Ligand-conjugated pH-sensitive polymeric micelles for the targeted delivery of gefitinib in lung cancers

Article abstract of DOI:10.1039/c5ra09931e

The aim of the present study was to investigate the tumor targeting potential of a mannose-conjugated pH-sensitive nanosystem for the effective delivery of gefitinib (Gnb) to lung cancers. We have successfully demonstrated the potential of mannose-tagged nanomicelles as an efficient vector to transport the anticancer drug. The micelles had nanosized particles with excellent dispersity index. The nanomicelles exhibited a pH-responsive nature with enhanced drug release in acidic pH conditions. Fluorescence and flow cytometer analysis showed a superior cellular uptake for the mannose-tagged nanomicelles. Confocal laser scanning microscopy study revealed a receptor-mediated cellular internalization process. The M-NP-Gnb showed a superior anticancer effect in A549 cancer cells and remarkably inhibited cancer cell proliferation. Furthermore, M-NP-Gnb significantly increased the proportion of cells in the apoptosis and necrosis region. Importantly, the half-life of Gnb encapsulated in nanomicelles increased by about 5-fold compared to that of free Gnb. The augmented half-life clearly indicates the maximum residence time of the drug in the systemic circulation. Consistently, M-NP-Gnb showed a 7-fold higher accumulation of drug in tumor tissues compared to that of free Gnb. Overall, PLGA/His-based nanomicelles could be a promising delivery system to increase the therapeutic efficiency of Gnb in lung cancers.

Full text of DOI:10.1039/c5ra09931e

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Page 1 of 31
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A Ligand-conjugated pH-sensitive Polymeric Micelles for the Targeted Delivery of
Gefitinib in Lung cancers
ShiꢀJiang Wang 1, ZhiꢀJun Huo2, Kai Liu3，Ning Yu4，Yan Ma1，YueꢀHong Qin5，XiaoꢀChen
Li6，ZhiꢀQi Wang7*
1Department of Radiation Oncology, Shandong Cancer Hospital and Institute, Ji'nan，
Shandong，250117，China
2Department of Breast Disease Center, Shandong Cancer Hospital and Institute, Ji’nan，
Shandong，250117，China
3Department of Gastrointestinal Surgery, Shandong Cancer Hospital and Institute, Ji’nan，
Shandong，250117，China
4Department of pathology, Affiliated hospital of Binzhou medical college, Binzhou, Shandong,
256603, China
5Department of Neurosurgery, Shandong Provincial Hospital, Ji’nan，Shandong，250021，
China
6Department of Internal Medicine, Affiliated Hospital of Shandong Academy of Medical
Sciences, Ji’nan，Shandong，250021，China
7Department of Head and Neck Surgery, Shandong Cancer Hospital and Institute, Ji'nan，
Shandong，250117，China
Corresponding author:
ZhiꢀQi Wang,
Department of Head and Neck Surgery,
Shandong Cancer Hospital and Institute,
No.440, Ji Yan Road, Jinan, 300162, Shandong, P.R.China
Tel & Fax: 0086ꢀ0531ꢀ67626141
Email:wzqbansg@hotmail.com

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Abstract
The aim of present study was to investigate the tumor targeting potential of mannoseꢀconjugated
pHꢀsensitive nanosystem for the effective delivery of Gefitinib (Gnb) to lung cancers. We have
successfully demonstrated the prospective of mannoseꢀtagged nanomicelles as an efficient vector
to ferry anticancer drug. The micelles exhibited a nanosized particle with excellent dispersity
index. The nanomicelles exhibited a pHꢀresponsive nature with enhanced drug release in the
acidic pH conditions. The fluorescence and flow cytometer analysis showed a superior cellular
uptake for mannoseꢀtagged nanomicelles. The CLSM study revealed a receptorꢀmediated cellular
internalization process. The MꢀNPꢀGnb showed a superior anticancer effect in A549 cancer cells
and remarkably inhibited the cancer cell proliferation. Furthermore, MꢀNPꢀGnb significantly
increased the proportion of cells in the apoptosis and necrosis region. Importantly, halfꢀlife of
Gnb encapsulated in nanomicelles increased by about 5ꢀfold comparing to that of free Gnb. The
augmented halfꢀlife clearly indicates the maximum residence time of drug in the systemic
circulation. Consistently, MꢀNPꢀGnb showed a 7ꢀfold higher accumulation of drug in tumor
tissues compared to that of free Gnb. Overall; PLGA/Hisꢀbased nanomicelles could be a
promising delivery system to increase the therapeutic efficiency of Gnb in lung cancers.
Keywords
Lung cancers, apoptosis, gefitinib, nanomicelles, pHꢀsensitive

