Dual Stimuli-Responsive Vesicular Nanospheres Fabricated by Lipopolymer Hybrids for Tumor-Targeted Photodynamic Therapy

Article abstract of DOI:10.1021/acs.biomac.5b01474

Smart delivery system of photosensitizer chlorin e6 (Ce6) has been developed for targeted photodynamic therapy (PDT). Simple self-assemblies of the mixtures comprising soybean lecithin derived phosphatidylcholine (PC), phosphatidylethanolamine-poly(l-hist

Full text of DOI:10.1021/acs.biomac.5b01474

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Article
Dual Stimuli-Responsive Vesicular Nanospheres Fabricated by
Lipopolymer Hybrids for the Tumor-Targeted Photodynamic Therapy
Johnson V. John, Chung-Wook Chung, Renjith P Johnson, Young-Il Jeong,
Kyu-Don Chung, Dae Hwan Kang, Hongsuk Suh, Hongyu Chen, and Il Kim
Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.5b01474 • Publication Date (Web): 04 Dec 2015
Downloaded from http://pubs.acs.org on December 16, 2015
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Dual StimuliꢀResponsive Vesicular Nanospheres
Fabricated by Lipopolymer Hybrids for the Tumorꢀ
Targeted Photodynamic Therapy
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†,¥
-
‡,¥,
†
∥
⊥
Johnson V. John, Chung Wook Chung,
Renjith P. Johnson, Young-Il Jeong, Kyu-Don
#
‡
§
∇
†,*
Chung, Dae Hwan Kang, Hongsuk Suh, Hongyu Chen, and Il Kim
†
BK21 PLUS Center for Advanced Chemical Technology, Department Polymer Science and
Engineering, Pusan National University, Busan 609ꢀ735, Republic of Korea
‡
Department of Gastroenterology, Pusan National University Hospital, Busan 602ꢀ739, Republic
of Korea)
⊥
Biomedical Research Institute, Pusan National University Hospital, Busan 602ꢀ739, Republic
of Korea
#
Department of Anesthesiology and Pain Medicine, College of Medicine, The Catholic
University, Seoul 137ꢀ701, Republic of Korea
§
Department of Chemistry and Chemistry institute for functional materials, Pusan National
University, Busan 609ꢀ735, Republic of Korea
∇
Division of Chemistry and Biological Chemistry, Nanyang Technological University, 21
Nanyang Link 637371 Singapore
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ABSTRACT
Smart delivery system of photosensitizer chlorin e6 (Ce6) has been developed for targeted
photodynamic therapy (PDT). Simple selfꢀassemblies of the mixtures comprised of soybean
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lecithin derived phosphatidylcholine (PC), phosphatidylethanolamine–poly(
LꢀHistidine) (PE–
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p(His) ) and folic acid (FA) conjugated phosphatidylethanolamineꢀpoly(Nꢀisopropyl
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acrylamide) (PE–p(NIPAM) –FA) in different ratios yield smart nanospheres characterized by
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i) stable and uniform particle size (~ 100 nm), ii) positive surface charge, iii) high hydrophobic
drug (Ce6) loading efficiency up to 45%, iv) covalently linked targeting moiety, v) low
cytotoxicity, and vi) smartness showing p(His) block oriented pH and p(NIPAM) oriented
temperature responsiveness. The Ce6ꢀencapsulated vesicular nanospheres (Ce6@VNS) were
used to confirm the efficiency of cellular uptake, intracellular distribution, and phototoxicity
against KB tumor cells comparing to free Ce6 at different temperature and pH conditions. The
Ce6@VNS system showed significant photodynamic therapeutic efficiency on KB cells than free
Ce6. A receptorꢀmediated inhibition study proved the siteꢀspecific delivery of Ce6 in targeted
tumor cells.
