assess the biological performance of MSNs as a drug delivery vehicle in relation 451
to its investigated kinetic release profiles. Potent treatments were derived from 48 -hour exposures 452
with the 5 -FU loaded MSN, in colon, breast and cervical cancer cells undergoing rapid apoptotic 453
events, and shifts in ce ll cycle distribution. Thus, there is therapeutic relevancy attached with the use 454
of polymeric coated MSNs loaded with 5 -FU for drug delivery in cancer therapy. 455
5. Conclusions 456
The selective and exaggerated burst -release until saturation is reached, allude s to the potential 457
use of these MSN loaded 5 -FU formulations in orally administered regimens for colorectal cancers , 458
or cancers of the gastrointestinal tract, as the small intestine has a more basic pH, which favours 5 – 459
FU release from MSN without triggerin g systemic toxicity in healthy tissue or accumulation in non – 460
targeted organs. Furthermore, MSN is a rel atively biodegradable material, that has been 461
commercially and clinically applied as additives in a variety of products with no adverse toxicities 462
noted. Thus, an oral administration of 5 -FU would be highly advantageous, as the matrix is capable 463
of sustained drug release through Fickian diffusion with little erosion or degradation of the polymeric 464
framework in vitro . Pharmacokinetic studies have further al luded to the safe excretion of silica from 465
the body once metabolised. Hence, further studies and in vivo investigations are warranted to enable 466
a transition to possible clinical applications. 467
468
Author Contributions: Conceptualization, T.M, M.S Supervision, Resources, Project Administration and 469
Funding Acquisition, Software, M.S; Methodology and Investigation, T .M, M.S; Visualisation, Data curation, 470
Formal analysis, Wri ting-Original Draft Preparation, T.M.; Writing -Review and Editing, T .M, M.S. 471
472
Funding: This research was partly funded by The National Research Foundation, South Africa, grant number 473
88195/81289. 474
475
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the 476
study; in the collection, analy ses, or interpretation of data; in the writing of the manuscript, or in the decision 477
to publish the results. 478
479
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