Non-ionic self-assembling amphiphilic polyester dendrimers as new drug delivery excipients

Article abstract of DOI:10.1039/c6ra28100a

Solubility enhancement of poorly soluble antibiotics via self-assembling nano systems could be a promising approach to effectively treat bacterial infections in the current scenario of evolving resistant species. The study in this paper reports the synthesis of novel biocompatible G2 and G3 polyester amphiphilic dendrimers (ADs) (GMOA-G2-OH, GMOA-G3-OH, GMS-G2-OH and GMS-G3-OH) and their application as: (i) solubility enhancers for fusidic acid (FSD) as a model antibiotic with poor aqueous solubility and (ii) as stearic stabilizers in the preparation of solid lipid nanoparticles (SLNs). Two different series of ADs from glycerol monostearate (GMS) and glycerol monooleate (GMOA) were synthesized and their structures were confirmed employing FT-IR, NMR (¹H and ¹³C) and HR-MS. The MTT assay confirmed their non-toxicity to mammalian cells. The critical aggregation concentration value order for ADs was GMS-G3-OH (5 × 10^?6 mol l^?1) < GMOA-G3-OH (7 × 10^?6 mol l^?1) < GMOA-G2-OH = GMS-G2-OH (5 × 10^?5 mol l^?1). All ADs formed micelles in the size range of 6.48 ± 0.04 nm to 12.38 ± 0.36 nm. At 1% w/w concentration FSD solubility enhancement in GMOA-G2-OH, GMOA-G3-OH, GMS-G2-OH and GMS-G3-OH was 43, 11, 9.1 and 6.8-fold respectively compared to water. As GMOA-G2-OH enabled the highest solubility of FSD, it was further evaluated for its antibacterial activity against Staphylococcus aureus and methicillin-resistant S. aureus (MRSA). The minimum inhibitory concentration values for FSD with and without GMOA-G2-OH against S. aureus were 0.23 μg ml^?1 and 0.53 μg ml^?1 respectively whereas the values were 0.23 μg ml^?1 and 0.39 μg ml^?1 against MRSA respectively. These results suggested that GM-OA-G2 not only enhanced the solubility but also enhanced antibacterial potency of FSD. Furthermore, these ADs showed their potential as promising pharmaceutical excipients as they acted as stearic stabilizers in the preparation of SLNs. Using these ADs stable SLNs with zeta potential value in the range of ?15.30 ± 1.44 to ?38.46 ± 3.04 were formed.

Full text of DOI:10.1039/c6ra28100a

RSC Advances
View Article Online
View Journal | View Issue
PAPER
Non-ionic self-assembling amphiphilic polyester
dendrimers as new drug delivery excipients†
Cite this: RSC Adv., 2017, 7, 14233
*
Dhiraj R. Sikwal, Rahul S. Kalhapure, Mahantesh Jadhav, Sanjeev Rambharose,
*
Chunderika Mocktar and Thirumala Govender
Solubility enhancement of poorly soluble antibiotics via self-assembling nano systems could be a promising
approach to effectively treat bacterial infections in the current scenario of evolving resistant species. The
study in this paper reports the synthesis of novel biocompatible G2 and G3 polyester amphiphilic
dendrimers (ADs) (GMOA-G2-OH, GMOA-G3-OH, GMS-G2-OH and GMS-G3-OH) and their application
as: (i) solubility enhancers for fusidic acid (FSD) as a model antibiotic with poor aqueous solubility and (ii)
as stearic stabilizers in the preparation of solid lipid nanoparticles (SLNs). Two different series of ADs
from glycerol monostearate (GMS) and glycerol monooleate (GMOA) were synthesized and their
structures were confirmed employing FT-IR, NMR (1H and 13C) and HR-MS. The MTT assay confirmed
their non-toxicity to mammalian cells. The critical aggregation concentration value order for ADs was
GMS-G3-OH (5 ꢀ 10ꢁ6 mol lꢁ1) < GMOA-G3-OH (7 ꢀ 10ꢁ6 mol lꢁ1) < GMOA-G2-OH ¼ GMS-G2-OH (5
ꢀ 10ꢁ5 mol lꢁ1). All ADs formed micelles in the size range of 6.48 ꢂ 0.04 nm to 12.38 ꢂ 0.36 nm. At 1%
w/w concentration FSD solubility enhancement in GMOA-G2-OH, GMOA-G3-OH, GMS-G2-OH and
GMS-G3-OH was 43, 11, 9.1 and 6.8-fold respectively compared to water. As GMOA-G2-OH enabled
the highest solubility of FSD, it was further evaluated for its antibacterial activity against Staphylococcus
aureus and methicillin-resistant S. aureus (MRSA). The minimum inhibitory concentration values for FSD
with and without GMOA-G2-OH against S. aureus were 0.23 mg mlꢁ1 and 0.53 mg mlꢁ1 respectively
whereas the values were 0.23 mg mlꢁ1 and 0.39 mg mlꢁ1 against MRSA respectively. These results
suggested that GM-OA-G2 not only enhanced the solubility but also enhanced antibacterial potency of
FSD. Furthermore, these ADs showed their potential as promising pharmaceutical excipients as they
acted as stearic stabilizers in the preparation of SLNs. Using these ADs stable SLNs with zeta potential
value in the range of ꢁ15.30 ꢂ 1.44 to ꢁ38.46 ꢂ 3.04 were formed.
Received 12th December 2016
Accepted 24th February 2017
DOI: 10.1039/c6ra28100a
rsc.li/rsc-advances
solubility.1–4 Among various classes of APIs, antimicrobial drugs
are one of the classes in which many antibiotics have poor or
1. Introduction
practically no aqueous solubility. Antibiotics with low aqueous
solubility include polyene antibiotics [amphotericin B (0.0819 mg
mlꢁ1),5 nystatin (3.6 mg mlꢁ1),6 pimaricin (0.39 mg mlꢁ1)6],
macrolide antibiotics [erythromycin (2 mg mlꢁ1),6 azithromycin
(0.6 mg mlꢁ1)7], quinolone antibiotics [nadioxacin and pruli-
oxacin (sparingly soluble)8] and steroidal antibiotics [fusidic
acid, helvolic acid and cephalosporin P (sparingly soluble)9]. In
the current scenario of development of resistance to antibiotics
by various bacteria, solubility enhancement via self-assembling
nano systems is one of the promising approaches to improve
the efficacy of poorly soluble antibiotics.10
Numerous techniques have been reported in the literature
for solubilization of poorly water soluble antibiotics which
includes (i) physical modication by particle size reduction11 (ii)
chemical modication such as salt formation,12 and (iii) nano-
carrier based strategies including formulation of nano-
suspension,13 nanoemulsion,14 polymeric nanocarriers,15
micellization,16 nanoplexes17 and use of dendrimers.18
Solubility, a phenomenon of dissolution of solute in solvent, is
considered as one of the most important parameters to achieve
a required concentration of drug in the systemic circulation for
a desired pharmacological action.1 Poor aqueous solubility of
many active pharmaceutical ingredients (APIs) limits their
bioavailability and absorption and thus has become a major
obstacle in the development of highly potent drugs.1–4 More than
40% of newly developed APIs are practically insoluble in water
and are rejected by the pharmaceutical industry due to inade-
quate or variable bioavailability and will never benet patients.
Another 17% of available drugs in the market also suffer from
suboptimal performance due to their limited or no aqueous
Discipline of Pharmaceutical Sciences, University of KwaZulu-Natal, Private Bag
X54001, Durban, 4000, KwaZulu-Natal, South Africa. E-mail: govenderth@ukzn.ac.
za; kalhapure@ukzn.ac.za; rahul.kalhapure@rediffmail.com; Fax: +27 31 260 7792;
Tel: +27 31 260 7358
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c6ra28100a
This journal is © The Royal Society of Chemistry 2017
RSC Adv., 2017, 7, 14233–14246 | 14233