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Introduction
Lung cancer is one of the leading causes of cancerꢀrelated death worldwide. The death rate due
to lung cancer far exceeds the death rate of prostate, colon, and breast cancers united.1,2 The 5ꢀ
year survival rate of lung cancer patients are below 15% despite the advancement in
technological field. The lung cancers could be divided into nonꢀsmall cell lung cancer (NSCLC)
and small cell lung cancer (SCLC).3 Nonꢀsmall cell lung cancer (NSCLC) comprises
approximately 75–80% of all respiratory lung cancers. The incidence and mortality of lung
cancer are increasing every year in China. At present, surgery, radiotherapy and chemotherapy
constitutes the main treatment mode for lung cancers.4,5
In this regard, tyrosine kinase inhibitors are shown to possess excellent antitumor activity against
lung cancer cells. Gefitinib (Gnb), a tyrosine kinase inhibitor has high affinity towards epithelial
growth factor receptor (EGFR) which is normally expressed in lung cancers.6,7 Gnb can
effectively kill lung cancer cells via promoting metastasis, angiogenesis, and apoptosis in tumor
cells. The main mechanism behind the anticancer effect of Gnb is by blocking the signal
transmission by competitive binding MgꢀATP situated on the catalytic domain of EGFRꢀTK.8,9
This will inhibit the activation of mitogen activated protein kinase and induce the apoptosis of
cancer cells. Despite its potential clinical application, Gnb is widely distributed in the body and
results in severe drugꢀrelated toxicity. Moreover, limited presence of drug in the cancer cells
further complicates the chemotherapy treatment.10 Therefore, new strategies have to be
developed to increase the chemotherapeutic efficacy of Gnb in lung cancers.
Nanotechnology has opened up new horizons in cancer drug delivery. Recent studies have
highlighted the role of nanotechnology in cancer therapy.11 Drugꢀloaded nanocarriers have been

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shown to increase the blood circulation of the anticancer small molecules, sustained the drug
release in the physiological conditions, and increased its accumulation in the tumor region.12
When antitumor drugs are loaded in nanoparticles with the size of 20–200 nm, their
biodistribution in heart and lung is largely decreased.13 In this study therefore, a novel pHꢀ
sensitive delivery system has been designed that will increase the drug concentration in the
cancer tissues while reducing its presence in the normal tissues. PLGA is one of the most
common polymers used as carrier for drug delivery applications due to inherent biodegradability
and low toxicity. Therefore, PLGAꢀPEG was selected in order to form a stable hydrophobic core
and PEG forms the outer shell which protects the carrier from immediate systemic clearance.
Polyꢀhistidine was synthesized and utilized in micelle formation in order to bring the pHꢀ
responsiveness to the carrier system.14,15 The so formed nanocarrier is able to extravasate solid
tumors into the tumor tissues via enhanced permeability and retention effect (EPR). However,
passive targeting of cancer cells is not found to be effective and warranted alternative therapeutic
approach.17,18 In this context, glycotargeting involves the interaction of lectin receptors to the
carbohydrate ligands. It has been reported that mannoseꢀcytostatics complexes exhibits selective
uptake in cancer cells.19 The preferential uptake was attributed to the receptorꢀmediated
internalization by lectin receptors at the tumor surface. In the present study therefore,
polysaccharideꢀmannose tagged PLGAꢀbased nanocarrier system has been developed to target
lung cancers.20
The main objective of present study was to explore the prospective of polysaccharide tagged pHꢀ
sensitive nanomicelles (PLGA/Histidine) for the selective delivery of gefitinib in lung cancers.
In this study, we firstly developed PLGA/pHistidineꢀbased nanomicelles which were then
conjugated with mannose as a cancer targeting moiety. The pHꢀsensitive nature of nanocarrier

