2. F.R. Ahmed et al. / Colloids and Surfaces B: Biointerfaces 135 (2015) 50–55 51
Fig. 1. (a) length distribution of HNTs ranging from <250 nm to >2000 nm. (b) Diameter distribution of HNTs from <50 nm to 250 nm. (c) Aspect ratio (length/diameter) of
HNTs from <3 to >15. Counts of HNTs are given as percentage (%) and standard deviation bars (SD) of total no. (>200) of nanotubes counted per sample (total samples; n = 3).
(d) TEM image of halloysite nanotubes (HNTs).
evaluated in-vitro of their cytotoxic potential at high doses against
two model cell lines (colorectal carcinoma cells HCT116; and hep-
atocellular carcinoma cells HepG2) [21,22] which represent the
earliest entry point and the first accumulating organ, respectively,
for xenobiotics and nanoparticles en-route to systemic circulation
after oral delivery [23,24]. Moreover, cytogenetic toxicity of hal-
loysite nanotubes has also been estimated in this study for the first
time by in-vitro mitotic index assay using peripheral blood human
lymphocytes cultures [25].
2. Materials and methods
2.1. Materials
Halloysite (premium grade; Al2Si2O5(OH)4·2H2O, 99.7%) was
received as a gift from New Zealand China Clays Ltd., (New Zealand)
and further it was sieved (125 m) to separate large agglomerates
[4]. Potassium chloride solution (≥99.5% AT), dimethyl sulfoxide
(DMSO anhydrous ≥99.9) and giemsa stain were purchased from
Sigma–Aldrich Chemical Co., Ltd., (Germany). Methanol (100%,
redistilled) and glacial acetic acid (100%) were procured from
Riedel-de Haen (Germany). Phosphate buffer saline tablets (pH 7.4)
were obtained from MP Biomedicals, LLC (France) and KaryoMAX®
Colcemid Solution (10 g/mL) was purchased from Invitrogen
(USA).
2.2. Culture media
The culture media for HCT116 (colorectal carcinoma cells;
ATCC CCL247) and HepG2 (hepatocellular carcinoma cells; ATCC
HB-8065) cell lines was prepared by supplementing high glu-
cose containing ‘Dulbecco’s modified eagle’s medium (DMEM-high
glucose)’ (GIBCO-Invitrogen, Grand Island, NY) with 10% (v/v)
fetal bovine serum (FBS; GIBCO, USA) and 100 IU/mL of Anti-
AntiR (GIBCO). The culture media for lymphocytes was based
on RPMI-1640 containing l-glutamine (Sigma–Aldrich, Germany)
supplemented with 10% (v/v) fetal bovine serum (GIBCO, USA),
100 IU/mL of Anti-AntiR (GIBCO, USA) and 1.5% phytohaemagglu-
tinin (GIBCO, USA).
2.3. Electron microscopy and particle size distribution
Transmission electron microscopy (TEM) was employed to
determine the length, diameter, and aspect ratio distribution of
halloysite nanotubes (HNTs). The samples were prepared by plac-
ing few drops of aqueous suspension of the halloysite samples
(10 mg/mL) on the carbon coated copper grid and then drying in
the air. TEM was then performed using JEOL JEM-2100 at an accel-
erating voltage of 15 kV. The length, diameter distribution analysis
of halloysite nanotubes was carried out by means of first measur-
ing minimum of 200 nanotubes at different places of grid in each
of the 3 TEM samples prepared using Adobe Acrobat 9 Pro software
3. 52 F.R. Ahmed et al. / Colloids and Surfaces B: Biointerfaces 135 (2015) 50–55
and then sorting and graphing the size distribution using Origin-
Pro 8 software [26,27]. The aspect ratio of HNTs was calculated by
dividing the length of the nanotube by its diameter.