KEYWORDS: Cancer therapy, Drug delivery, Lipopolymer, Photodynamic therapy, Stimuli
responsive, Vesicle
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INTRODUCTION
To date, various cancerous tumors have been treated by conventional anticancer therapies
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such as chemotherapy and photodynamic therapy (PDT). The PDT using photosensitizers (PSs)
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has received more attention as a versatile therapeutic cancer treatment. Specific wavelengths of
light activate the photosensitizer to generate highly reactive singlet oxygen species which nonꢀ
invasively treat diseased tissues, while causing minimal nonspecific damage to normal tissues.
Additionally, the PDT offers easy detection and tracking of tumors both in vitro and in vivo;
fluorescence emission from the photosensitizers aids the imaging and quantification of
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photosensitizers during the treatment. Even though the PDTꢀbased clinical trials are promising,
a few problems such as the weak absorption of PSs in the red region of visible light and their
poor water solubility need to be overcome. Moreover, the majority of PSs have an absorption
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band below 650 nm. Among PDT candidates, chlorin e6 (Ce6) possesses advantages such as a
strong absorption band at 663 nm, low cytotoxicity, and almost no side effects to skin. It
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penetrates deeply into tissue and produces higher reactive oxygen species (ROS).
The selective accumulation of the PSs in tumors strongly dependent on the interaction
between the hydrophobic macrocycle and lowꢀdensity lipoprotein (LDL) in the blood due to the
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highly expressed LDL receptors in the tumor cells. The lipid formulation of PSs has been
attempted to circumvent the drawbacks of reticuloendothelial system (RES) trapping and renal
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clearance of PSsꢀLDL copmlex. Most of the liposomal formulations showed lack of selectivity
and drug leakage during cancer treatment either by chemotherapy or PDT. The leakage of the
drug by Fickian diffusion could not avoid by the incorporation of polyethylene glycol (PEG) into
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nanocarriers (NCs) as a longevity moiety. Additionally, PEG does not provide any selectivity of
drug carriers in delivering therapeutics into the cancer cells. Recently, our group reported a
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poly(
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ꢀhistidine) (p(His)) incorporated lipopolymer for the controlled delivery of doxorubicin
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(Dox). The resulting selfꢀassembled NCs showed a reasonable circulation stability (72 h) and a
fusogenicity, where the pH sensitive p(His) block disrupted the membranes of acidic subcellular
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endosomes because it enhances the endosomal escape. In this aspect, p(His)ꢀbased hybrid
materials have great developmental potential as pH responsive liposomal NCs in therapeutic
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delivery systems.
The multiꢀresponsive delivery vehicles responding pH, temperature, redox, and/or magnet
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have recently emerged as a promising platform for tuning the “smartness” of nanocarriers.
Specifically pH/temperature dual responsive NCs have been widely studied because they exploit
the intrinsic differences in the relative acidity and temperature between solid tumors and the
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surrounding normal tissues. Poly(Nꢀisopropylacrylamide) (p(NIPAM)) has been widely
adopted as a temperature responsive moiety since its lower critical solution temperature (LCST)
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is close to the physiological temperature of the human body.
Accordingly extensive study has
been made for the development of stimuli responsive polymeric drug carriers for the controlled
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delivery of anticancer agents; however, little effort has gone towards developing stimuli
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responsive NCs for the controlled delivery of PSs for PDT.
Along with the stimuli responsive NCs, targeting species decorated NCs enhance the
endocytosis in the controlled delivery of the therapeutics with respect to malignancies. In
particular, the wellꢀknown tumorꢀassociated folate receptor is a good target and is overexpressed
in many tumors of the breast, lung, kidney, and brain due to the high affinity for folate in the
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receptor of cancer cells. Reports have also addressed the importance of cationic liposomes that
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have high affinity for angiogenic endothelial and tumor cells. The cationic liposomes
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significantly enhanced the endocytic uptake of PS into cells in the presence of serum. In
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addition nanoparticles decorated by the zwitterionic phosphoryl choline also showed enhanced
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cellular internalizations than unmodified counterparts.
This high affinity allows
cationic/choline enriched liposomes to deliver the drug into tumor cells, effectively inducing
damage to cancer cells.