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was asserted by means of particle size analysis and drug release kinetics study. In vitro cellular
uptake of targeted and nonꢀtargeted NP was analyzed by fluorescence and flow cytometry
(FACS) and observed by confocal laser scanning microscopy (CLSM). The
methylthiazoltetrazolium (MTT) assay was employed to examine the in vitro cytotoxicity of the
free drug as well as drugꢀloaded micelles. Annexin V/PIꢀbased apoptosis analysis was performed
in A549 lung cancer cells. Finally, systemic performance of free drug and drugꢀloaded micelles
were performed in experimental rats.
Results and Discussion
Lung cancer is one of the leading causes of cancerꢀrelated death worldwide. In this regard,
Gefitinib (Gnb), a tyrosine kinase inhibitor could effectively kill lung cancer cells via promoting
metastasis, angiogenesis, and apoptosis in tumor cells. Therefore, in the present study, we have
developed PLGA/pHistidineꢀbased nanomicelles to target the Gnb to lung cancer cells (Figure
1).
Preparation of M-NP-Gnb nanomicelles
The pHISꢀPEG was synthesized by ring opening polymerization process. The poly (Lꢀhistidine)
was conjugated with PEG via an amide bond. In case of PLGAꢀPEGꢀNH2, PEGꢀdiamine was
reacted with the PLGAꢀNHS and resulting product was reacted with mannose. The
mannosylation was performed by ring opening of mannose and subsequent reaction of its
aldehyde group with free amine functional group of PLGAꢀPEGꢀNH2 of the nanoparticle
surface. This leads to the formation of Schiff’s base (ꢀNꢀCHꢀ). The main objective of present
study was to explore the prospective of polysaccharide tagged pHꢀsensitive nanomicelles

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(PLGA/Histidine) for the selective delivery of gefitinib in lung cancers. The PLGAꢀPEG and
HistidineꢀPEG was synthesized in order to formulate mixed micelles wherein PLGA will form
the inner hydrophobic core and PEG forms the outer shell. The mannose was conjugated to
PLGAꢀPEG polymer block and present in the outer nanoparticle surface. The presence of
mannose on the outer nanoparticle surface will increase the targeting and accumulating
efficiency of chemotherapeutic drug.
Characterization of M-NP-Gnb
The particle size and zeta potential of all the nanoparticles were evaluated by dynamic light
scattering method. The average particle size of NPꢀGnb was about 120 nm and the particle size
increased for the mannose conjugation (168 nm) (Figure 2a). The increase in the size of
nanoparticles was due to the presence of mannose conjugation to the outer surface. Nevertheless,
particle size of <200 nm would be much suitable for tumor targeting applications.21 The micelles
were negative charged with ꢀ18.5 mV. The negative surface charge of nanoparticles would
increase the physiological stability in the blood circulation. During in vivo applications, negative
surface charge would reduce the undesirable clearance by reticuloendothelial system (RES)
which would increase its accumulation in the tumor tissues. The particle morphology was
observed using TEM imaging. TEM showed clear spherical shaped particles for MꢀNPꢀGnb
which is uniformly dispersed in the carbonꢀcoated copper grid (Figure 2b). The particle size
observed form TEM imaging was consistent with the DLS particle size.
pH-sensitivity of M-NP-Gnb micelles
The pHꢀsensitive nature of MꢀNPꢀGnb was investigated by means of size, size distribution, and
zeta potential analysis. The histidine present in the micellar structure is mainly responsible for

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the pHꢀresponsiveness of the system. As shown in Table 1, zeta potential increased with the
decrease in the pH of suspended medium indicating the effective protonation of the histidine
residue. Especially, micelles were stable at pH 7.4, however when the pH decreased to 5.5,
abundant protonation of histidine resulted in low zeta potential of ꢀ3 mV. Consistently, particle
size of the micelles increased due to the disassembly and aggregation of the multiple polymers.
Drug loading and in vitro drug release study
Knowledge of drug entrapment efficiency is very important for drug delivery applications. The
MꢀNPꢀGnb exhibited a high entrapment efficiency of >95% with a high drug loading of ~24.7%.
The pHꢀsensitive nature of NPꢀGnb and MꢀNPꢀGnb was evaluated from the release kinetics. It is
expected that presence of Histidine in the mixed micelle would provide the pHꢀresponsive nature
to the nanocarrier system. The pHꢀresponsiveness would increase the swelling and dissociation
of micellar system resulting in the release of drug. It can be seen from Figure 3 that both NPꢀGnb
and MꢀNPꢀGnb exhibited a typical pHꢀsensitive release pattern. Approximately, 60% of drug
released in pH 5.5 conditions compared to 35% of drug released in pH 7.4 conditions. The
release trend continued up to 48h where in almost 95% of drug released in acidic pH and 50% of
drug released in pH 7.4 conditions. It should be noted that no initial burst release phenomenon
was observed suggesting that all of drug were tightly packed in the hydrophobic core of micelles.
Although the rate of drug release from NPꢀGnb and MꢀNPꢀGnb was almost similar, a slight
decrease in the release rate from MꢀNPꢀGnb was ascribed to the presence of the protective coat
of mannose on the SLNs surface, which impedes the drug release.22 Overall, enhanced release