2.4. Statistical analysis
All the graphs in this study were plotted using OriginPro 8
software (OriginLab Corporation, Northampton, MA, USA) while
the statistical calculations were performed with computer soft-
ware IBM SPSS Statistics 22 (SPSS Inc., Chicago, IL, USA). The mean
and standard deviations of treatments in WST-1 were compared
with respective controls by means of student-t test (<0.05) while
one-way ANOVA and Duncan multiple range tests were applied
to calculate the pair-wise variance among different treatments for
mitotic index assay with respect to control (<0.05) [28].
2.5. In-vitro cytotoxicity study
The in-vitro cell viabilities of both, HCT116 (colorectal carcinoma
cells) and HepG2 (hepatocellular carcinoma cells) against halloysite
nanotubes were tested using a WST-1 assay (Biovision, CA, USA)
[29–31]. Initially, cells from continuous passage numbers of 12 and
7, respectively, were each seeded into three 96-well plates (Flat
Bottom Costar, Corning, NY, USA) at a density of 2.5 × 104 cells per
well for HCT116 and 2.0 × 104 cells per well for HepG2. The plates
were then incubated at 37 ◦C, 5% CO2 and 95% humidified air for
24 h. After incubation, the cells were washed with PBS (pH 7.4),
followed by the addition of 100 L of halloysite suspensions in cul-
ture media at various final concentrations of 10, 50, 100, 250, 500,
and 1000 g/mL. Non-treated cells containing only culture media
served as the control. Sample media with halloysite without cells
served as blank. These plates were then further incubated for 24 h,
48 h, and 72 h. The plates especially for 48 h and 72 h incubation
were observed for any exhaustion of culture media periodically
after every 24 h as can be visualized by the change in color and were
replaced gently with fresh media with or without final concentra-
tion of HNTs accordingly in the respective non treated and treated
cells. Afterwards the cells were gently washed thrice with PBS (pH
7.4) and then 100 L of solution of WST-1 dye in culture media
(1:10) was added to each well. The plates were allowed to incu-
bate for an hour in the dark and then the absorbance was taken on
scan mode at a wavelength of 460 nm (620 nm was used as a refer-
ence wavelength) using SpectraMaxM5e (Molecular Devices, USA)
plate reader. For calculations, absorbance values of media contain-
ing wells were subtracted from the values of corresponding treated
wells. The percent value indicating the cell viability was obtained
by dividing values of treated cells by those of untreated cells as
control.
2.6. In-vitro mitotic activity assay
Human blood lymphocytes culture was established after blood
taken (with informed consent) from three healthy, non-smoking
male volunteers who were not exposed to any medicine and radi-
ation in the past 1 month and 6 months, respectively.
The human blood lymphocyte cultures were prepared as
reported by Surrallés et al. [32] and the assay was performed
according to the method mentioned by Eroglu et al. [25] with few
modifications. Initially human venous blood (5 mL) was collected
in an anticoagulant containing vacutainer (5 mL) by venipuncture
[32,33].
Then five tubes containing 0.5 mL of blood were added with
4.5 mL of lymphocyte culture medium at ambient temperature fol-
lowed by incubation for 24 h in an incubation chamber (37 ◦C in 5%
CO2 and 95% humidified air). After that, these tubes were added
with lymphocyte culture medium (control) and four concentra-
tions of halloysite suspensions in culture medium, so that final
concentrations of 10, 100, 500, 1000 g/mL of halloysite may be
achieved. This was followed by incubation at slanting (oblique)
position for further 48 h with occasional gentle shaking of the
tubes. To arrest the cells in metaphase, colcemid solution (100 l;
colchicine 10 g/ml) was added in the sample and incubated for
further 1.5 h. The samples were finally centrifuged at 1000 rpm for
8 min and the resulting pellet was re-suspended with gentle vor-
texing. Pre-warmed (37 ◦C) hypotonic solution (75 mM KCl; 5 mL)
was gradually added into it and incubated in water bath (37 ◦C)
for 20 min followed by centrifugation at 1000 rpm for 8 min. The
supernatant was removed without disturbing the Buffy coat. The
freshly prepared ice cold fixative solution (5 mL; methanol:glacial
acetic acid 3:1) was added gradually followed by centrifugation at
1000 rpm for 8 min. This step was repeated a few times until a clear
pellet was obtained. This pellet was re-suspended in fixative solu-
tion (250 l) and placed overnight at 4 ◦C. The cell suspension was
then dropped (2–3 drops) onto the pre-cleaned cold microscopic
slide. These microscopic slides were air dried, stained with Giemsa
stain (2%, 5 min), washed with de-ionized water and dried at room
temperature. For scoring the cells at least ∼1000 cells/microscopic
slide were counted for the presence of interphase and metaphase
stages in control and various treatments. The images were acquired
at 20× magnification using Nikon compound microscope and pro-
cessed in Windows Photo Gallery.