View Article Online
RSC Advances
Paper
Fusidic acid (FSD) a steroidal antibiotic, isolated from biomedical applications necessitates the search for new AD
fungus Fusidium coccineum. It is primarily active against Gram- scaffolds. ADs have only been explored as materials for micellar
positive bacteria such as Staphylococcus, Streptococcus, and delivery and solubility enhancement despite of their other
Corynebacterium species.12 FSD has also been reported as an potential applications for various purposes in pharmaceutical
effective antibiotic against methicillin resistant Staphylococcus and biomedical sciences. To the best of our knowledge, there
aureus (MRSA),19–21 a bacteria resistant to many antibiotics. are no literature reports on use of ADs as steric stabilizers for
However, its applicability is limited by the fact that it is spar- colloidal drug delivery systems (DDS). Therefore, investigating
ingly soluble in water and tends to partition into oil in in vitro the applicability of these ADs as stearic stabilizers will provide
oil/water partition experiments.12 It is therefore prepared as the new insights that will assist in the development of ADs as
diethanolamine salt for intravenous administration and as colloidal drug delivery excipients. Therefore, the synthesis of
sodium salt for oral administration.22 Different nanocarrier new ADs will widen the pool of available structures for appli-
systems such as microemulsion,23 liposomes,24,25 polymeric cation as new pharmaceutical excipients with enhanced
micropheres26 and polymeric nanobers27 were reported for the performance.
delivery of FSD alone or in combination with other antibiotics.
The study in this paper reports the synthesis of novel
However, all these formulations need tedious preparation biocompatible G2 and G3 polyester ADs and their application as
procedures and use of organic solvents. On the contrary, solubility enhancers for poorly soluble antibiotics and as
amphiphilic dendrimers (ADs) can easily self-assemble into a stearic stabilizer for colloidal DDS. Two different series of ADs
ultra-small micelles by simple mixing with drug without the use from glycerol monostearate (GMS) and glycerol monooleate
of any organic solvent28 thus leading to easy and green formu- (GMOA) were synthesized. The hydrophobic tail in GMS and
lations. The ultra-small size features of these micelles will GMOA series consisted of saturated (stearic acid) and unsatu-
further provide a larger surface area to interact with the bacte- rated (oleic acid) fatty acid respectively. Whilst the hydrophilic
rial cell membrane thereby possibly, improving the antibacte- head was made up of 2,2-bis(hydroxymethyl)propionic acid (Bis-
rial potency of an antibiotic. Thus, strategies to enhance MPA) based polyester dendrons in both types. Bis-MPA was
solubility of poorly soluble antibiotics using ADs would be selected as the hydrophilic scaffold because of their character-
benecial to improve their pharmacokinetic properties and istic features such as (i) in vitro and in vivo biocompatibility, (ii)
subsequently antibacterial activity to control emergence of new solubility in biological media, (iii) biodegradability by enzy-
resistant bacteria.
matic and non-enzymatic hydrolysis and (iv) ease of function-
The concept of nano drug delivery is of great interest as it alization.37 The other structural components, stearic acid and
provides improved pharmacokinetic properties such as oleic acid, are dietary fatty acids and are generally regarded as
enhanced solubility, increased half-life and improved bioavail- safe materials38 for pharmaceutical and cosmetic prepara-
ability to existing drugs.29 The use of non-ionic amphiphilic tions.39 The aim of this study was therefore to synthesize and
polymeric materials to form self-assembled nanostructures characterize novel biocompatible GMS and GMOA derived ADs
have gained importance in the eld of nanomedicine and for pharmaceutical applications by evaluating their ability to (i)
nanobiotechnology due to the formation of various morpho- enhance solubility and antibacterial activity of FSD (ii) form
logical structures including micelles, vesicles and complex self-assembled nanoaggregates and (iii) act as stearic stabilizers
hierarchical structures such as bers, ribbons and helices.30 for vancomycin, a glycopeptide antibiotic, loaded solid lipid
Various poly(ethylene glycol) based amphiphiles for example nanoparticles (SLNs). The originality and novelty of this study
Cremophore, Pluronics, and Tween have been extensively used can be specied as (i) most of the literature on ADs reports on
as solubilizing agents for hydrophobic drugs.31 However, it is synthesis of ADs with polyglycerol dendrons and single or
reported that nonionic surfactants leads to the formation of double saturated C11, C16 and C18 aliphatic chains with
unstable, diverse and ill-dened micellar structure with high aromatic linkers. ADs in this study are synthesized using simple
polydispersity properties.32 Therefore, there is a need to develop esterication reaction between polyester dendrons and C18
nonionic amphiphiles which can generate structurally uniform saturated/unsaturated aliphatic chains, (ii) unlike other reports
micelles that can act as stable nanocarriers for solubility in the literature, we have demonstrated the biosafety of the
enhancement of hydrophobic drugs. Recently much attention synthesized ADs using three different cell lines, (iii) besides
has been focused on a novel class of amphiphilic systems their use as solubility enhancer and micellar carrier, we have
known as amphiphilic dendrimers (ADs). Multiple functional extended their applicability by proving the concept that ADs
groups at hydrophilic end and single aliphatic hydrocarbon could be used as promising stearic stabilizers for colloidal drug
chain on hydrophobic side makes these structures align at the delivery systems by preparing stable SLNs.
interface of conventional surfactants and amphiphilic poly-
In terms of signicance and impact, the ndings presented
mers. Owing to their unique structural arrangement, the AD in this study can be applied to facilitate the design of new
micelles can harbor a large void space within inner cores for biocompatible ADs for multiple biological applications. This
high drug loading.33 The eld of ADs is comparatively new and study will also serve as a directive for the design and synthesis of
there are very few reports on their applicability as nanocarriers novel ADs as substitutes for conventional non-ionic surfactant
for hydrophobic drug molecules,28 carriers for gene therapy,34 and gemini surfactants for application in various elds such as
photo-responsive delivery,35 and solubility enhancement.36 The catalysis,40 pharmaceuticals,41 environmental sciences,42 deter-
limited number of biocompatible ADs for pharmaceutical and gents, cosmetics, textile and dyeing.43
14234 | RSC Adv., 2017, 7, 14233–14246
This journal is © The Royal Society of Chemistry 2017

View Article Online
Paper
RSC Advances
Broth (MHB) and Mueller–Hinton Agar (MHA) were obtained
from Biolab (South Africa). The bacterial cultures used Staphy-
lococcus aureus (S. aureus) ATCC 25923 and MRSA (Rosenbach
ATCC BAA 1683). Cell lines A549 (ATCC® CCL-185™), Hep G2
(ATCC® HB-8065™), MCF7 (ATCC® HTB-22™) were originally
purchased from ATCC and donated to us as generous gi by Dr
2. Material and methods
2.1. Materials
GMS, puried powder was purchased from Alfa-Aesar (Ger-
many). Dowex®50WX2 hydrogen form (50–100 mesh), Bis-MPA,
4-(dimethylamino)pyridine (DMAP) and oleic acid (99%) were
purchased from Sigma-Aldrich (USA). The 2,2-dimethox-
ypropane was obtained from Sigma-Aldrich (Germany), DL-1,2-
isopropylideneglycerol from Sigma-Aldrich (Spain), 1-(3-dime-
thylaminopropyl)-3-ethylcarbodiimide$HCl (EDC) from Carbo-
synth (UK), p-toluenesulfonic acid monohydrate (PTSA) from
Sigma-Aldrich (France) and 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) was purchased from
Merck Chemicals (Germany). Pluronic F-68 (PF-68) from Sigma-
Aldrich (USA) and fusidic acid was purchased from Aopharm
Co. (China). Compritol 888 ATO was obtained as a generous gi
S
Naidoo (Department of Therapeutics and Medicinal
Management, University of KwaZulu-Natal). All other reagents
and chemicals were of analytical grade purchased from Merck
Chemicals (Germany).
2.2. Synthesis of GMS and GMOA ADs
G1, G2 and G3 GMS/GMOA ADs were synthesized from GMS and
GMOA through iterative generation growth by acetonide pro-
tected Bis-MPA and subsequent deprotection by Dowex® acidic
resin as depicted in Schemes 1 and 2.
´
from Gattefosse (France) and vancomycin (VCM) was purchased
from Sinobright Import and Export Co. Ltd. (China). For thin
layer chromatography (TLC), Merck precoated silica gel 60F254
plates (Germany) were used. Puried water used during the
study was obtained with a Milli-Q purication system (Millipore
corp., USA) in the laboratory. Nutrient Broth, Mueller–Hinton
2.2.1. Synthesis of GMS ADs (Scheme 1)
2.2.1.1. General procedure for esterication (I). Synthesis of
acetonide protected Bis-MPA was undertaken using a previously
reported procedure.44 The esterication reaction between ace-
tonide protected Bis-MPA and terminal hydroxyl group was
Scheme 1 Synthesis of GMS-G1, G2 and G3 ADs. Reagents and conditions: (a) Bis-MPA 2, DMAP, EDC, MDC, room temperature, 12 h; (b)
Dowex® H+ resin (10% w/w), MeOH, room temperature, 20 min–3 h.
This journal is © The Royal Society of Chemistry 2017
RSC Adv., 2017, 7, 14233–14246 | 14235

View Article Online
RSC Advances
Paper
Scheme 2 Synthesis of GMOA ADs. Reagents and conditions: (a) DMAP, EDC, MDC, room temperature, 12 h; (b) Bis-MPA 2, DMAP, EDC, MDC,
room temperature, 12 h; (c) Dowex® H+ resin (10% w/w), MeOH, room temperature, 20 min–3 h.
performed using Steglich esterication.45 The hydroxyl termi- intermediate product aer column chromatographic purica-
nated dendrimer, acetonide protected Bis-MPA and DMAP were tion (hexane/ethyl acetate, 60 : 40) (20.08 g, 88.73%). GMS-G2-
dissolved in dried methylene dichloride (MDC) and stirred Me (20.00 g, 0.016 mol) was then deprotected as per the
under nitrogen for 30 minutes. EDC was then added and the general procedure II using MeOH (100 ml) to give GMS-G2-OH 5
reaction mixture was further stirred under nitrogen and at room as a white solid (17.00 g, 97.92%).
temperature for 12 h. The reaction was monitored by TLC, and
the crude product obtained was puried by column chroma- 0.006 mol), 2 (15.85 g, 0.09 mol) and DMAP (1.04 g, 0.09 mol) in
tography using silica gel #60-200. dry MDC (40 ml), EDC (17.44 g, 0.09 mol) was added and the
2.2.1.5. GMS-G3-OH 6. To a solution of compound 5 (6.00 g,
2.2.1.2. General procedure for acetonide deprotection (II). reaction was continued as per the general procedure I. Aer
Acidic Dowex® H+ resin (10% w/w) was added to a methanolic completion of the reaction, the crude product was puried by
solution of acetonide protected dendrimer and stirred at room column chromatography (hexane/ethyl acetate, 50 : 50) to get
temperature until completion of the reaction. The resin was GMS-G3-Me as a colorless oil (11.20 g, 85.5%). GMS-G3-Me
removed by ltration and MeOH was evaporated under reduced (11.00 g, 0.004 mol) was dissolved in MeOH (80 ml) and
pressure to obtain nal product.
deprotected as per the general procedure II to give compound 6
2.2.1.3. GMS-G1-OH 4. GMS 1 (8.00 g, 0.022 mol), Bis-MPA 2 as a white solid (8.30 g, 87.7%).
(9.71 g, 0.056 mol), DMAP (1.36 g, 0.011 mol) were dissolved in
dry MDC (40 ml) and EDC (10.69 g, 0.056 mol) was added to the
2.2.2. Synthesis of GMOA ADs (Scheme 2)
2.2.2.1. GMOA (10). EDC (11.19 g, 0.06 mol) was added to
reaction mixture under stirring. The reaction was further a solution of oleic acid (OA) 7 (15.00 g, 0.05 mol), DL-1,2-iso-
continued according to the general procedure I, and the crude propylideneglycerol 8 (7.70 g, 0.06 mol) and DMAP (6.48 g, 0.05
product was puried by column chromatography (hexane/ethyl mol) in dry MDC (50 ml) and the reaction was continued
acetate, 80 : 20) to obtain GMS-G1-Me 3 as a colorless viscous oil following the general procedure I. Intermediate crude product,
(13.88 g, 92.78%). GMS-G1-Me, 3 (13.00 g, 0.019 mol) was dis- GMOA-Me 9, was puried by chromatography (hexane/ethyl
solved in MeOH (80 ml) and subjected to acetonide depro- acetate, 80 : 20) (20.80 g, 98.85%). GMOA-Me 9 (20.50 g, 0.05
tection following the general procedure II to obtain GMS-G1-OH mol) was dissolved in MeOH (250 ml) and deprotected as per
4 as a white solid (11.05 g, 96.50%).
the general procedure II to obtain GMOA 10, a colorless oil
2.2.1.4. GMS-G2-OH 5. Compound 4 (11.00 g, 0.019 mol), 2 (18.00 g, 96.2%).
(17.84 g, 0.102 mol), DMAP (1.14 g, 0.009 mol) and EDC (19.63 g,
2.2.2.2. GMOA-G1-OH 12. Compound 9 (10.00 g, 0.028 mol),
0.102 mol) were reacted in dry MDC (50 ml) as per the general 2 (12.22 g, 0.07 mol), DMAP (1.71 g, 0.014 mol) were dissolved in
esterication procedure
I to obtain GMS-G2-Me as an dry MDC (50 ml) and EDC (13.45 g, 0.07 mol) was added. The
14236 | RSC Adv., 2017, 7, 14233–14246
This journal is © The Royal Society of Chemistry 2017