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rate at lower pH would be beneficial for antiꢀcancer application since tumor tissue is
characterized by an acidic microenvironment
Cellular uptake efficiency
The cellular uptake efficiency as a function of targeting agent has been studied on CHO (control
cells) and A549 lung cancer cells. For this purpose, rhodamineꢀb was loaded in the nanomicellar
formulations to observe the fluorescence intensity or fluorescence measurement. The cellular
uptake of targeted and nonꢀtargeted nanoparticles was studied in these two cell lines by means of
flow cytometer (Figure 4a). In case of CHO cells (which don’t express lectin receptors), cellular
uptake of NP and MꢀNPꢀGnb were not markedly different. In case of Aꢀ549 cells however,
remarkable difference were observed between targeted and nonꢀtargeted nanoparticles. It could
be clearly seen that MꢀNPꢀGnb had significantly higher cellular uptake than NP after 2h of
incubation. The mannoseꢀtagged micelles could enter the cells more quickly than the nonꢀ
targeted NP. Mannosylated NP’s could not be taken up by CHO cells via receptorꢀmediated
endocytosis because mannose receptors are not expressed on their membrane. Having shown the
specificity of mannosylated NP towards lectin receptor expression cancer cells, we have carried
all our experiments in Aꢀ549 cells.
The cellular uptake was further studied by fluorescence experiments. The cancer cells were
exposed with NPꢀGnb and MꢀNPꢀGnb and incubated for different time period up to 4h (Figure
4a). The results clearly revealed a timeꢀdependent cellular uptake. Importantly, mannoseꢀtagged
formulations showed a significant increment in the cellular uptake compared to that of nonꢀ
targeted nanosystem. For example, at the end of 4h, MꢀNP showed ~70% fluorescent intensity
compared to that of ~38% fluorescent intensity of nonꢀtargeted NP. The high fluorescent

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fluorescence intensity associated with the MꢀNP system was mainly attributed to the lectin
receptorꢀmediated internalization of nanomicelles. It could be noticed that nonꢀtargeted NP also
showed some uptake which is possibly due to the endocytosis mediated nonꢀspecific
internalization.23 Such high uptake of mannoseꢀtargeted system could increase the intracellular
concentration as well the cytotoxic effect of the anticancer cancer drugs.
Cellular internalization
The result from the cellular uptake analysis indicates the mannoseꢀtagged nanomicelles could
effectively internalize the cancer cells than the nonꢀtargeted nanomicelles. We further
investigated the mechanism of cellular uptake in A549 cancer cells. CLSM was used to evaluate
the cellular uptake. For this purpose, we have stained the lysosome with Lysotracker Green
(green color) and nucleus was stained with DAPI (blue color). As mentioned above,
nanomicelles were loaded with rhodamineꢀb to impart the fluorescence intensity. As seen (Figure
5), MꢀNP treated cells showed bright red fluorescence compared to that of NP treated cell group
indicating the enhanced cellular internalization in case of ligandꢀtagged formulations. The MꢀNP
was very well colocalized in the lysosomal region indicating a typical lectin receptorꢀmediated
endocytosis uptake. In contrast, NP did not colocalize appreciably due to simple gradientꢀ
mediated uptake mechanism. It could expect that nanomicelles are triggered in the acidic
endosomal/lysosomal compartments and leave endosomal/lysosomal compartments via simple
diffusion to the nuclear region. Earlier, it has been reported that micellar formulations were
internalized by caveolaeꢀdependent manner wherein nanoparticles rapidly colocalize in the acidic
compartments and the released drug will be travelling to the nuclear region.

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Cytotoxicity assay
The cytotoxic potential of free Gnb and Gnbꢀloaded nanomicelles were evaluated by means of
MTT assay. The results clearly showed a timeꢀdependent and concentrationꢀdependent cytotoxic
action of Gnb (free and encapsulated form) in A549 lung cancer cells. The cytotoxic potential of
different formulations was consistent with the cell uptake assays (Figure 6a,b). The IC50 value
was calculated to quantitate the cytotoxic action of individual formulations. The IC50 value (the
concentration that inhibited cell growth by 50%) of MꢀNPꢀGnb was 0.85 µg/ml comparing to
2.35 µg/ml for NPꢀGnb and 4.12 µg/ml for free Gnb after 24h incubation. The superior cytotoxic
action of MꢀNPꢀGnb was mainly attributed to the ligandꢀreceptor interactions. The enhanced
internalization, high accumulation of nanomicelles due to lectinꢀmannose interaction, sustained
release characteristics of formulations within the cells could be responsible for the augmented
cytotoxic effect.24
The Hoechst staining was carried out to observe the cellular morphology. As seen, control cells
were intact with clear morphology (Figure 6c). The drug treatment however induced remarkable
changes in cell morphology. Especially, MꢀNPꢀGnb showed marked changes in cellular and
nuclear morphology, including chromatin condensation, membrane blebbing, nuclear
breakdown, and the appearance of membraneꢀassociated apoptotic bodies.
Apoptosis assay
The apoptosis effect of individual formulations was studied by FACS analysis using Annexin
V/PI staining (Figure 7). The results clearly showed that number of apoptotic and necrotic cells
increased upon treatment with the formulation. The free Gnb slightly induced cell apoptosis with
marginal amount of cells in the necrotic. The NPꢀGnb however showed higher percentage of