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The goal of this study was is to develop a new lipopolymer PDT formulation to
circumvent the drawbacks of the conventional liposomal formulations showing low retention and
low specificity of NCs. For this dualꢀresponsive lipopolymer hybrid systems bearing
phosphatidylcholine (PC), pH responsive phosphatidylethanolamine (PE)
−p(His) conjugate, and
temperature responsive FA conjugated PE p(NIPAM) conjugate have been developed. The
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resulting Ce6ꢀencapsulated vesicular nanospheres (Ce6@VNS) are intensively tested to confirm
the efficiency of cellular uptake, intracellular distribution, and phototoxicity against KB tumor
cells comparing to free Ce6 at different temperature and pH conditions.
EXPERIMENTAL SECTION
Materials. 1ꢀDodecanethiol (98.5%), tetramethylammonium bromide (98.5%), carbon
disulfide (99.9%), triethylamine (TEA, 99.5%), 4ꢀ(dimethylamino)pyridine (DMAP, 99%), 1ꢀ
ethylꢀ3ꢀ(3ꢀdimethylaminopropyl)carbodiimide (EDC, 99.0%), FA (97%), 2,2’ꢀazobis(2ꢀ
methylpropionitrile) (AIBN 98%), propargyl alcohol (98%), 5ꢀbromovalerate (97%),
bromotris(triphenylphosphine)copper(I) (Cu(PPh ) Br), Leupeptin, 2’,7’ꢀdichlorofluorescein
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diacetate (DCFHꢀDA), 3ꢀ[4,5ꢀdimethylthiazolꢀ2ꢀyl]ꢀ2,5ꢀdiphenyltetrazolium bromide (MTT) and
propidium iodide (PI) were purchased from SigmaꢀAldrich and used without further purification.
NꢀIsopropylacrylamide (NIPAM, 97% Acros Organics) was recrystallized from hexane and
solvents such as N,Nꢀdimethylformamide (DMF), 1,4ꢀdioxane, acetone and chloroform were
purified according to standard procedures before use. Ce6 was obtained from Frontier Scientific,
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Inc. (Logan, USA) and NꢀtertꢀbutoxycarbonylꢀNꢀbenzylꢀLꢀhistidine (BocꢀLꢀHisꢀ(Bzl)ꢀOH) was
purchased from Bachem. Fluorescein isothiocyanate (FITC)ꢀAnnexin V was obtained from Santa
Cruz Biotechnology, Inc. (Santa Cruz, USA). Cell culture materials were purchased from
Invitrogen (Carlsbad, USA). PC and PE were simultaneously extracted from soybean lecithin
(Solae Co., South Korea) by using a mixed solvent consisting of ethanol and chloroform
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according to the reported procedure.
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Instrumentation and measurements. H NMR (400 MHz), C NMR (100 MHz), and
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P NMR (161.9 MHz) spectra were recorded on a Varian INOVA 400 NMR spectrometer. The
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temperatureꢀdependent H NMR measurements were performed after the sample tube was
allowed to equilibrate at each preset temperature for approximately 10 min. Chemical shifts are
presented in parts per million (ppm) relative to the residual solvent peaks as the internal standard.
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Peak multiplicities in H NMR spectra are abbreviated as s (singlet), d (doublet), t (triplet), m
(multiplet), and br (broad). Column chromatography was performed using a CombiꢀFlash
Companion purification systems (Teledyne ISCO) using 300−400 mesh silica gel. UVꢀvis
turbidimetry experiments were carried out for LCST determination on a Shimadzu UVꢀ1650 PC
equipped with a temperature controller. Fourier transform infrared (FTꢀIR) spectra were recorded
on a Shimadzu IR prestige 21 spectrometer. The spectra were taken on KBr discs in the range
−
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000−600 cm . The molecular weight (MW) and polydispersity index (Đ) of the polymers were
measured on a Waters GPC system, which was equipped with a Waters 1515 HPLC solvent
pump, a Waters 2414 refractive index detector, and three Waters Styragel High Resolution
columns (HR4, HR2, HR1, with effective MW ranges of 5000−500,000, 500−20,000, and
100−5000 g/mol, respectively) at 40 °C using highꢀperformance liquid chromatography (HPLC)ꢀ
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grade tetrahydrofuran (THF) containing 0.1 N LiBr or HPLCꢀgrade DMF as eluent at a flow rate
of 1.0 mL/min. Monodisperse polystyrenes were used to generate the calibration curve.