The mitotic index was calculated according to the following for-
mula,
Mitotic Index % =
Cells in Metaphase
Cells in Metaphase and Interphase
× 100
3. Results and discussion
Halloysite nanotubes were first analyzed for their length, diam-
eter, and aspect ratio distribution by means of TEM. The results
suggest that more than 50% of halloysite tubes were in the length
range of 500 nm and the rest mainly within sub-micrometer size
range (see Fig. 1a) while ∼90% of these had diameter of less than
150 nm with more than 60% below 100 nm (Fig. 1b). Calculation
of the aspect ratios of halloysite nanotubes revealed that almost
all (>90%) had low aspect ratios (<12; Fig. 1c) in contrast to higher
aspects ratios of tens to hundreds and thousands which are known
to exert high toxicity in-vitro [34]. These results are consistent with
other studies reporting the size range of halloysite nanotubes in the
range of 500–2000 nm and aspect ratios from 1 to 10 [1,4].
To evaluate the toxic potential of halloysite nanotubes at higher
concentrations, they were first subjected to WST-1 in-vitro cyto-
toxicity testing against the two cell lines HCT116 and HepG2
representing, respectively, the epithelial lining of the major absorp-
tive site for drugs administered via oral route; and the cells of the
first major organ where the nanomaterials are generally localized
and accumulated after absorption [29,31]. A total of six different
final concentrations of 10, 50, 100, 250, 500, and 1000 g/mL of
halloysite nanotubes were employed for this purpose (see Fig. 2).
Halloysite nanotubes exhibited similar profile against both the cell
lines and statistically relevant decline in cellular viability (100,
250, 500, and 1000 g/mL) was found to be concentration depen-
dent. Interestingly, the usually considered toxic concentrations
of 100, 250 and 500 g/mL for many nanomaterials [35], were
found to have no major anti-proliferative activity upon the two
cell types and only cytotostatic effect was observed at concentra-
tions of 250–500 g/mL, as evidenced by the ∼14–28% inhibition
of proliferation over the course of 72 h incubation in both the
cell lines [36,37]. The highest concentration of halloysite nano-
tubes of 1000 g/mL, however was found to exert significant
anti-proliferative activity in both the cases and significant decline
4. F.R. Ahmed et al. / Colloids and Surfaces B: Biointerfaces 135 (2015) 50–55 53
Fig. 2. WST-1 cytotoxicity assay of HCT116 and HepG2 cells treated with different concentrations of HNTs from 10–1000 g/mL at 24 h, 48 h, and 72 h time periods. (* = p < 0.05
compared to the respective controls; ˛ = p < 0.05 compared to cell viability with preceding concentration; n = 3).
Fig. 3. Graph showing mitotic index (MI) of human peripheral blood lymphocytes treated against various concentrations of HNTs compared to control. (n = 3). Duncan
multiple range test* = p < 0.05. Representative compound microscope image (1000×) of geimsa stained peripheral lymphocytes treated with HNTs (1000 g/mL) and showing
metaphase spread and various cells in interphase (scale bar = 50 m).
in viability (∼48–70% from 24–72 h) was observed which were also
significant as compared to preceding concentrations (see Fig. 3;
˛ = p < 0.05). These results represent similar and comparable cyto-
compatibility and safety profile of halloysite nanotubes with the
results demonstrated by Vergaro et al. against HeLa and MCF-7 cells
[12].