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cells in the apoptosis and necrotic region. Notably, MꢀNPꢀGnb showed significantly higher
proportions of cells in the apoptosis and necrotic stage indicating the superior anticancer effect of
mannoseꢀtagged formulations. The enhanced apoptosis of cancer cells were consistent with the
higher cellular internalization and higher cell killing effect.
Pharmacokinetic and Biodistribution analysis
The pharmacokinetic study has been performed to investigate the systemic performance of free
drug as well the drugꢀloaded nanomicelles. The in vivo study clearly revealed that
pharmacokinetics of free drug significantly altered after encapsulation in the nanomicelles. As
seen (Figure 8a), free Gnb was immediately cleared from the systemic circulation within 4ꢀ6h.
The NPꢀGnb and MꢀNPꢀGnb significantly prolonged the residence time of Gnb up to 24h. The
halfꢀlife of Gnb encapsulated in nanomicelles increased by about 5ꢀfold comparing to that of free
Gnb. The augmented halfꢀlife clearly indicates the maximum residence time of drug in the
systemic circulation. The AUC0–24 and AUC0–∞ values of free DOX (18.45ꢁg h/mL), NPꢀGnb
(65.92ꢁg h/mL) and MꢀNPꢀGnb (68.12ꢁg h/mL) further demonstrated the long circulation
property of nanomicelles. The long circulation property of NPꢀGnb and MꢀNPꢀGnb was
attributed to the presence of PEG on the outer surface of nanocarrier. The PEG will effectively
evade the macrophage uptake and augment the halfꢀlife of drug in the nanocarrier system. The
prolonged blood circulation profile of mannoseꢀconjugated nanomicelles would be crucial for
tumor targeting. The MꢀNPꢀGnb would be preferentially accumulated in tumor tissues via
receptorꢀmediated endocytosis in mannose receptor overexpressed A549 tumor tissues.
The biodistribution study was conducted to further investigate the targeting ability of mannoseꢀ
targeted nanosystem. As shown in Figure 8b, significantly higher concentration of Gnb was

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accumulated in tumor tissues from MꢀNPꢀGnb therapeutic system across all the time points.
Approximately, 7ꢀfold higher accumulation of drug was observed in tumor tissues than
comparing to free drug. At the end of 24h, ~10 ng/gm of tissue was observed in MꢀNPꢀGnb
treated mice group whereas none was observed for free drug treated group. The ability of MꢀNPꢀ
Gnb to localize preferentially in the tumor tissue indicates its reduced entry on other vital organs.
Moreover, it should be noted that drug level in MꢀNPꢀGnb treated group kept increasing until 4h.
This may be explained on the basis of the presence of lectin receptors on tumor tissues that
favored selective entry of MꢀNPꢀGnb in tumors. The presence of lectin receptors on the tumor
surface presents an interesting platform and facilitates targeting. This strategy will help to
augment the therapeutic effectiveness by lowering the dose of therapeutic moiety.
Materials and Methods
Materials
PLGA ([η] = 0.32–0.44 dL/g; 50:50) and dꢀmannose was purchased from SigmaꢀAldrich
(China). PEGꢀdiamine was purchased from Laysan Bio Inc (Arab, AL). 1ꢀethylꢀ3 (3ꢀ
dimethylaminopropyl) carbodimide (EDC) was purchased from Sigma Aldrich, China. Nꢀ(tꢀ
butyloxycarboryl)ꢀ1ꢀ(2,4ꢀdinitrophenyl)ꢀLꢀHistidine [BocꢀHis(DNP)ꢀOH] was purchased from
Sigma Aldrich (St, Louis, USA). All other chemicals were of reagent grade and used as such
without further modification.
Preparation of Gefitinib-loaded nanomicelles