Dynamic light scattering (DLS) measurements were carried out using a Nano ZS90 zeta
potential analyzer (Malvern Instruments, Ltd., U.K.) with a He−Ne laser (633 nm), 90°
collecting optics, and a thermoelectric Peltier temperature controller. Block copolymer solutions
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(2 mg/mL) were filtered through a 0.5ꢀꢁm filter prior to use. Particle morphology was analyzed
by transmission electron microscopy (TEM). TEM was performed using a JEOL−1299EX
electron microscope with an accelerating voltage of 80 keV. TEM samples were prepared in
grids with formvar film and treated with oxygen plasma (from a Harrick plasma
cleaner/sterilizer) for 15 s to make their surface hydrophilic. A TEM grid was then floated on top
of the bead with the hydrophilic face in contact with the solution; the aqueous solution was
blotted away with a strip of filter paper and the samples were dried overnight at room
temperature.
Synthesis of benzylꢀNꢀcarboxyꢀLꢀhistidine anhydride (BnꢀHisꢀNCA). BocꢀLꢀHis(Bn)ꢀ
OH (2.5g) was suspended in anhydrous 1,4ꢀdioxane (10 mL), to which a solution of PCl (1.8 g)
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in 1,4ꢀdioxane (20 mL), was added to form BnꢀHisꢀNCA at 25 °C under stirring. Within a few
minutes, a clear solution was obtained and filtered through a glass filter. Crystals of BnꢀHisꢀ
NCA were obtained after the addition of the filtrate to an excess of diethyl ether. The product
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was subsequently washed and dried under vacuum at 50 °C. H NMR (400 MHZ, DMSO d6) δ =
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.78 (d, 2H, HCꢀCH –C), 4.83 (t, 1H, NꢀCHꢀCH –), 5.15 (s, 2H, NꢀCH –C), 6.5–7.4 (m, 2H,
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N=CHꢀN–, NꢀCH=C–), and 7.15–7.4 (m, 5H, ArꢀH) ppm.
Synthesis of PE−p(His) conjugate. To a flameꢀdried nitrogenꢀpurged schlenk tube,
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.69 g (0.1 mmol) PEꢀNH was added under nitrogen, followed by the addition of 5 mL
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anhydrous DMF/Chloroform (3/2 v/v) to solubilize the lipid initiator. A DMF solution (5 mL)
containing predetermined amount of Bn−His−NCA was added to the PE−NH solution using a
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transfer needle under nitrogen, and the reaction mixture was allowed to proceed for 72 h under
nitrogen atm. After polymerization, the reaction solution was concentrated under high vacuum,
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the product precipitated in cold diethyl ether, and dried under vacuum to yield PE
For the deꢀprotection of the benzyl groups, a roundꢀbottomed flask was charged with a solution
of PE p(Bn His) (100 mg) in trifluoroacetic acid (10 mL). To this was added a 4ꢀfold molar
excess of a 33 wt% solution of hydrobromic acid (HBr) in acetic acid in cold diethyl ether to
precipitate PE p(His) . The obtained pale yellow solid was reꢀdissolved in 10 mL of DMF and
−p(Bn−His) .