View Article Online
Paper
RSC Advances
reaction mixture was stirred and continued as described in the 633 nm at an angle of 173ꢃ for AD concentration ranging from 1
general procedure I. Crude product was puried by column ꢀ 10ꢁ6 mol lꢁ1 to 1 ꢀ 10ꢁ3 mol lꢁ1 prepared from aqueous stock
chromatography (hexane/ethyl acetate, 80 : 20) to give GMOA- solution (1% w/v) for each derivative. Intensity measurements
G1-Me 11 as colorless oil (17.55 g, 93.55%). Compound 11 in kilo cycles per second (kcps) were performed in polystyrene
(17.00 g, 0.025) was deprotected following the general proce- cuvettes at 25 ꢃC. A graph of kcps versus concentration was
dure II using MeOH (100 ml) as a reaction medium to obtain 12 plotted and an intersection point of two straight lines drawn
as white sticky solid (14.00 g, 93.58%).
was recorded as CAC.
2.2.2.3. GMOA-G2-OH 13. To the solution of compound 11
(13.00 g, 0.022 mol), 2 (19.23 g, 0.11 mol) and DMAP (2.70 g,
0.022 mol) in dry MDC (80 ml), EDC (21.16 g, 0.110 mol) was
added and the reaction proceeded as described in the general
procedure I. Crude intermediate product (GMOA-G2-Me) was
puried with column chromatography (hexane/ethyl acetate,
60 : 40) to obtain GMOA-G2-Me as a thick oil (25.90 g, 96.68%).
GMOA-G2-Me (25.00 g, 0.020 mol) was subjected to depro-
tection in MeOH (250 ml) according to the general procedure II
to obtain GMOA-G2-OH 13 as a white solid (20.00 g, 92.25%).
2.2.2.4. GMOA-G3-OH 14. GMOA-G2-OH, 12 (10.00 g, 0.01
mol), 2 (26.46 g, 0.152 mol), EDC (29.12 g, 0.152 mol) and DMAP
(1.16 g, 0.009 mol) were dissolved in dry MDC (80 ml) and
reacted as per the general procedure I. GMOA-G3-Me was
puried by column chromatography (hexane/ethyl acetate,
50 : 50) to oily product (19.30 g, 88.30%). GMOA-G3-Me (19.00 g,
0.008 mol) was dissolved in MeOH (200 ml) and deprotected as
per the general procedure II to obtain GMOA-G3-OH 14 as
a white solid (13.00 g, 79.51%).
2.7. Self-assembly and characterization
Self-assembly of GMS-G2-OH, GMS-G3-OH, GMOA-G2-OH,
GMOA-G3-OH and PF-68 was studied by dissolving the respec-
tive amphiphiles in Milli-Q water at a concentration of 1% w/v.
The formulated micelles were characterized in terms of size,
poly dispersity index (PDI) and zeta potential (ZP) by DLS (Nano
ZS, Malvern, UK).
2.8. Solubility studies
Solubilization of FSD in GMS-G2-OH, GMS-G3-OH, GMOA-G2-
OH and GMOA-G3-OH ADs and PF-68, a commercialized solu-
bilizing agents, was studied by a simple shaking method.36
Excess (ca. 10 mg) of FSD was added in screw-top glass vials
containing 2 ml of amphiphiles dispersion (0.2 and 1% w/w) in
Milli-Q water. The obtained suspensions were incubated at
ꢃ
37 C in a shaking incubator (100 rpm) for 24 h. Aer incuba-
tion, undissolved FSD was removed by ltering through syringe
lters (cellulose acetate membrane, 0.2 mm, GVS lter tech-
nology, USA). FSD content was determined by a reported High
Performance Liquid Chromatography (HPLC) method48 using
an Agilent HPLC (Shimadzu Prominence DGU-20A3) (HPLC
procedure details are given in ESI†).
2.3. Structural characterization
FT-IR spectra of all the compounds were recorded on a Bruker
Alpha-p spectrometer with diamond ATR (Germany) as per
standard protocols. 1H NMR and 13C NMR measurements were
performed using a Bruker 400/600 Ultrashield™ (United
Kingdom) NMR spectrometer. HRMS was performed on
a Waters Micromass LCT Premier TOF-MS (United Kingdom).
2.9. In vitro antibacterial activity
In vitro antibacterial study was performed by using broth dilu-
tion method against S. aureus and MRSA. Both the bacterial
cultures were grown in Mueller–Hinton Broth (MHB) and
further diluted to 5 ꢀ 105 colony forming units per ml (CFU
mlꢁ1). To determine the minimum inhibitory concentration
(MIC) values for FSD in the presence and absence of GMOA-G2-
OH (test substances) against both cultures, serial dilutions of
drug and micelles were prepared in MHB broth and incubated
with bacterial cultures for 18 h in a shaking incubator at 37 ꢃC
and 100 rpm. Dilutions (10 ml) were spotted on Mueller–Hinton
Agar (MHA) plates and incubated further for 18 h. The
minimum concentration at which no visible bacterial growth
was observed was considered as the MIC.
2.4. In vitro cytotoxicity
MCF 7, Hep G2 and A549 human carcinoma cells were selected
to investigate the in vitro cytotoxicity using MTT assay.46 The
details of the MTT assay are provided in the ESI.†
2.5. Determination of log Poctanol/water and HLB values
The log Poctanol/water value of all ADs were calculated using ACD/
ChemSketch version 2016, Advanced Chemistry Development,
Inc., Toronto, ON (Canada), whereas HLB values were calcu-
lated by method described by Davies and Rideal47 using the
following formula:
HLB ¼ Ʃ (hydrophilic group numbers)
ꢁ Ʃ (lipophilic group numbers) + 7
2.10. Bacterial growth kinetics
To perform bacterial growth kinetics study, 50 ml of bacterial
suspension of S. aureus and MRSA (5 ꢀ 105 CFU mlꢁ1) in Lur-
iya–Bertani (LB) media was incubated with bare FSD and FSD
2.6. Determination of critical aggregation concentration
(CAC)
ꢃ
with GMOA-G2-OH at MIC in sterilized bottles at 37 C. Plain
The CAC of samples were determined by dynamic light scat- bacterial cultures were incubated as the untreated control, 3 ml
tering (DLS),46 using a zeta sizer nano ZS (Malvern, UK) equip- of samples were withdrawn from the bottles at different time
ped with 4 mm helium–neon laser operating at wavelength of points (0, 2, 4, 6, 8, 12, 18 and 24 h) and absorbance was
This journal is © The Royal Society of Chemistry 2017
RSC Adv., 2017, 7, 14233–14246 | 14237