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The PLGAꢀPEG polymer block was fabricated using carbodiimide chemistry. In brief, PLGA
(10 g) was dissolved in DMSO and to this solution, EDC/NHS was added and the reaction was
allowed to continue for 24h. The soꢀformed PLGAꢀNHS was collected by the addition of cold
ether and filtered. The PLGAꢀNHS was reacted with PEG diamine at a ratio of 6:1 in DMSO and
further stirred for 20h at room temperature. The final product PLGAꢀPEGꢀNH2 was precipitated
by excessive acetonitrile and washed with diethyl ether. The unreacted PEGꢀdiamine was
separated by dialysis method. The final product Folate–PEG–PLA was obtained by freezeꢀdrying
and stored in refrigerator (4°C).26
mPEGꢀPolyHistidine was synthesized from the precursor BocꢀHis(DNP)ꢀOH. In brief, 10 g of
BocꢀHis(DNP)ꢀOH was dissolved in dioxane and dichloro sulfoxide was added and stirred for
1h. The soꢀformed DNPꢀNCAꢀHCl was obtained after washed being by dioxane and dried in
vacuum. Now, specific amount of DNP.NCA.HCl was reacted with sodium carbonate in DMF
solution for 1h. The mPEGꢀNH2 (in DMF) was then added dropꢀwise to the above mixture and
stirred for 48h at room temperature. The soꢀformed mPEGꢀPoly(NimꢀDNPꢀhistidine) was
obtained after filtrated and dried in vacuum. Then, mPEGꢀPoly(NimꢀDNPꢀhistidine) (0.5g) was
dissolved in 8 ml of DMSO and reacted with 2–mercaptoethanol and reacted for 12h at room
temperature. The final product mPEGꢀPolyHistidine was obtained after filtrated and washed with
ethanol and ether. The isolated product was dialyzed against distilled water (molecular weight
cutꢀoff (MWCO) = 3500 Da), and then freezeꢀdried to give the final product. 27
The precipitate was redissolved in DMF The drugꢀloaded nanomicelles were prepared by dialysis
method. Briefly, Gnb (5 mg), PLGAꢀPEGꢀNH2 (20 mg), pHIS–PEG (10 mg) was dissolved in 4
ml of DMSO and stirred for 20h.The solution was then dialyzed in a large quantity of water
(dialysis membrane Mw~3500) for 48h. The drugꢀloaded nanomicelles was collected and

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subsequently conjugated with dꢀmannose. Dꢀmannose (10 µM) was dissolved in sodium acetate
buffer (pH 4.0) and stirred for 24h. The mannosylated nanomicelles were further dialyzed for
48h to remove the unconjugated mannose. The final product was collected and purified as
mentioned above and stored at cold temperatures.
Particle size and zeta potential analysis
The particle size, size distribution, and zeta potential surface charge of nanomicelles were
evaluated by photon correlation spectroscopy (PCS) method. Zetasizer (Malvern Instruments,
England) was used to measure the particle size and zeta potential. Samples were suitably diluted
and analyzed at a fixed angle of 90°. For zeta potential measurement, the samples were diluted
with phosphate buffered saline (pH 7.4).
In vitro release study
The drug release profile of Gnb from MꢀNPꢀGnb and NPꢀGnb was evaluated by means of
dialysis method. For this purpose, 1 mg equivalent of Gnb in formulations were loaded in a
dialysis tube (MW~3500 Da), sealed at both the end and suspended in a Falcon tube containing
20 ml of release media containing 1% Tween 80 to increase the solubility of drug in the medium.
The medium was continuously stirred at 100 rpm and the whole assembly was placed at 37°C. At
specific time point, samples were withdrawn from the medium and replaced with equal amount
of fresh release medium. The samples were analyzed using HPLC method. Shimadzu HPLC
system consisting of Cꢀ18 column was used. 0.02M dipottasium hydrogen ortho phosphate and
methanol at a fixed ratio of 10:90 was used as a mobile phase. The mobile phase was run at 1
ml/min and quantified at 246 nm.