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further purified by dialysis against deionized water. Water was removed by freezeꢀdrying to
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yield a white solid product. H NMR (400 MHz, DMSO d6) δ (ppm): 0.96−1.63 (br, CH , CH ),
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.3 (br, CH C=O−O), 3.01 (d, nH, CH ), 3.95 (t, nH, CH), 6.87 (br, nH, ArꢀH), 6.97 (br, nH, Arꢀ
2 2
H), 8.10 (br, nH, NH), and 9.35 (br, ArꢀNH) ppm. M = 6800 (by GPC); Đ = 1.24 (by GPC).
n
Synthesis of chain transfer agent (CTA) for reversible additionꢀfragmentation chain
transfer (RAFT) polymerization. Trithiocarbonate CTA, Sꢀ1ꢀdodecylꢀS'ꢀ(α,α'ꢀdimethylꢀα''ꢀ
acetic acid) trithiocarbonate bearing acid functionality was synthesized according to the
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literatures. In brief, 4.04 g of dodecanethiol (20 mmol), 10 mL of acetone, and 0.26 g of
tetrabutyl ammonium bromide (0.8 mmol) were added to a 50ꢀmL flask, and nitrogen gas was
bubbled through the solution for 30 min at 10 °C. An aqueous solution of 1.68 g of 50 wt %
sodium hydroxide (21 mmol) was subsequently slowly added at temperature below 10 °C. After
stirring for 15 min, a carbon disulfide solution in acetone was added in a dropꢀwise manner (CS2:
1.525 g, 20 mmol; acetone: 2.015 g, 34.5 mmol). The system was stirred for another 15 min prior
to the addition of 2.4 mL of chloroform (30 mmol) and 8 g of 50 wt % sodium hydroxide (100
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mmol) at temperature below 10 °C. The ice bath was removed 30 min later, and the reaction was
allowed to proceed for 12 h before the addition of 30 mL of distilled water and 5 mL of
hydrochloric acid (6.8 M). After 30 min, the system was distilled under reduced pressure to
remove the volatile solvents, resulting in the appearance of a yellow precipitate which was
collected by filtration. The precipitate was dissolved in 100 mL of isopropanol under strong
stirring, and the undissolved residue was removed by filtration. The filtrate was subsequently
distilled under reduced pressure to remove isopropanol, and the remaining residue was
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recrystallized in hexane and dried in vacuum for one day. At the end, 4.3 g of trithiocarbonate
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CTA was obtained as pale yellow solid (Yield = 45%) . H NMR (400 MHz, CDCl ) δ (ppm) =
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0.87 (t, 3H, CH C H CH –), 1.26–1.71 (m, 20H, CH C H CH –), 1.72 (s, 6H,–S(CS)S–
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C(CH ) –COOH), 3.28 (t, 2H, CH C H CH –). C NMR (100 MH , CDCl ) δ (ppm) = 14.8,
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22.8, 23.2, 28.9, 30.0, 30.3, 31.1, 32.5, 34.3, 55.1, 172.5, 220.3.
Synthesis of alkyneꢀterminated CTA. Alkyneꢀterminated Sꢀ1ꢀdodecylꢀS'ꢀ(α,α'ꢀ
dimethylꢀα''ꢀacetic acid) trithiocarbonate (CTA−alkyne) was synthesized by esterification of Sꢀ
ꢀdodecylꢀS'ꢀ(α,α'ꢀdimethylꢀα''ꢀacetic acid) trithiocarbonate and propargyl alcohol. Sꢀ1ꢀ
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DodecylꢀS'ꢀ(α, α'ꢀdimethylꢀα''ꢀacetic acid) trithiocarbonate (2 g, 5.5 mmol), EDCꢀHCl (2.12 g,
11 mmol), and DMAP (0.067 g, 0.55 mmol) were mixed in 20 mL of dry CH Cl . Propargyl
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alcohol (0.635 mL, 11 mmol) was added dropwise to the mixture at 0 °C, and the resulting
transparent yellow solution was stirred at room temperature for 48 h. The mixture was
subsequently washed three times with distilled water. The ester was further purified with silica
column chromatography (eluent: a mixture of petroleum ether and ethyl acetate with a volume
1
ratio of 1:1), and the CTA−alkyne was obtained as a plae yellow liquid (Yield = 51%). H NMR
(
400 MHz, CDCl ) δ (ppm) = 0.87 (t, 3H, CH C H CH –), 1.26–1.71 (m, 20H,
3 3 10 20 2
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CH C H CH –), 1.72 (s, 6H,–S(CS)S–C(CH ) –COOH), 3.28 (t, 2H, CH C H CH –). C
3
10 20
2
3 2
3
10 20
2
NMR (100 MHZ, CDCl ) δ (ppm) = 14.8, 22.8, 23.2, 28.9, 30.0, 30.3, 31.1, 32.5, 36.3, 53.4
3
56.1, 76.2, 78.3 172.5, 221.2.