Halloysite nanotubes (HNTs) were further assessed for their
cytogenetic toxicity by determining their activity against human
peripheral lymphocytes by means of mitotic index assay. For this
purpose four different concentrations (10, 100, 500, 1000 g/mL) of
halloysite nanotubes were incubated with the peripheral lympho-
cyte culture. In accordance with the WST-1 assay results against
the two cell lines, mitotic index assay demonstrated slight but
statistically relevant inhibition of proliferation of lymphocytes at
only 1000 g/mL concentration (see Fig. 3 and Table 1). How-
ever, the lower concentrations (10, 100, and 500 g/mL) did not
induce mitotic inhibition. It could be attributed to the fact that
the two cell lines are more sensitive towards nanoparticles as
compared to peripheral lymphocytes, thus even lower concentra-
tions of 250–500 g/mL of nanoparticles exhibit anti-proliferative
effect [38]. This might further substantiate the results of WST-1
assay where only 1000 g/mL concentration was found to exert
cytotoxicity. While discussing the prospects of halloysite nano-
tubes two things must be kept in mind, the first being the fact
that other nanoclay materials are known to exhibit lower toxicity
in-vivo as compared to in-vitro results [14]. Secondly, for vari-
ous conventional dosage forms especially tablets, the diluents are
used in the range of minimum 20% for tablets having large dose
sizes (∼400–500 mg) to maximum 90% in tablets having low dose
sizes (≤25 mg), respectively [39]. If the average percentage quan-
tity of diluent of ∼50% is considered for a conventional large tablet
of 250 mg dose than even this would not account for more than
250 mg (50% of total weight 500 mg) of diluent [40–42]. More-
over, upon dilution in GI fluids this supposedly maximum amount
of 250 mg becomes diluted to a concentration of approximately
125–165 g/mL in an average content of 1.5–2.0 L of gastric secre-
tions and food content especially in postprandial situations [43].
It is also pertinent to discuss that as per the FDA Redbook (2007)
guidance to test the toxicological potential of supposedly nontoxic
ingredients in food, nutrition, and pharmaceuticals, the highest
concentration applicable for insoluble substances which in this
case is 1000 g/mL, was used since at the highest concentration
of 2000 g/mL, recommended for soluble substances, HNTs tend
to sediment significantly and interfere with the testing conditions
[44].
These results are indicative of the potential of halloysite nano-
tubes to be used in various oral drug delivery systems; particularly
as diluent/filler material in tablets, capsules, and suspensions;
without causing toxicity to the absorptive sites and first accumu-
lating organ. This study primarily aims to shed light upon the safety
profile of this novel clay mineral, prevalently used in Chinese tradi-
tional medicine, that could save pharmaceutical industry hundreds
5. 54 F.R. Ahmed et al. / Colloids and Surfaces B: Biointerfaces 135 (2015) 50–55
Table 1
Mitotic index (MI) scoring of human peripheral blood lymphocytes against various concentrations of HNTs compared to control. More than 1000 cells were counted each
time (n = 3).
HNT concentration(g/mL) Human lymphocytes count Metaphase scored Mitotic index(MI) Cumulative (MI)
10 1005 1001 1003 59 61 68 5.35 5.73 5.42 5.50 ± 0.20
100 1000 1000 1010 60 56 58 5.66 5.30 5.43 5.46 ± 0.10
500 1008 1000 1020 60 61 70 5.62 5.75 6.42 5.93 ± 0.25
1000 1020 1026 1000 45 42 42 4.22 3.94 4.03 4.06 ± 0.08*
Control 1000 1000 1000 62 66 57 5.83 6.20 5.39 5.81 ± 0.23
*
Duncan multiple range test (p < 0.05).
of millions of dollars in coming years, especially in generic indus-
try [5]. However, these results must be followed by detailed in-vivo
toxicity evaluation of the nanotubes.