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Cell viability assay
A549 lung epithelial cancer cell line was cultured in DMEM medium supplemented with 10%
heatꢀinactivated fetal bovine serum (FBS), 1% streptomycin, 3 mM glutamine in a 37°C
humidified incubator and 5% CO2 atmosphere. The cell viability of A549 cancer cell was
investigated using 3ꢀ(4,5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀ diphenyltetrazolium bromide (MTT) assay.
The cells were seeded into 96ꢀwell plate at a density of 5×104 cells per well and allowed to attach
for 24h at 37°C. The cells were then treated with free Gnb, NPꢀGnb and MꢀNPꢀGnb and
incubated for 24h and 48h, respectively. The media was removed and washed twice with PBS
buffer. Cells were treated with MTT solution (10 mg/mL) and incubated for 4 h at 37 °C,
facilitating MTT to be reduced by viable cells with the formation of purple formazan crystals.
DMSO was added to each well to dissolve the formazan crystals and the absorbance of
individual wells was noted at 570 nm using an ELISA plate reader.
Cellular uptake analysis
The cellular uptake of targeted and nonꢀtargeted nanomicelles was determined by fluorescence
microscopy. Briefly, cells were seeded in 96ꢀwell plate, allowed to attach, and exposed with
rhodamineꢀb loaded NP and MꢀNP. The formulations were incubated in a timeꢀdependent
manner. The cells were washed twice with PBS to remove the nanocarriers adhering on the cell
surface. The uptake was calculated from the absorbance of individual wells which was noted at
570 nm via an ELISA plate reader.
The cellular uptake was further confirmed by fluorescence activated cell sorters (FACS)
instrument (BD Biosciences FACS Aria, Germany). Briefly, cells were seeded in 96ꢀwell plate,
allowed to attach, and exposed with rhodamineꢀb loaded NP and MꢀNP. The formulations were

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incubated in a timeꢀdependent manner. The cells were washed and trypsinized (0.1% w/v). The
cells were pelleted by centrifuging at 1200 rpm/5 min. The pellets were again reꢀdispersed in
PBS buffer and cellꢀassociated fluorescence was measured by fluorescence activated cell sorters
(FACS) instrument.
Cell apoptosis analysis
The apoptosis effect of individual formulation was evaluated by AnnexinꢀV/PI double staining
assay using flow cytometer. The A549 cells were seeded in a 12ꢀwell plate and incubated
overnight. Following day, cells were exposed with free Gnb, NPꢀGnb and MꢀNPꢀGnb
formulations and incubated for 18h. Untreated cells were considered as a control. Next day, cells
were washed with PBS twice, extracted, and reꢀsuspended with binding buffer. Cells were then
treated with 5 µL of Annexin VꢀFITC and 5 µL of propidium iodide for 5 minutes in the dark at
room temperature, and apoptosis was then detected by flow cytometry (BD Biosciences FACS
Aria, Germany).
Qualitative apoptosis analysis
The A549 cells were seeded in a 12ꢀwell plate (2×105 cells/well) and treated with free Gnb, NPꢀ
Gnb and MꢀNPꢀGnb formulations and incubated for 24h. The cells were fixed and exposed with
Hoechst 33382 nuclear staining dye. The cellular and nuclear morphology was observed using a
fluorescence inversion microscope.
Cellular internalization
The cellular internalization of targeted and nonꢀtargeted nanomicelles was studied by means of
confocal laser scanning microscopy (CLSM). The cells were seeded in a 6ꢀwell plate and

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allowed to attach for 24h. The cells were then exposed with rhodamineꢀb loaded NP and MꢀNP
and incubated for 1h. After washing with PBS, cells were stained with Lysotracker Green for
lysosomes. The cells were fixed with 4% paraformaldehyde and washed. The cells were again
stained with DAPI as a nuclear staining dye. The cellular internalization process was investigated
using confocal microscope (Nikon, Japan).
Pharmacokinetic and biodistribution study
Male Balb/c mice, 4–5 weeks old, weighing 10–20 g was selected to perform the
pharmacokinetic analysis. The animal study protocol was approved (Ref no. 2015ANZQ268) by
Institutional Animals Ethical Committee (IAEC), Shandong Cancer Hospital and Institute,
China. The entire study was carried out in accordance with the standard institutional guiding
principles of IAEC. As per the reported protocol, A549 tumors were generated on the right flank
of mice. The mice were divided into 3 groups with 6 mice in each group. The free Gnb, NPꢀGnb,
and MꢀNPꢀGnb were administered at a fixed dose of 5 mg/kg via tail vein. For biodistribution
study, animals were sacrificed at specific time intervals and tumor was extracted and stored at ꢀ
80°C until further analysis. For pharmacokinetic assay, blood samples were collected and
centrifuged at 5000rpm for 10 min and plasma was separated. To 100 µl of plasma, 100 µl of
acetonitrile was added and protein was precipitated. The drug was extracted from the organic
mixture using centrifugation. The supernatant was collected, filtered through 0.45 µm filter and
injected into the HPLC column. The amount of drug present in the blood as well as in the
individual organ was determined using HPLC method.