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Synthesis of p(NIPAM) via RAFT polymerization using CTA−alkyne. A Schlenk
tube containing a reaction mixture of NIPAM (1.1760 g, 10.4 mmol), CTA−alkyne (66.1 mg, 0.1
mmol), and AIBN (3.344 mg, 0.02 mmol) in DMF (4 mL) was degassed by three
freeze−pump−thaw cycles, and the solution was stirred at 70 °C under N for 24 h. Precipitation
2
in ethyl ether twice yielded alkyneꢀterminated p(NIPAM) [p(NIPAM) −alkyne] (n = 25, 40, 60)
n
1
as a white powder. H NMR (400 MHz, CDCl ) δ (ppm) = 0.98 (t, 3H, CH C H CH –), 1.28
3
3
10 20
2
(
br, CH(CH ) , 1.30−1.96 (br, 20H, CH C H CH –), 2.52 (br, CH ), 3.47 (br, CH), 3.94 (br,
3 2 3 10 20 2 2
1
3
CH), 4.40 (m, 2H, OCH ), 6.21 (br, NH). C NMR (100 MHz, CDCl ) δ (ppm) = 14.08, 20.9,
2
3
2
1.6 23.1, 23.81, 23.1, 28.9, 30.3, 30.9, 31.7, 32.5, 34.1, 39.4, 40.3,41.3, 45.6, 52.8, 67.5, 174.1,
74.5, 221.7.
1
Synthesis of azideꢀfunctionalized PE. Lecthin PE (0.2 g, 0.25 mmol) and succinimidyl
5
ꢀazidovalerate (0.028 mmol) were dissolved in chloroform (14 mL) in a 100ꢀmL roundꢀbottom
flask with a magnetic stirrer. A solution of TEA (54 mg, 0.67 mmol) in 2 mL of chloroform was
added to the reaction mixture and stirred at 30 °C for 2 h. The reaction mixture was subsequently
washed with water (3 × 30 mL). The solvent was removed under reduced pressure and flash
column silica gel chromatography was performed using an 80:20 mixture of chloroform:
methanol as an eluent for the separation of azideꢀterminated lipids (PE–N ) with yield 64%.
3
Synthesis of PE−p(NIPAM)40−FA conjugate. Together was mixed 0.07 g (0.1 mmol) of
PE
−
N with 10.0 mL of CHCl , 0.41 g of p(NIPAM) −alkyne (M = 4100, 0.1 mmol), and 0.5
3
3
40
n
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mol% of Cu(PPh ) Br. The reaction was allowed to proceed for 24 h under inert atmosphere at
3
3
room temperature. The click conjugation between the p(NIPAM)40 and PE was verified the
−
1
complete disappearance of azide stretching in the FT−IR spectrum at 2230 cm . The reaction
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mixture was passed through silica gel to remove the catalyst and recrystallized in nꢀhexane to
yield a white powder. Aminolysis removal of the thiocarbonyl moiety was accomplished as
2
4
previously described. PE
nitrogen atmosphere in the presence of nꢀhexylamine and tributylphosphine. The resulting thiolꢀ
terminated PE p(NIPAM)40 SH precipitated as a white powder in cold ether, and was dried
−p(NIPAM) was dissolved in chloroform and stirred for 2 h under
40
−
−
under reduced pressure. Its identity was confirmed by the complete disappearance of the RAFT
entity at 308 nm by UVꢀVis. For the conjugation of FA moiety via click reaction, FA was subject
2
5
to esterification to yield allyl folate according to previous literature. The resulting allyl folate
was dissolved in DMF and a solution of PE p(NIPAM)40 SH, and TEA dissolved in DMF was
−
−
added dropꢀwise. The solution was stirred at room temperature for 12 h under nitrogen
atmosphere. The product was precipitated in iceꢀcold ether twice, and the FAꢀconjugated
lipopolymer (PE−p(NIPAM)40−FA) was dissolved in DMF and dialyzed for 6 h using a
membrane with molecular weight limit of 1000 Da, and then was lyophilized.