4. Conclusion
Halloysite nanotubes decrease cellular viability of HepG2 and
HCT116 cells in a concentration dependent manner with cyto-
static activity at 250 g/mL and 500 g/mL and cytotoxicity at
1000 g/mL. The mitotic index assay against human peripheral
lymphocytes demonstrates that halloysite nanotubes exert statis-
tically relevant cytogenetic toxicity at only 1000 g/mL by blocking
the passage of cell cycle. The safety profile against the two model
cell lines and human peripheral lymphocytes strongly advocate
and justify for in-vivo toxicity study of halloysite nanotubes (HNTs)
and make an important case for commercial pharmaceutical and
biomedical applications based on earlier evidence in literature of
its nature to sustain the release of loaded drugs.
References
[1] M. Du, B. Guo, D. Jia, Newly emerging applications of halloysite nanotubes: a
review, Polym. Int. 59 (2010) 574–582.
[2] Y.M. Lvov, D.G. Shchukin, H. Möhwald, R.R. Price, Halloysite clay nanotubes
for controlled release of protective agents, ACS Nano 2 (2008) 814–820.
[3] E. Joussein, S. Petit, J. Churchman, B. Theng, D. Righi, B. Delvaux, Halloysite
clay minerals — a review, Clay Miner. 40 (2005) 383–426.
[4] S.R. Levis, P.B. Deasy, Characterisation of halloysite for use as a microtubular
drug delivery system, Int. J. Pharm. 243 (2002) 125–134.
[5] Chi Shi Zhi (Halloysite, Kaolin)—Chinese Herbal Medicine, in: Yin Yang House,
2014.
[6] K. Krejcova, P.B. Deasy, M. Rabiskova, Optimization of diclofenac sodium
profile from halloysite nanotubules, Ceska Slov. Farm. 62 (2013) 71–77.
[7] S.R. Levis, P.B. Deasy, Use of coated microtubular halloysite for the sustained
release of diltiazem hydrochloride and propranolol hydrochloride, Int. J.
Pharm. 253 (2003) 145–157.
[8] J. Forsgren, E. Jamstorp, S. Bredenberg, H. Engqvist, M. Stromme, A ceramic
drug delivery vehicle for oral administration of highly potent opioids, J.
Pharm. Sci. 99 (2010) 219–226.
[9] Y.F. Shi, Z. Tian, Y. Zhang, H.B. Shen, N.Q. Jia, Functionalized halloysite
nanotube-based carrier for intracellular delivery of antisense
oligonucleotides, Nanoscale Res. Lett. 6 (2011) 608.
[10] H. Cornejo-Garrido, A. Nieto-Camacho, V. Gómez-Vidales, M.T. Ramírez-Apan,
P. del Angel, J.A. Montoya, M. Domínguez-López, D. Kibanova, J. Cervini-Silva,
The anti-inflammatory properties of halloysite, Appl. Clay Sci. 57 (2012)
10–16.
[11] Refusal Details as Recorded in OASIS by FDA for Refusal FS2-1092363-7/1/24,
in: Import Refusal Report-US Food and Drug Adminitration, 2009.
[12] V. Vergaro, E. Abdullayev, Y.M. Lvov, A. Zeitoun, R. Cingolani, R. Rinaldi, S.
Leporatti, Cytocompatibility and uptake of halloysite clay nanotubes,
Biomacromolecules 11 (2010) 820–826.
[13] N.G. Veerabadran, R.R. Price, Y.M. Lvov, Clay nanotubes for encapsulation and
sustained release of drugs, Nano 02 (2007) 115–120.
[14] S. Maisanaba, S. Pichardo, M. Puerto, D. Gutiérrez-Praena, A.M. Cameán, A. Jos,
Toxicological evaluation of clay minerals and derived nanocomposites: a
review, Environ. Res. 138 (2015) 233–254.