View Article Online
RSC Advances
Paper
recorded at 600 nm by UV spectrophotometer (Shimadzu UV of the focal point was achieved through deprotection of the
1601, Japan).
acetonide group by using acidic Dowex® H+ resin. Synthesis of
these amphiphiles is simple and cost effective as overall yield of
nal compounds ranged from 52.13% to 77.79% even aer
multiple synthetic steps and therefore can be easily manufac-
tured on a commercial scale.
2.11. Formulation of SLNs using ADs as stearic stabilizer
Application of synthesized ADs as stearic stabilizer was evalu-
ated by formulating SLNs using VCM as model drug, Compritol
as lipid and ADs as surfactants. SLNs were prepared by reported
ultrasound dispersion technique49 with slight modications.
Briey, Compritol 888 ATO (0.3%) and VCM (0.1%) were heated
in water bath at 80 ꢃC and surfactant solution was prepared by
adding ADs (0.3%) in 15 ml of Milli-Q water and heated at the
same temperature as that of lipid phase. Hot surfactant solution
was added to the melted lipid drug mixture and homogenized
for 5 min at 6000 rpm with an Ultra Turrax T-25 homogenizer
(IKA Labortechnik, Germany). The resultant emulsion was
immediately subjected to high intensity probe sonication at
30% amplitude for 5 min under same temperature and then
cooled to 20 ꢃC to obtain SLNs. Volume of formulation was
adjusted to 10 ml if necessary. Blank formulations were
prepared using similar method without VCM. For comparison
purpose, SLNs were also prepared using PF-68 as a standard
non-ionic surfactant.
As the synthesis of these ADs involved formation of esters
and aliphatic alcohols, it was easy to monitor the progress of the
reaction using TLC, FT-IR and NMR. The formation of an ester
bond between acetonide protected Bis-MPA acid and hydroxyl
terminated dendron was conrmed by disappearance of the
peak at 3300 cmꢁ1 of –OH and appearance of the characteristic
ester peak at 1730 cmꢁ1. Deprotection of acetonide group of Bis-
MPA could be identied by disappearance of acetonide (C–O
stretch) peak at 1077 cmꢁ1 and appearance of peaks at 1036
cmꢁ1 (O–H bending) and 3326 cmꢁ1 (O–H stretching) of the free
hydroxyl group (Fig. S2 of ESI†). Deprotection of acetonide
1
could also be conrmed by H NMR spectroscopy as shown in
Fig. 1. The two peaks at 1.37 and 1.41 d ppm corresponding to
terminal CH3 of acetonide disappeared aer deprotection. To
test the chemical identity of ADs, elemental combustion anal-
ysis was performed on a ThermoScientic Flash2000 Elemental
Analyser. For GMS-G3-OH the observed C/H values were
consistent with the theoretical values. However, for GMOA-G2-
OH, GMOA-G3-OH and GMS-G2-OH the observed %C value
were lower than theoretical values (Table S1 and Fig. S3–S6 of
ESI†). This could be due to the incomplete adsorption and
combustion of gases and moisture of the samples.51 It has been
reported in the literature that elemental analysis for polymers
could give wrong results such as low carbon content.51,52
Further, High Resolution Mass spectrometry (HRMS) analysis
was performed on a Waters Micromass LCT Premier TOF-MS
(United Kingdom). The accurate mass recorded for all ADs
matched with the theoretical mass which gave conrmation of
their structural purity (Fig. S23, S31, S51 and S59 of ESI†).
3.1.1. GMS-G1-Me 3. FT-IR n: 2923.21, 2853.57, 1736.87,
1079.12. 1H-NMR (CDCl3) d (ppm): 0.81 (t, 3H), 1.04 (s, 3H), 1.07
(s, 3H), 1.21 (m, 28H), 1.28 (s, 6H), 1.34 (s, 6H), 1.51 (m, 2H),
2.24 (t, 2H), 3.54–3.58 (m, 4H), 3.63–3.76 (m, 4H), 4.01–4.33 (m,
4H), 5.20 (m, 1H). 13C-NMR (CDCl3) d (ppm): 14.12, 17.13, 17.87,
18.49, 29.14–29.70, 30.92, 31.92, 33.92, 42.08, 42.23, 46.85,
49.82, 65.05, 65.98, 66.04, 98.15, 98.26, 173.51, 174.99. HRMS
(ESI-TOF) m/z: [M + Na]+ – calculated 693.4554 (C37H66O10Na),
found 693.4549.
2.12. Characterization of SLNs
The particle size, PDI and ZP were measured by DLS technique
using zeta sizer (Nano ZS, Malvern, UK). All measurements were
performed in triplicate by diluting 300 ml of SLNs dispersion in
to 10 ml of Milli-Q water. The percentage entrapment efficiency
(%EE) was determined by a previously reported method50 using
following formula.
%EE ¼ (weight of VCM in SLN/weight of VCM added) ꢀ 100%
The morphology of formulated SLNs was determined by
using scanning electron microscopy (SEM). Briey, samples
were prepared by placing a few drops of appropriately diluted
SLNs on glass coverslip placed on carbon tape. The SLNs were
allowed to air dry, sputter coated with gold and images were
captured by eld-emission gun SEM (ZEISS FEGSEM Ultra Plus,
Germany) at an accelerated voltage of 10 kV.
2.13. Statistical analysis
GraphPad Prism® 5 (GraphPad Soware Inc., USA) was used to
statistically analyze the data. The data is representative of at
least three replicates and reported as mean ꢂ standard devia-
tion (SD). The results were analyzed using one-way ANOVA and
unpaired t-test followed by Welch's correction and the differ-
ence was considered signicant when p < 0.05.
3.1.2. GMS-G1-OH 4. FT-IR n: 3405.63, 2917.88, 2851.41,
1728.73, 1036.43. 1H-NMR (CDCl3) d (ppm): 0.87 (t, 3H), 1.07 (d,
6H), 1.24 (m, 28H), 1.60 (m, 2H), 2.31 (t, 2H), 3.69–3.72 (m, 4H),
3.81–3.86 (m, 4H), 4.19–4.48 (m, 4H), 5.36 (m, 1H). 13C-NMR
(CDCl3) d (ppm): 14.11, 17.16, 17.26, 22.68, 24.83, 29.12–29.69,
31.91, 34.02, 49.62, 49.74, 61.77, 62.99, 67.54, 70.01, 173.51,
174.93, 175.34. HRMS (ESI-TOF) m/z: [M + Na]+ – calculated
613.3928 (C31H58O10Na), found 613.3925.
3. Results and discussion
3.1. Characterization
3.1.3. GMS-G2-Me. FT-IR n: 2925.15, 2855.11, 1735.43,
1078.86. 1H-NMR (CDCl3) d (ppm): 0.81 (t, 3H), 1.04–1.07 (d,
The synthetic methods involved simple protection and depro- 18H), 1.22 (m, 28H), 1.28 (s, 12H), 1.34 (s, 12) 1.51 (m, 2H), 2.24
tection sequences, where the hydrophilic dendron of ADs were (t, 2H), 3.54–3.67 (m, 8H), 4.06–4.18 (m, 8H), 4.19–4.27 (m, 8H),
grown divergently with acetonide protected Bis-MPA. Activation 4.29–4.43 (m, 4H), 5.26 (m, 1H). 13C-NMR (CDCl3) d (ppm):
14238 | RSC Adv., 2017, 7, 14233–14246
This journal is © The Royal Society of Chemistry 2017

View Article Online
Paper
RSC Advances
Fig. 1 1H NMR data confirming deprotection of acetonide group of GMOA-G1-Me to GMOA-G1-OH.
14.11, 17.13, 17.63, 17.82, 18.37, 18.49, 22.68, 24.79, 29.14– 4.26 (m, 24H), 4.34–4.41 (m, 4H), 5.20 (m, 1H). 13C-NMR (CDCl3)
29.69, 30.91, 31.91, 33.92, 42.09, 42.22, 46.84, 49.82, 61.59, d (ppm): 14.11, 17.13, 17.48, 17.67, 17.80, 18.31, 18.38, 18.50,
64.88, 65.98, 66.04, 67.71, 98.11, 98.14, 98.26, 173.47, 173.53, 21.03, 21.88, 22.09, 22.67, 24.79, 25.14, 25.30, 28.07, 29.18–
174.97. HRMS (ESI-TOF) m/z: [M + Na]+ – calculated 1237.7073 30.90, 31.91, 33.88, 41.47, 42.02, 42.18, 46.64, 46.83, 49.89,
(C63H106O22Na), found 1237.7097.
60.38, 64.81, 65.90, 65.95, 66.03, 98.24, 171.16, 171.80, 174.88,
3.1.4. GMS-G2-OH 5. FT-IR n: 3285.22, 2921.6, 2852.17, 177.57. HRMS (ESI-TOF) m/z: [M + Na]+ – calculated 2326.2113
1730.06, 1039.20. 1H-NMR (CDCl3) d (ppm): 0.81 (t, 3H), 1.01 (s, (C115H186O46Na), found 2326.2119.
12H), 1.18 (m, 34H), 1.52 (m, 2H), 2.25 (t, 2H), 3.61–3.64 (m,
3.1.6. GMS-G3-OH 6. FT-IR n: 3355.28, 2921.20, 1721.30,
1
8H), 3.70–3.73 (m, 8H), 4.19–4.30 (m, 8H), 4.35–4.39 (m, 4H), 1030.16. H-NMR (CDCl3) d (ppm): 0.85 (t, 3H), 1.00 (d, 24H),
5.22 (m, 1H). 13C-NMR (CDCl3) d (ppm): 14.13, 17.12, 18.05, 1.19–1.23 (m, 38H), 1.50 (m, 2H), 2.28 (t, 2H), 3.40–3.47 (m,
22.69, 24.79, 29.14–29.40, 31.92, 33.95, 46.53, 46.58, 49.87, 32H), 4.12–4.2 (m, 12H), 4.31–4.36 (m, 4H), 4.60–4.62 (m, 12H),
49.93, 63.11, 64.75, 66.59, 66.70, 70.24, 172.19, 172.62, 175.04. 5.20 (m, 1H). 13C-NMR (CDCl3) d (ppm): 13.92, 16.66, 16.96,
HRMS (ESI-TOF) m/z: [M + Na]+ – calculated 1077.5821 17.09, 22.05, 24.27, 28.35, 28.67, 29.01, 31.25, 33.25, 46.10,
(C51H90O22Na), found 1077.5806. Anal calcd for C51H90O22: C, 46.23, 49.43, 50.20, 63.61, 63.81, 64.35, 171.78, 173.99, 176.57.
58.05; H, 8.60. Found: C, 57.31; H, 8.62.
HRMS (ESI-TOF) m/z: [M + Na]+ – calculated 2005.9609
3.1.5. GMS-G3-Me. FT-IR n: 2929.61, 2872.05, 1731.20, (C91H154O46Na), found 2005.9673. Anal calcd for C91H154O46: C,
1
1078.19. H-NMR (CDCl3) d (ppm): 0.81 (t, 3H), 1.02–1.17 (m, 55.09; H, 7.82. Found: C, 55.08; H, 8.06.
42H), 1.18–1.21 (m, 28H), 1.28 (s, 24H), 1.34 (s, 24H), 1.51 (m,
2H), 2.25 (t, 2H), 3.53–3.67 (m, 16H), 4.02–4.11 (m, 16H), 4.20– 1084.29. H-NMR (CDCl3) d (ppm): 0.81 (t, 3H), 1.20–1.23 (m,
3.1.7. GMOA-Me 9. FT-IR n: 2923.74, 2854.00, 1739.83,
1
This journal is © The Royal Society of Chemistry 2017
RSC Adv., 2017, 7, 14233–14246 | 14239