Formulation and fabrication of dualꢀresponsive NCs. NCs consisting of PE−p(His) ,
40
PE
dialysis. Initially, a dry lipid film (20 mg) consisting of PE
PC was prepared by rotary evaporation of 20 mL of chloroform/ethanol (6:4) solution. The
weight ratio of PC/PE p(His) /PE p(NIPAM)40 FA was modulated to 50/25/25, 60/20/20,
0/15/15, 80/10/10, and 90/5/5, respectively. The formulated film was further dried under high
−p(NIPAM)40
−FA and PC were fabricated by simultaneous heatingꢀcooling followed by
−
p(His) , PE p(NIPAM)40 FA, and
−
−
4
0
−
−
−
40
7
vacuum for at least 4 h to remove solvent traces. 10 mg of film was dissolved in 8 mL mixed
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solvent of ethanol and DMSO (5:1 v/v), then 2 mL water was added dropꢀwise into the solution,
and the resulting solution was heated to 50 °C for 1 h. The clear solution mixture was then
−1
allowed to cool slowly at a rate of 1 °C min in an automated thermalꢀcycler to 25 °C. The
resulting solution was transferred into a dialysis membrane (molecular weight cutꢀoff (MWCO)
of 1000) at 25 °C and dialyzed against 10 mM PBS, pH 7.4. The outer phase was replaced with
fresh buffer solution at 1, 2, 4, 6, and 12 h. After 24 h, the polymers inside the membrane were
selfꢀassembled into mixed vesicular nanosphere (VNS). The solution was subsequently
lyophilized after filtering through an 800 nm syringe filter. The yield (wt %) of VNSs was
calculated by weighing the lyophilized VNS powder.
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To obtain photosensitizerꢀloaded hybrid VNSs, 4 mL aliquots of ethanol/DMSO (5:1 v/v)
was used to dissolve Ce6 (20 mg) and dehydrated film (10 mg) and then combined; for a
preparation of VNSs, the resulting solution was heated to 50 °C for 1 h and cooled slowly at a
−
1
rate of 1 °C min to 25 °C in an automated thermalꢀcycler and then dialyzed against 10 mM
PBS, pH 7.4, at room temperature for 24 h. The final mixed VNSs were used after filtration
through an 800 nm syringe filter (58% yield).
For quantitative analysis of drug encapsulation, aliquots of the drugꢀloaded VNS solution
were lyophilized and dissolved in 2 mL of DMSO and analyzed using UVꢀVis spectroscopy. The
characteristic absorbance of Ce6 (663 nm) was recorded and compared to five point standard
curve of Ce6 from 0 to 100 mg/mL in DMSO. The percentages of DLC and DLE were calculated
using the following equations:
weightꢀofꢀtheꢀdrugꢀinꢀtheꢀVNS
DLCꢀꢁ%ꢂ =
X100
weightꢀofꢀdrugꢀloadedꢀVNS
weightꢀofꢀtheꢀdrugꢀinꢀtheꢀVNS
DLEꢀꢁ%ꢂ =
X100
weightꢀofꢀtheꢀdrugꢀforꢀtheꢀpreparationꢀofꢀdrugꢀloadedꢀVNS
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Cell culture. KB cells were cultured in RPMI 1640 medium (Gibco, Grand Island, NY,
USA) supplemented with 10% fetal bovine serum (FBS) (Invitrogen) and 1% antibiotics at 37 °C
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in a humidified atmosphere of 5% CO . Human normal skin fibroblast CDDꢀ986Sk cells (Korean
2
Cell Line Bank, Seoul, Korea) were cultured in IMEM medium (Gibco) containing 10% FBS
and 1% antibiotics. Cells were subꢀcultured twice per week.