[15] Y.J. Suh, D.S. Kil, K.S. Chung, E. Abdullayev, Y.M. Lvov, D. Mongayt, Natural
nanocontainer for the controlled delivery of glycerol as a moisturizing agent,
J. Nanosci. Nanotechnol. 11 (2011) 661–665.
[16] N. Verma, E. Moore, W. Blau, Y. Volkov, P. Ramesh Babu, Cytotoxicity
evaluation of nanoclays in human epithelial cell line A549 using high content
screening and real-time impedance analysis, J. Nanopart. Res. 14 (2012)
1–11.
[17] G.I. Fakhrullina, F.S. Akhatova, Y.M. Lvov, R.F. Fakhrullin, Toxicity of halloysite
clay nanotubes in vivo: a Caenorhabditis elegans study, Environ. Sci. Nano 2
(2015) 54–59.
[18] J. Cervini-Silva, A. Nieto-Camacho, E. Palacios, J.A. Montoya, V. Gomez-Vidales,
M.T. Ramirez-Apan, Anti-inflammatory and anti-bacterial activity, and
cytotoxicty of halloysite surfaces, Colloids Surf. B Biointerfaces 111C (2013)
651–655.
[19] M. Guo, A. Wang, F. Muhammad, W. Qi, H. Ren, Y. Guo, G. Zhu, Halloysite
nanotubes, a multifunctional nanovehicle for anticancer drug delivery, Chin. J.
Chem. 30 (2012) 2115–2120.
[20] M. Massaro, C.G. Colletti, R. Noto, S. Riela, P. Poma, S. Guernelli, F. Parisi, S.
Milioto, G. Lazzara, Pharmaceutical properties of supramolecular assembly of
co-loaded cardanol/triazole-halloysite systems, Int. J. Pharm. 478 (2015)
476–485.
[21] J.A. Sergent, V. Paget, S. Chevillard, Toxicity and genotoxicity of nano-SiO2 on
human epithelial intestinal HT-29 line cell, Ann. Occup. Hyg. 56 (2012)
622–630.
[22] K. Kawata, M. Osawa, S. Okabe, In vitro toxicity of silver nanoparticles at
noncytotoxic doses to HepG2 human hepatoma cells, Environ. Sci. Technol. 43
(2009) 6046–6051.
[23] C. Schleh, M. Semmler-Behnke, J. Lipka, A. Wenk, S. Hirn, M. Schaffler, G.
Schmid, U. Simon, W.G. Kreyling, Size and surface charge of gold
nanoparticles determine absorption across intestinal barriers and
accumulation in secondary target organs after oral administration,
Nanotoxicology 6 (2012) 36–46.
[24] B. Ballarin-Gonzalez, F. Dagnaes-Hansen, R.A. Fenton, S. Gao, S. Hein, M. Dong,
J. Kjems, K.A. Howard, Protection and systemic translocation of siRNA
following oral administration of chitosan/siRNA nanoparticles, Mol. Ther.
Nucleic Acids 2 (2013) e76.
[25] H.E. Eroglu, A. Aksoy, E. Hamzaoglu, U. Budak, S. Albayrak, Cytogenetic effects
of nine Helichrysum taxa in human lymphocytes culture, Cytotechnology 59
(2009) 65–72.
[26] W.D. Pyrz, D.J. Buttrey, Particle size determination using TEM: a discussion of
image acquisition and analysis for the novice microscopist, Langmuir 24
(2008) 11350–11360.
[27] John E. Bonevich, Wolfgang K. Haller, Measuring the size of nanoparticles
using transmission electron microscopy (TEM), in: NIST-NCL Joint Assay
Protocol, PCC-7, National Cancer Institute-Frederick, Nanotechnology
Characterization Laboratory, 2010, pp. 2–11.
[28] T. Lialiaris, E. Lyratzopoulos, F. Papachristou, M. Simopoulou, C. Mourelatos, N.
Nikolettos, Supplementation of melatonin protects human lymphocytes
in vitro from the genotoxic activity of melphalan, Mutagenesis 23 (2008)
347–354.