View Article Online
RSC Advances
Paper
Fig. 2 Results of MTT assay showing % cell viability vs. concentration of GMOA (A, B, C) and (D, E, F) ADs against MCF 7, A549 and Hep G2 cell
lines respectively.
20H), 1.30 (s, 3H), 1.36 (s, 3H), 1.56 (m, 2H), 1.93 (m, 4H), 2.27 (t, 2H), 3.69–3.72 (m, 4H), 3.80–3.85 (m, 4H), 4.20–4.29 (m, 2H),
(t, 2H), 3.65–3.68 (m, 2H), 4.00–4.11 (m, 2H), 4.24 (m, 1H), 5.27 4.37–4.48 (m, 2H), 5.34 (m, 2H), 5.37 (m, 1H). 13C-NMR (CDCl3)
(t, 2H). 13C-NMR (CDCl3) d (ppm): 14.10, 22.67, 24.88, 25.39, d (ppm): 14.10, 17.17, 17.27, 22.67, 24.82, 29.11–29.68, 31.91,
26.68, 27.15, 27.21, 29.08–29.76, 31.90, 34.09, 64.52, 66.35, 34.01, 49.69, 49.80, 61.80, 62.91, 66.69, 67.03, 67.09, 67.21,
73.66, 109.80, 129.71, 130.00, 173.59.
3.1.8. GMOA 10. FT-IR n: 3385.13, 2921.99, 2833.21, calculated 611.3771 (C31H56O10Na), found 611.3744.
1720.19, 1036.12. 1H-NMR (CDCl3) d (ppm): 0.81 (t, 3H), 1.20 (m,
3.1.11. GMOA-G2-Me. FT-IR n: 2927.70, 2857.58, 1735.40,
69.90, 173.51, 174.93, 175.37. HRMS (ESI-TOF) m/z: [M + Na]+ –
1
20H),1.56 (m, 3H), 1.95 (m, 4H), 2.28 (t, 2H), 3.51–3.87 (m, 2H), 1078.56. H-NMR (CDCl3) d (ppm): 0.81 (t, 3H), 1.07 (d, 12H),
4.10–4.16 (m, 2H), 4.85 (m, 1H), 5.27 (t, 2H). 13C-NMR (CDCl3) 1.22 (m, 26H), 1.28 (s, 12H), 1.34 (s, 12H), 1.51 (m, 2H), 1.93 (m,
d (ppm): 14.10, 22.68, 24.90, 27.15, 27.22, 29.10–29.76, 31.90, 4H), 2.23 (t, 2H), 3.53–3.67 (m, 8H), 4.10–4.16 (m, 8H), 4.21–4.27
34.14, 63.33, 65.17, 70.27, 129.71, 130.04, 174.33.
(m, 8H), 4.28–4.33 (m, 4H), 5.22 (m, 1H), 5.27, (m, 2H). 13C-NMR
3.1.9. GMOA-G1-Me 11. FT-IR n: 2925.25, 2855.25, 1736.47, (CDCl3) d (ppm): 14.10, 17.13, 17.63, 17.68, 17.82, 18.37, 18.49,
1078.78. 1H-NMR (CDCl3) d (ppm): 0.87 (t, 3H), 1.17 (s, 3H), 1.20 21.14, 21.72, 21.93, 22.03, 22.67, 22.87, 24.40, 24.78, 25.19,
(s, 3H), 1.26 (m, 20H), 1.37 (s, 3H), 1.41 (s, 3H), 1.58 (m, 2H), 25.29, 25.42, 26.00, 27.17, 27.21, 29.11–29.75, 30.91, 31.90,
2.00 (m, 4H), 2.30 (t, 2H), 3.60–3.64 (m, 4H), 4.14–4.18 (m, 4H), 33.88, 66.18, 67.69, 70.00, 98.10, 129.70, 130.00, 173.47, 173.53,
4.19–4.24 (m, 2H), 4.32–4.47 (m, 2H), 5.33 (m, 2H), 5.37 (m, 1H). 174.96.
13C-NMR (CDCl3) d (ppm): 14.11, 17.13, 17.22, 22.68, 24.81,
3.1.12. GMOA-G2-OH 13. FT-IR n: 3350.65, 2926.27,
27.17, 27.22, 29.10–29.76, 30.92, 31.90, 33.99, 41.90, 42.00, 2855.28, 1728.74, 1036.93. 1H-NMR (CDCl3) d (ppm): 0.87 (t,
61.75, 62.37, 67.58, 67.96, 68.03, 68.32, 70.02, 98.12, 98.29, 3H), 1.06 (d, 12H), 1.26 (m, 26H), 1.59 (m, 2H), 2.00 (m, 4H),
129.71, 130.02, 173.44, 174.16, 175.24.
2.31 (t, 2H), 3.67–3.78 (m, 8H), 4.14–4.17 (m, 8H), 4.28–4.36 (m,
3.1.10. GMOA-G1-OH 12. FT-IR n: 3399.39, 2923.29, 8H), 4.43–4.46 (m, 4H), 5.29 (m, 1H), 5.33 (m, 2H). 13C-NMR
2853.51, 1726.59, 1036.42. 1H-NMR (CDCl3) d (ppm): 0.88 (t, (CDCl3) d (ppm): 14.12, 17.12, 22.67, 24.78, 27.18, 27.22,
3H), 1.09 (s, 6H), 1.27 (m, 20H), 1.59 (m, 2H), 2.00 (m, 4H), 2.31 29.12–29.75, 31.89, 33.93, 46.55, 49.87, 64.68, 66.25, 69.93,
14240 | RSC Adv., 2017, 7, 14233–14246
This journal is © The Royal Society of Chemistry 2017