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Statistical analysis
Statistical analysis of the data was performed using SPSS 16.0 (SPSS Corp., USA) with oneꢀway
ANOVA followed by Tukey’s post hoc test. P<0.05 was considered statistically significant. The
data are presented as mean±SD.
Conclusion
In this study, we have successfully prepared a mannoseꢀconjugated pHꢀsensitive nanosystem for
the effective delivery of Gefitinib to lung cancer cells. We have successfully demonstrated the
prospective of mannoseꢀtagged nanomicelles as an efficient vector to ferry anticancer drug. The
micelles exhibited a nanosized particle with excellent dispersity index. The nanomicelles
exhibited a pHꢀresponsive nature with enhanced drug release in the acidic pH conditions. The
fluorescence and flow cytometer analysis showed a superior cellular uptake for mannoseꢀtagged
nanomicelles. The CLSM study revealed a receptorꢀmediated cellular internalization process.
The MꢀNPꢀGnb showed a superior anticancer effect in A549 cancer cells and remarkably
inhibited the cancer cell proliferation. Furthermore, MꢀNPꢀGnb significantly increased the
proportion of cells in the apoptosis and necrosis region. Importantly, halfꢀlife of Gnb
encapsulated in nanomicelles increased by about 5ꢀfold comparing to that of free Gnb. The
augmented halfꢀlife clearly indicates the maximum residence time of drug in the systemic
circulation. Consistently, MꢀNPꢀGnb showed a 7ꢀfold higher accumulation of drug in tumor
tissues compared to that of free Gnb. Overall; PLGA/Hisꢀbased nanomicelles could be a
promising delivery system to increase the therapeutic efficiency of Gnb in lung cancers.

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Acknowledgement
This work supported by Traditional Chinese medicine science and technology development plan
of Shandong Province（No. 2013ꢀ208).
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Figure captions
Figure 1: Synthesis of PLGAꢀPEGꢀNH2, HistidineꢀPEG, PLGAꢀPEGꢀMannose.
Figure 2: Characterization of drugꢀloaded nanoparticles. (a) Particle size distribution of MꢀNPꢀ
Gnb, (b) transmission electron microscope (TEM) of MꢀNPꢀGnb.
Figure 3: Release profile of NPꢀGnb and MꢀNPꢀGnb in phosphate buffered saline (pH 7.4) and
acetate buffered saline (pH 5.5). The release study was performed at 37°C (containing 0.1%
Tween 80).
Figure 4: Cellular uptake study on A549 cells. (a) Fluorescence analysis of NPꢀGnb and MꢀNPꢀ
Gnb in cancer cells. The cellular uptake analysis was performed at various time points (b) flow
cytometer analysis of NPꢀGnb and MꢀNPꢀGnb.
Figure 5: Cellular internalization of NPꢀGnb and MꢀNPꢀGnb in A549 cancer cells. The cells
were stained with Lysotracker Green (green color) and DAPI (blue color).
Figure 6: (a) Cytotoxic effect of free Gnb, NPꢀGnb and MꢀNPꢀGnb in A549 cancer cells. The
cells were incubated for 24h (a) and 48h (b) at 37°C. The cytotoxic effect of individual
formulations was evaluated by means of MTT assay.
Figure 7: Annexin V/PIꢀbased apoptosis assay via flow cytometer (FACS). Annexin V was used
for apoptosis analysis and PI was used for necrosis analysis.
Figure 8: (a) Plasma concentrationꢀtime profile of free Gnb, NPꢀGnb and MꢀNPꢀGnb. The study
was carried up to 24h. The Gnb was administered at a fixed dose of 5 mg/kg via tail vein. (b)
Biodistribution of free Gnb, NPꢀGnb and MꢀNPꢀGnb in tumors at various time intervals up to
24h.

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Table 1: pHꢀsensitive nature of MꢀNPꢀGnb by DLS analysis
pH
8.5
7.4
6.5
5.5
Size (nm)
146.8±2.16
165.7±1.68
210.2±2.61
325.5±4.65
PDI
Zeta potential (mV)
ꢀ24.5±1.65
0.118±0.018
0.157±0.025
0.265±0.034
0.398±0.042
ꢀ18.2±1.12
ꢀ8.32±1.89
ꢀ3.26±1.46

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Figure 1
260x170mm (300 x 300 DPI)

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Figure 2
167x73mm (300 x 300 DPI)

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Figure 3
123x79mm (300 x 300 DPI)

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Figure 4
208x159mm (300 x 300 DPI)

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Figure 5
185x88mm (300 x 300 DPI)

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Figure 6
244x164mm (300 x 300 DPI)

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Figure 7
117x134mm (300 x 300 DPI)