[29] A.R. Kim, F.R. Ahmed, G.Y. Jung, S.-W. Cho, D.-I. Kim, S.H. Um, Hepatocyte
cytotoxicity evaluation with zinc oxide nanoparticles, J. Biomed. Nanotechnol.
9 (2013) 926–929.
[30] S. Dey, M. Das, V.K. Balla, Effect of hydroxyapatite particle size, morphology
and crystallinity on proliferation of colon cancer HCT116 cells, Mater. Sci. Eng.
C 39 (2014) 336–339.
[31] Z. Hanif, F.R. Ahmed, S.W. Shin, Y.-K. Kim, S.H. Um, Size- and dose-dependent
toxicity of cellulose nanocrystals (CNC) on human fibroblasts and colon
adenocarcinoma, Colloids Surf. B: Biointerfaces 119 (2014) 162–165.
[32] J. Surralles, E. Carbonell, R. Marcos, F. Degrassi, A. Antoccia, C. Tanzarella, A
collaborative study on the improvement of the micronucleus test in cultured
human lymphocytes, Mutagenesis 7 (1992) 407–410.
[33] C.E. Alakoc, Halil Erhan, Determining mitotic index in peripheral lymphocytes
of welders exposed to metal arc welding fumes, Turkish J. Biol. 35 (2011)
325–328.
[34] W.S. Journeay, S.S. Suri, H. Fenniri, B. Singh, High-aspect ratio nanoparticles in
nanotoxicology, Integr. Environ. Assess. Manag. 4 (2008) 128–129.
[35] P.C. Ray, H. Yu, P.P. Fu, Toxicity and environmental risks of nanomaterials:
challenges and future needs, J. Environ. Sci. Health C Environ. Carcinog.
Ecotoxicol. Rev. 27 (2009) 1–35.
[36] V.N. Sumantran, Cellular chemosensitivity assays: an overview, Methods Mol.
Biol. (Clifton, N.J.) 731 (2011) 219–236.
[37] A. Narang, D. Desai, Anticancer drug development, in: Y. Lu, R.I. Mahato (Eds.),
Pharmaceutical Perspectives of Cancer Therapeutics, Springer, US, 2009, pp.
49–92.
[38] M.M. Joseph, S.R. Aravind, S. Varghese, S. Mini, T.T. Sreelekha, PST-Gold
nanoparticle as an effective anticancer agent with immunomodulatory
properties, Colloids Surf. B: Biointerfaces 104 (2013) 32–39.
[39] United States Pharmacopeia and National Formulary (USP 32-NF 27), in:
Uniformity of Dosage Units, United States Pharmacopeia Convention,
Rockville, MD, 2011.
6. F.R. Ahmed et al. / Colloids and Surfaces B: Biointerfaces 135 (2015) 50–55 55
[40] Tablets Obtained by Direct Compression, in: G. BASF (Ed.) Generic Drug
Formulations, BASF, 2005.
[41] WHO, Training Workshop on Pharmaceutical Development with focus on
Paediatric Formulations, in: P. Development (Ed.), vol. 2014, World Health
Organization, Tallin, Estonia, 2007, Workshop.
[42] Cellulose, Microcrystalline, in: Raymond C. Rowe, Paul J. Paul Sheskey, Marian
E. Quinn (Eds.), Handbook of Pharmaceutical Excipients, Pharmaceutical Press
and American Pharmaceutical Association, London, 2009, pp. 129–132.
[43] Susan A. Charman, William N. Charman, Oral modified-release delivery
systems, in: Michael J. Rathbone, Jonathan Hadgraft, Michael S. Roberts (Eds.),
Modified-Release Drug Delivery Technology, Marcel Dekker, Inc., New York,
Basel, 2003, pp. 1–10.
[44] Guidance for Industry and Other Stakeholders: Toxicological Principles for the
Safety Assessment of Food Ingredients, REDBOOK 2000, in: U. FDA (Ed.), US
Deparment of Health and Human Services, Silver Spring, MD 2007,
pp. 286.