View Article Online
Paper
RSC Advances
Table 1 Calculated log P and HLB values for synthesized ADs
Table 3 Size, PDI and ZP of ADs after 30 days at 4 ꢃC
ADs
Log Poctanol/water
HLB ADs
Size (nm)
PDI
ZP (mV)
GMS-G1-OH
GMOA-G1-OH
GMS-G2-OH
GMOA-G2-OH
GMS-G3-OH
GMOA-G3-OH
5.42 ꢂ 0.71
4.90 ꢂ 0.71
4.19 ꢂ 0.93
3.67 ꢂ 0.93
0.99 ꢂ 1.07
0.47 ꢂ 1.07
8.5
8.5
15.7
15.7
34.9
34.9
GMS-G2-OH
8.93 ꢂ 0.08
12.45 ꢂ 0.86
8.16 ꢂ 0.61
13.32 ꢂ 0.39
0.400 ꢂ 0.011
0.685 ꢂ 0.018
0.291 ꢂ 0.011
0.381 ꢂ 0.015
ꢁ1.43 ꢂ 0.483
ꢁ0.000 ꢂ 0.005
ꢁ6.62 ꢂ 0.868
ꢁ6.86 ꢂ 0.337
GMS-G3-OH
GMOA-G2-OH
GMOA-G3-OH
non-toxic and biodegradable building blocks used for their
synthesis.
129.69, 130.04, 172.58, 173.39, 174.88. HRMS (ESI-TOF) m/z: [M
+ Na]+ – calculated 1075.5665 (C51H88O22Na), found 1075.5623.
Anal calcd for C51H88O22: C, 58.16; H, 8.42. Found: C, 55.88; H,
8.64.
3.3. Determination of log Poctanol/water and HLB values
Calculated log Poctanol/water and HLB values for all GMS and
3.1.13. GMOA-G3-Me. FT-IR n: 2930.19, 2876.40, 1730.29, GMOA ADs are presented in Table 1. Calculations showed that
1
1077.99. H-NMR (CDCl3) d (ppm): 0.81 (t, 3H), 1.06 (m, 24H), log Poctanol/water values decreased with increasing generation of
1.20 (m, 38H), 1.27 (s, 24H), 1.33 (s, 24H), 1.52 (m, 2H), 1.93 (m, polyester dendrimeric head group of ADs making G2 and G3
4H), 2.24 (t, 2H), 3.59–3.64 (m, 16H), 4.01–4.11 (m, 16H), 4.19– derivatives more hydrophilic than G1. GMOA ADs exhibited
4.25 (m, 24H), 4.27–4.37 (m, 4H), 5.20 (m, 1H), 5.26 (m, 2H). 13C- lower log Poctanol/water values than their respective GMS counter
NMR (CDCl3) d (ppm): 14.18, 17.13, 17.48, 17.67, 17.82, 18.33, parts. The order of log Poctanol/water was GMOA-G3-OH < GMS-
18.50, 21.03, 21.89, 22.07, 22.66, 24.66, 24.78, 25.15, 25.28, G3-OH < GMOA-G2-OH < GMS-G2-OH < GMOA-G1-OH < GMS-
27.21, 28.01, 29.13–29.75, 30.91, 31.90, 33.85, 41.52, 42.03,
G1-OH. HLB calculations showed that the values increased
42.17, 46.64, 46.83, 49.88, 60.39, 64.81, 65.91, 65.95, 66.00, from 8.5 to 34.9 for G1 to G3 respectively with an increase in
98.09, 98.34, 129.69, 129.99, 171.18, 171.81, 173.46, 178.04. hydrophilicity. G2 and G3 ADs showed HLB values more than 15
HRMS (ESI-TOF) m/z: [M + Na]+ – calculated 2324.2320 indicating they can used as solubilizing agents.49
(C115H184O46Na), found 2326.2119.
3.1.14. GMOA-G3-OH 14. FT-IR n: 3326.62, 2927.71,
3.4. Determination of CAC
1723.28, 1033.67. 1H-NMR ((CD3)2SO) d (ppm): 0.85 (t, 3H), 1.01
Avoidance of the inuence of an external probe makes non-
invasive methods as the most advantageous methods for
determination of CAC.46,56 DLS, one of the non-invasive tech-
niques, was successfully used for the determination CAC of
GMS-G2-OH, GMS-G3-OH, GMOA-G2-OH and GMOA-G3-OH
ADs. DLS measures time-dependent change in intensity of
scattered light from particles in the Brownian motion within the
colloidal system.46,57 Intensity of scattered light for concentra-
tion below CAC is low and similar to that of Milli-Q water. Once
concentration reaches the CAC, amphiphiles self-assemble to
form micelles which results in an increased intensity of scat-
(s, 24H), 1.16 (m, 38H), 1.5 (m, 2H), 2.0 (m, 4H), 2.28 (t, 2H),
3.39–3.47 (m, 16H), 4.11–4.25 (m, 16H), 4.31–4.37 (m, 24H), 5.21
(m, 1H), 5.32 (m, 2H). 13C-NMR ((CD3)2SO) d (ppm): 13.90,
16.66, 16.81, 17.10, 22.04, 24.25, 26.52, 26.57, 28.39–29.06,
31.23, 33.12, 46.09, 46.21, 46.23, 50.20, 63.61, 64.34, 129.59,
131.48, 171.78, 172.50, 173.99. HRMS (ESI-TOF) m/z: [M + Na]+ –
calculated 2003.9452 (C91H152O46Na), found 2003.9434. Anal
calcd for C91H152O46: C, 55.14; H, 7.73. Found: C, 54.62; H, 7.87.
3.2. In vitro cytotoxicity
The MTT results indicated a high percentage of cell viability for tered light.46,57 First generation derivatives (GMS-G1-OH and
all derivatives tested (>78%) across the concentration range GMOA-G1-OH) were very slightly soluble, and were therefore not
(Fig. 2). No concentration dependent trend was observed across used in the study. In the case of G2 and G3 ADs, there was
all cell lines studied. The results obtained in this study are in a decrease in CAC value with an increase in the generation. This
line with previous studies which report on materials that exhibit decrease in CAC could be attributed to an increase in the
a toxicity prole that are independent of the concentration.53,54 number of hydrophilic polyester functions with an increase in
The test material displaying percentage cell viability >70% can the generation. The CAC order was GMS-G3-OH (5 ꢀ 10ꢁ6 mol
be considered as biologically safe and nontoxic to mammalian lꢁ1) < GMOA-G3-OH (7 ꢀ 10ꢁ6 mol lꢁ1) < GMOA-G2-OH ¼ GMS-
cells.55 The biosafety of all ADs can be attributed to selection of G2-OH (5 ꢀ 10ꢁ5 mol lꢁ1) (Table 2). The results obtained are in
Table 2 CAC, size, PDI and ZP of ADs
ADs
CAC (mol lꢁ1
)
Size (nm)
PDI
ZP (mV)
GMS-G2-OH
GMS-G3-OH
GMOA-G2-OH
GMOA-G3-OH
5 ꢀ 10ꢁ5
5 ꢀ 10ꢁ6
5 ꢀ 10ꢁ5
7 ꢀ 10ꢁ6
7.60 ꢂ 0.13
6.48 ꢂ 0.04
8.62 ꢂ 0.48
12.38 ꢂ 0.36
0.263 ꢂ 0.053
0.620 ꢂ 0.060
0.272 ꢂ 0.028
0.339 ꢂ 0.019
ꢁ2.61 ꢂ 0.121
ꢁ0.011 ꢂ 0.053
ꢁ5.51 ꢂ 0.760
ꢁ7.89 ꢂ 0.763
This journal is © The Royal Society of Chemistry 2017
RSC Adv., 2017, 7, 14233–14246 | 14241

View Article Online
RSC Advances
Paper
Fig. 3 Solubilization of FSD by ADs and PF-68 at 0.2% w/w (A) and at 1% w/w (B).
line with previous ndings where a similar trend of a decrease all ADs at 0.2% w/w concentration showed higher solubilization
in CAC with an increase in generation number was observed for of FSD [GMS-G2-OH (1.7-fold), GMOA-G2-OH (5.6-fold), GMS-
polyglycerol dendrimers.28 When comparing GMS and GMOA G3-OH (1-fold) and GMOA-G3-OH (2.9-fold) increase in solu-
ADs, dendrimers of same generation had no signicant differ- bility compared to water]. Aer increasing the amphiphile
ence in their CAC values. This suggests that unsaturation in the concentration to 1% w/w, there was an enhancement in the
aliphatic chain had no effect on CAC value.
solubility of FSD [GMOA-G2-OH (43-fold), GMOA-G3-OH (11-
fold), GMS-G2-OH (9.1-fold) and GMS-G3-OH (6.8-fold)].
However, at both concentrations FSD showed signicantly
lower solubility (p ¼ 0.0001) in the standard solubilizing
excipient PF-68 (0.8-fold and 3.5-fold at 0.2 and 1% respec-
tively). These results conrmed the superiority of the synthe-
sized ADs over the marketed non-ionic surfactant. The results of
the solubilization of FSD by PF-68 are in line with previous
reports where PF-68 showed low solubilization enhancement
compared to non-ionic glycerol based dendritic amphiphiles
studied for poorly soluble anticancer drug sagopilone.36 The
order of solubilization of FSD in all amphiphiles was GMOA-G2-
OH > GMOA-G3-OH > GMS-G2-OH > GMS-G3-OH > PF-68
(Fig. 3). The higher solubility of FSD in GMOA-G2-OH ADs can
be attributed to the chain kink due to cis conrmation of
unsaturated bond in oleic acid thereby leading to lower curva-
tures.58 This spatial arrangement could have assisted in
accommodating more FSD molecules leading to its higher
entrapment.
3.5. Self-assembly and characterization
Aggregation behavior of all synthesized ADs was evaluated by
dissolving 1% w/w in Milli-Q water (concentration well above
CAC). DLS study results showed the existence of smaller
aggregates for G2 and G3 derivatives of GMS and GMOA with
average size in the range of 6.48 to 12.38 nm and narrow PDI
(Table 2). In the case of GMOA ADs, the G2 derivative formed
signicantly smaller aggregates (p ¼ 0.0016) than G3 with low
PDI, whilst with GMS ADs, no signicant (p ¼ 0.0835) difference
was observed for average size in between G2 and G3 derivative.
Amongst all ADs, GMOA derivatives formed larger aggregates
compared to GMS. The average particle size for GMOA G2 and
GMOA G3 was 8.62 ꢂ 0.48 nm and 12.38 ꢂ 0.36 nm respectively
whereas for GMS G2 and G3 the size was 7.6 ꢂ 0.13 nm and 6.48
ꢂ 0.04 nm respectively. ZP for G2 and G3 GMS and GMOA ADs
were ꢁ2.61, ꢁ0.011, ꢁ5.51 and ꢁ7.87 respectively. Further-
more, stability of all micelles were carried out for 30 days at 4 ꢃC
by measuring particle size, PDI and ZP. There was a signicant
(p < 0.05) change observed in PDI and ZP of GMS ADs whereas
no signicant difference was observed in these values in the
case of GMOA ADs aer 1 month storage (Table 3). These results
indicated that, though particle sizes for GMOA G2 and G3 ADs
were high, they were more stable as compared to their respec-
tive GMS derivatives aer 30 days.
Effect of FSD on size, PDI and ZP of ADs and PF-68 aggre-
gates were also studied. The size, PDI and ZP values for FSD
loaded aggregates are presented in Table S2 of ESI.† The results
showed that FSD loading signicantly increases size of
Table 4 MIC of FSD with and without GMOA-G2-OHa
MIC (mg mlꢁ1
)
3.6. Solubility studies
Formulation
S. aureus
MRSA
Solubilization results of FSD in G2 and G3 ADs and in PF-68 are
presented in Fig. 3. The results were expressed as factor of
solubilization (F solubilization, where F is x fold increase in
solubility of FSD compared to water). PF-68 was selected in the
study as a commercial non-ionic surfactant for comparison
purposes. FSD showed 0.0003% w/v solubility in water, whilst
FSD
0.53
NA
0.23*
0.39
NA
0.23*
GMOA-G2-OH
FSD with GMOA-G2-OH
a
NA ¼ no activity, * ¼ results statistically signicant (p < 0.05)
compared to control (FSD).
14242 | RSC Adv., 2017, 7, 14233–14246
This journal is © The Royal Society of Chemistry 2017

View Article Online
Paper
RSC Advances
Fig. 4 Bacterial growth kinetics curve for (A) S. aureus (B) MRSA (n ¼ 3).
aggregates except in the case of GMOA-G2-OH where the size activity against S. aureus and MRSA. The minimum inhibitory
slightly decreased from 8.62 ꢂ 0.48 to 7.20 ꢂ 0.09. There was concentration (MIC) values for FSD with and without GMOA-G2-
a slight increase in PDI and decrease in ZP values of aggregates OH against S. aureus were 0.23 mg mlꢁ1 and 0.53 mg mlꢁ1
aer FSD solubilization. In case of PF-68 aggregates, size respectively whereas the values were 0.23 mg mlꢁ1 and 0.39 mg
increased form 28.67 ꢂ 4.87 to 142.6 ꢂ 9.86 nm with a bimodal mlꢁ1 against MRSA respectively (Table 4). These results sug-
distribution curve indicating its instability aer FSD loading. gested that GM-OA-G2 not only enhanced the solubility but also
Aer noting the differences in ZP values of plain and FSD enhanced the antibacterial potency of FSD. There was a signi-
loaded micelles, we hypothesized that there could be a presence cant difference between MIC of FSD with GMOA-G2-OH and
of unencapsulated FSD in ltered solution. The drug's positive bare FSD (p < 0.05). This enhanced activity could be ascribed to
or negative charge can affect the zeta potential value.59 To increased solubility of FSD which enabled its better diffusion.
support this statement, we determined the encapsulation effi- The results were in good agreement with a previous reported
ciency (%EE) and drug loading (%DL) of FSD in all amphiphiles nding where self-assembled b-cyclodextrin enhanced the
using an ultraltration method following a literature reported antimicrobial potency of chlorhexidine.63
procedure60 with detailed procedure provided in the ESI.† It was
observed that %EE and %DL for FSD was in the range of 4.5 ꢂ
3.8. Bacterial growth kinetics
0.9 to 25.76 ꢂ 0.1% and 0.009 ꢂ 0.001 to 0.35 ꢂ 0.001 (Table S3
of ESI†). The unencapsulated percentages (74.24 to 95.5%) of
FSD could have been solubilized by one or combination of one
or more mechanisms such as (i) reduction in surface tension,1
(ii) wetting and disintegration of solids forming ne particles61
and; (iii) incorporation of FSD onto the micellar surface.62
Investigation of bacterial growth kinetics in LB media quanti-
tatively evaluated the antibacterial potency of FSD GMOA-G2-
OH micelles against S. aureus and MRSA. Fig. 4 shows the
optical density at 600 nm for different incubation times. The
control group achieved a cell concentration of 0.120 OD and
0.103 OD for S. aureus and MRSA respectively at 4 h of incu-
bation time. Aer 18 h of incubation, the control group growth
curve in both strains achieved exponential growth with
3.7. In vitro antibacterial activity
As FSD exhibited highest solubilization in GMOA-G2-OH, it was maximum bacterial concentration of 1.388 OD and 1.308 OD. In
selected for further evaluation to assess the effect of these ADs bare FSD treated samples, bacterial cells started growing at
on antibacterial activity of FSD. GMOA-G2-OH exhibited no incubation of 12 h with concentration of 0.410 OD and 0.414 OD
Table 5 Size, PDI and ZP of blank SLNs using different ADs
Formulation
Size (nm)
PDI
ZP (mV)
DF-GMOA-G2-OH SLNs
DF-GMS-G2-OH SLNs
DF-GMOA-G3-OH SLNs
DF-GMS-G3-OH SLNs
DF-PF-68 SLNs
252.56 ꢂ 12.53
250.6 ꢂ 5.41
365.53 ꢂ 3.86
273.1 ꢂ 2.81
161.76 ꢂ 3.60
0.154 ꢂ 0.017
0.160 ꢂ 0.011
0.233 ꢂ 0.011
0.204 ꢂ 0.029
0.325 ꢂ 0.035
ꢁ26.06 ꢂ 2.04
ꢁ15.3 ꢂ 1.44
ꢁ38.46 ꢂ 3.04
ꢁ31.23 ꢂ 1.98
4.16 ꢂ 0.14
This journal is © The Royal Society of Chemistry 2017
RSC Adv., 2017, 7, 14233–14246 | 14243

View Article Online
RSC Advances
Paper
Table 6 Size, PDI and ZP of drug loaded SLNs using different ADs
Formulation
Size (nm)
PDI
ZP (mV)
%EE
DL-GMOA-G2-OH SLNs
DL-GMS-G2-OH SLNs
DL-GMOA-G3-OH SLNs
DL-GMS-G3-OH SLNs
133.93 ꢂ 3.44
192.80 ꢂ 4.44
291.17 ꢂ 22.02
278.97 ꢂ 8.26
0.296 ꢂ 0.008
0.338 ꢂ 0.042
0.379 ꢂ 0.018
0.485 ꢂ 0.055
ꢁ25.08 ꢂ 0.65
ꢁ22.33 ꢂ 1.02
ꢁ34.10 ꢂ 1.83
ꢁ26.13 ꢂ 3.16
12.81 ꢂ 1.02
10.46 ꢂ 0.57
10.68 ꢂ 1.72
8.34 ꢂ 1.04
attending maximum concentration of 1.225 OD and 1.231 OD properties of ADs suggested their potential to be new and
for S. aureus and MRSA respectively at 24 h of incubation time. It attractive biocompatible pharmaceutical excipients. Consid-
was observed that GMOA-G2-OH treated samples effectively ering the poor solubility of antibiotics, one of the reasons
controlled the bacterial growth for both the strains throughout limiting their effectiveness against bacteria and subsequently
the study period. At the end of 24 h, the OD values for FSD resulting in antibiotic resistance, these new ADs were evaluated
GMOA-G2-OH treated samples were 0.388 and 0.130 against S. for their solubility enhancement of FSD, a poorly soluble anti-
aureus and MRSA respectively. These growth curve kinetic biotic effective against MRSA. The solubility results suggested
studies demonstrated that the growth of both strains was that all ADs were able to signicantly improve solubility of FSD
signicantly (p < 0.05) inhibited by FSD ADs, thus proving its compared to water and the commercial PF-68. In addition, it
superiority over bare FSD.
was observed that during the solubilization process, ADs self-
assembled to form nano micelles with % FSD encapsulation
in the range of 1.1 to 3%. This conrmed their applicability in
the formulation of nano drug delivery systems. Amongst all
ADs, FSD exhibited highest solubility in GMOA-G2-OH with
formation of nano micelles of 8.62 ꢂ 0.48 nm particle size. In
vitro antibacterial activity and cell kill kinetic studies conrmed
that ADs enhanced the antibacterial activity of FSD against S.
aureus and MRSA. The ADs were further evaluated for their
application as stearic stabilizers for SLNs by using them as
surfactants. The results from formulation studies proved that
ADs are capable of providing sufficient stability to SLNs, as
conrmed by higher zeta potential values.
The overall results of the study direct that ADs reported in
this study could be promising excipients for pharmaceutical
applications. The ndings reported in this paper will also be
a guide towards the use of simple and efficient synthetic
methods to obtain biocompatible amphiphilic ADs which can
self-assemble to ultra-small aggregates and can acts as stearic
stabilizer for colloidal systems for various applications in
pharmaceutical and biomedical sciences.
3.9. Formulation and characterization of SLNs
SLNs were successfully formulated by using Compritol 888 ATO
as a solid lipid ADs as surfactants using ultrasound dispersion
technique. For comparative evaluation PF-68 was chosen as
commercial non-ionic surfactant. All results of particle size, PDI
and ZP for blank formulation are summarized in Table 5. The
PDI values were <0.3 and ZP values were between ꢁ15.3 ꢂ
1.44 mV to ꢁ38.46 ꢂ 3.04 mV for SLNs prepared using ADs
whereas PDI was >0.3 and ZP was 4.16 ꢂ 0.14 mV for SLNs
prepared using PF-68 (Table 5). The results showed that SLNs
formulated from GMOA ADs were more stable than those
prepared using PF-68 though their particle sizes were higher.
VCM loading produced SLNs (containing ADs as stabilizers)
with size, PDI and ZP values in the range of 133.93 ꢂ 3.44 to
291.17 ꢂ 22.02 nm, 0.296 ꢂ 0.008 to 0.485 ꢂ 0.055 and ꢁ22.33 ꢂ
1.02 mV to ꢁ34.10 ꢂ 1.83 mV respectively (Table 6). The %EE of
VCM in all the formulations was in between 8.34 ꢂ 1.04 to 12.81
ꢂ 1.02%. This low %EE could be ascribed to the hydrophilicity
of VCM. The results are consistent with previous ndings where
low %EE (16.81 ꢂ 3.64) was observed for VCM.50
Acknowledgements
Morphological investigation showed that SLNs formulated
from ADs were homogenous and spherical in shape (Fig. S3 and
S4 of ESI†). Overall results showed that ADs can formulate nano-
sized particulate systems with lower PDI and higher ZP making
them attractive stearic stabilizers for SLNs and other colloidal
drug delivery systems.
The authors acknowledge College of Health Sciences (CHS),
University of KwaZulu-Natal (UKZN), UKZN Nanotechnology
Platform and the National Research Foundation (NRF) of South
Africa for nancial support (NRF Grant No. 87790 and 88453).
References
4. Conclusion
1 K. T. Savjani, A. K. Gajjar and J. K. Savjani, ISRN Pharm.,
2012, 2012, 195727.
2 H. Chen, C. Khemtong, X. Yang, X. Chang and J. Gao, Drug
Discovery Today, 2011, 16, 354–360.
3 P. Khadka, J. Ro, H. Kim, I. Kim, J. T. Kim, H. Kim, J. M. Cho,
G. Yun and J. Lee, Asian J. Pharm. Sci., 2014, 9, 304–316.
4 S. Svenson and A. S. Chauhan, Nanomedicine, 2008, 3, 679–
702.
In this paper, new G2 and G3 ADs were synthesized by conju-
gation of polyester based dendrons to aliphatic hydrocarbon
chains. These ADs which were found to be non-toxic to
mammalian cells were further characterized for their CAC to
determine their micelle forming ability. All the G2 and G3 ADs
exhibited lower CAC values and were able to form self-
assembled nano structures as conrmed by DLS. These
14244 | RSC Adv., 2017, 7, 14233–14246
This journal is © The Royal Society of Chemistry 2017

View Article Online
Paper
RSC Advances
5 S. A. Adediran, T. P. Day, D. Sil, M. R. Kimbrell, 33 T. Wei, C. Chen, J. Liu, C. Liu, P. Posocco, X. Liu, Q. Cheng,
H. J. Warshakoon, S. S. Malladi and S. A. David, Mol.
Pharm., 2009, 6, 1582–1590.
S. Huo, Z. Liang and M. Fermeglia, Proc. Natl. Acad. Sci. U. S.
A., 2015, 112, 2978–2983.
6 P. Weiss, M. Andrew and W. Wright, Antibiot. Chemother., 34 S. Malhotra, H. Bauer, A. Tschiche, A. M. Staedtler, A. Mohr,
´
1957, 7, 374–377.
M. Calderon, V. S. Parmar, L. Hoeke, S. Sharbati,
7 M. Aucamp, R. Odendaal, W. Liebenberg and J. Hamman,
Drug Dev. Ind. Pharm., 2015, 41, 1100–1108.
R. Einspanier and R. Haag, Biomacromolecules, 2012, 13,
3087–3098.
8 Y. Cheng, H. Qu, M. Ma, Z. Xu, P. Xu, Y. Fang and T. Xu, Eur. 35 C. Kordel, C. S. Popeney and R. Haag, Chem. Commun., 2011,
J. Med. Chem., 2007, 42, 1032–1038. 47, 6584–6586.
9 M. C. Carey, J. C. Montet and D. M. Small, Biochemistry, 1975, 36 A. Richter, A. Wiedekind, M. Krause, T. Kissel, R. Haag and
14, 4896–4905. C. Olbrich, Eur. J. Pharm. Sci., 2010, 40, 48–55.
10 S. Ranghar, P. Sirohi, P. Verma and V. Agarwal, Braz. Arch. 37 A. Carlmark, E. Malmstrom and M. Malkoch, Chem. Soc.
Biol. Technol., 2014, 57, 209–222. Rev., 2013, 42, 5858–5879.
11 A. Chaudhary, U. Nagaich, N. Gulati, V. Sharma, R. Khosa 38 A. C. Rustan and C. A. Drevon, in eLS, John Wiley & Sons, Ltd,
¨
and M. U. Partapur, J. Adv. Pharm. Educ. Res., 2012, 2, 32–67.
12 J. Turnidge, Int. J. Antimicrob. Agents, 1999, 12, S23–S34.
13 D. Zhang, T. Tan, L. Gao, W. Zhao and P. Wang, Drug Dev.
Ind. Pharm., 2007, 33, 569–575.
2001, DOI: 10.1038/npg.els.0003894.
39 F.-Q. Hu, S.-P. Jiang, Y.-Z. Du, H. Yuan, Y.-Q. Ye and S. Zeng,
Colloids Surf., B, 2005, 45, 167–173.
40 H. Y. Rhyoo, H.-J. Park, W. H. Suh and Y. K. Chung,
Tetrahedron Lett., 2002, 43, 269–272.
14 S. Vatsraj, K. Chauhan and H. Pathak, J. Nanosci., 2014, 2014.
15 G. Mohammadi, H. Valizadeh, M. Barzegar-Jalali, 41 J. Eastoe and R. F. Tabor, in Colloidal Foundations of
F. Lotpour, K. Adibkia, M. Milani, M. Azhdarzadeh,
F. Kiafar and A. Nokhodchi, Colloids Surf., B, 2010, 80, 34–39.
Nanoscience, ed. G. Palazzo, Elsevier, Amsterdam, 2014, pp.
135–157, DOI: 10.1016/B978-0-444-59541-6.00006-0.
16 P. A. Bhat, A. A. Dar and G. M. Rather, J. Chem. Eng. Data, 42 F. Volkering, A. M. Breure and W. H. Rulkens,
2008, 53, 1271–1277. Biodegradation, 1997, 8, 401–417.
17 W. S. Cheow and K. Hadinoto, J. Colloid Interface Sci., 2012, 43 N. Kumar and R. Tyagi, J. Dispersion Sci. Technol., 2014, 35,
367, 518–526. 205–214.
18 K. Winnicka, M. Wroblewska, P. Wieczorek, P. T. Sacha and 44 H. Ihre, A. Hult, J. M. J. Frechet and I. Gitsov,
E. A. Tryniszewska, Molecules, 2013, 18, 8607–8617. Macromolecules, 1998, 31, 4061–4068.
19 P. Grohs, M.-D. Kitzis and L. Gutmann, Antimicrob. Agents 45 B. Neises and W. Steglich, Angew. Chem., Int. Ed. Engl., 1978,
Chemother., 2003, 47, 418–420. 17, 522–524.
20 R. C. Moellering, G. R. Corey and M. L. Grayson, Clin. Infect. 46 Surfactant micelle characterization using dynamic light
´
Dis., 2011, 52, S467–S468.
scattering,
http://www.malvern.com/en/pdf/secure/
21 M. Whitby, Int. J. Antimicrob. Agents, 1999, 12(2), S67–S71.
22 D. Reeves, J. Antimicrob. Chemother., 1987, 20, 467–476.
AN101104SurfactantMicelleCharacterization.pdf, accessed
23 January 2016.
23 T. Chhibber, S. Wadhwa, P. Chadha, G. Sharma and 47 J. T. Davies and E. K. Rideal, Interfacial Phenomenon,
O. P. Katare, J. Drug Targeting, 2015, 23, 943–952. Academic Press, New York, 1963, pp. 343–450.
24 D. Nicolosi, S. Cupri, C. Genovese, G. Tempera, R. Mattina 48 A. H. Hikal, A. Shibl and S. El-Hoofy, J. Pharm. Sci., 1982, 71,
and R. Pignatello, Int. J. Antimicrob. Agents, 2015, 45, 622–
626.
25 S. Wadhwa, B. Singh, G. Sharma, K. Raza and O. P. Katare,
Drug Delivery, 2016, 23, 1204–1213.
26 C. Yang, D. Plackett, D. Needham and H. M. Burt, Pharm.
Res., 2009, 26, 1644–1656.
1297–1298.
49 R. S. Kalhapure and K. G. Akamanchi, Colloids Surf., B, 2013,
105, 215–222.
50 R. S. Kalhapure, C. Mocktar, D. R. Sikwal, S. J. Sonawane,
M. K. Kathiravan, A. Skelton and T. Govender, Colloids
Surf., B, 2014, 117, 303–311.
27 S. E. Gilchrist, D. Lange, K. Letchford, H. Bach, L. Fazli and 51 Y. Yang, Q. Zhang, S. Zhang and S. Li, RSC Adv., 2014, 4,
H. M. Burt, J. Controlled Release, 2013, 170, 64–73. 5568–5574.
28 B. Trappmann, K. Ludwig, M. R. Radowski, A. Shukla, 52 J. X. Jiang, F. Su, A. Trewin, C. D. Wood, N. L. Campbell,
¨
A. Mohr, H. Rehage, C. Bottcher and R. Haag, J. Am. Chem.
H. Niu, C. Dickinson, A. Y. Ganin, M. J. Rosseinsky,
Y. Z. Khimyak and A. I. Cooper, Angew. Chem., Int. Ed.,
2007, 46, 8574–8578.
Soc., 2010, 132, 11119–11124.
29 T. M. Allen and P. R. Cullis, Science, 2004, 303, 1818–1822.
30 A. Sorrenti, O. Illa and R. M. Ortuno, Chem. Soc. Rev., 2013, 53 S. Di Gioia, C. Sardo, G. Belgiovine, D. Triolo, M. d'Apolito,
42, 8200–8219.
S. Castellani, A. Carbone, I. Giardino, G. Giammona,
G. Cavallaro and M. Conese, Int. J. Pharm., 2015, 491, 359–
366.
31 E. V. Batrakova and A. V. Kabanov, J. Controlled Release,
2008, 130, 98–106.
32 M. J. Schick, Nonionic surfactants: physical chemistry, Marcel 54 M. D. Romic, M. S. Klaric, J. Lovric, I. Pepic, B. Cetina-
Dekker, New York, 1987.
Cizmek, J. Filipovic-Grcic and A. Hafner, Eur. J. Pharm.
Biopharm., 2016, 107, 67–79.
This journal is © The Royal Society of Chemistry 2017
RSC Adv., 2017, 7, 14233–14246 | 14245

View Article Online
RSC Advances
Paper
55 S. Rambharose, R. S. Kalhapure, K. G. Akamanchi and 60 L. L. Cai, P. Liu, X. Li, X. Huang, Y. Q. Ye, F. Y. Chen,
T. Govender, J. Mater. Chem. B, 2015, 3, 6662–6675.
56 U. Anand, C. Jash and S. Mukherjee, J. Colloid Interface Sci.,
2011, 364, 400–406.
H. Yuan, F. Q. Hu and Y. Z. Du, Int. J. Nanomed., 2011, 6,
3499–3508.
61 L. Di and E. H. Kerns, Drug-like properties: concepts, structure
design and methods from ADME to toxicity optimization,
Academic Press, 2015.
¨
57 O. Topel, B. A. Çakır, L. Budama and N. Hoda, J. Mol. Liq.,
2013, 177, 40–43.
58 S. M. Sagnella, C. E. Conn, I. Krodkiewska, M. Moghaddam, 62 D. Everett, Pure Appl. Chem., 1972, 31, 577–638.
ˆ
J. M. Seddon and C. J. Drummond, Langmuir, 2010, 26, 3084– 63 A. M. L. Denadai, K. I. Teixeira, M. M. Santoro,
´
3094.
A. M. C. Pimenta, M. E. Cortes and R. D. Sinisterra,
59 S. Hassani, A. Laouini, H. Fessi and C. Charcosset, Colloids
Surf., A, 2015, 482, 34–43.
Carbohydr. Res., 2007, 342, 2286–2296.
14246 | RSC Adv., 2017, 7, 14233–14246
This journal is © The Royal Society of Chemistry 2017