Cytotoxicity of formulated VNSs. KB and CDDꢀ986Sk cells were seeded into 96ꢀwell
plates. For starvation, the cells were incubated in RPMI or IMEM medium containing 0.1% FBS
for 24 h. After removing the medium, the cells were washed with phosphateꢀbuffered saline
(PBS). Next, 100 µL of fresh serumꢀfree RPMI or IMEM medium containing various
concentrations of polymer (0ꢀ100 µg/ml) was added to each well. The cells were then incubated
at 37 °C for 24 h. Finally, the KB or CCDꢀ986Sk cells were washed twice with PBS and the
cytotoxicity was measured using the MTT assay.
Dark toxicity of Ce6 or Ce6@VNS (Ce6@VNS). KB cells were seeded into a 96ꢀwell
plate and treated with various concentrations of Ce6 or Ce6@VNS (0.0–1.0 µM) at both pH 6.6
and 7.4 in serumꢀfree medium for 3 h. After removing the medium, the cells were washed twice
with PBS. To each well, 100 µL of the RPMI medium containing 10% FBS was added and the
cells were incubated for additional 24 h. Cell viability was measured using the MTT assay.
Intracellular uptake of Ce6 or Ce6@VNS. KB cells were seeded into 96ꢀwell plates at a
4
density of 2 × 10 cells per well and incubated for 24 h. The cells were washed twice with PBS
and incubated for 3 h with serumꢀfree medium containing 0.1 µg/mL of Ce6 or Ce6@VNS at
different pH (pH 6.6 or 7.4) or culture temperature conditions (25 or 37 °C). After washing twice
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with PBS, the cells were solubilized in 50 µL of cell lysis buffer (Gen DEPOT, USA). Ce6
fluorescence showed excitation at 407 nm and emission at 664 nm using an Infinite M200 pro
microplate reader (Tecan, Switzerland).
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Fluorescence microscope. Intracellular Ce6 was observed using fluorescence microscopy.
5
Approximately 2 × 10 cells were seeded onto a cover glass in a 6 well plate. The cells were then
treated with 0.5 µg/mL Ce6 or Ce6@VNS for 3 h at different pH (pH 6.6 or 7.4) or culture
temperature conditions (25 or 37 °C) in serumꢀfree media. Afterwards, the medium was
discarded and the cells were washed with PBS. The cells on the cover glass were observed using
fluorescence microscope equipped with a 590 nm excitation filter and a 615 nm filter to detect
Ce6 fluorescence.
Photodynamic therapy. KB cells were seeded into a 96 well plate and cultured for 24 h.
The cells were then washed twice with PBS, and 100 µL of serumꢀfree medium, pH 6.6 or 7.4,
containing 0.1 µg/mL Ce6 or Ce6@VNS was added to each well. The cells were then incubated
for 3 h at either 25 or 37 °C, the medium removed, and the cells washed twice with PBS. Next,
100 µL of fresh serumꢀfree RPMI was added and the cells exposed to an expanded homogenous
2
beam of 664 nm radiation (SH Systems, Korea) at a dose of 1.0 J/cm measured by a photoꢀ
radiometer (DeltaOhm, Italy). After irradiation, the cells were incubated for an additional 24 h.
Cell viability was determined using the MTT assay.
Folic acid inhibition. KB cells were seeded into a 96 well plate and cultured for 24 h.
After washing the cells with PBS, 100 µL of serumꢀfree RPMI media containing folic acid (10
µg/mL) was added to each well and the cells were incubated for 1 h at pH 6.6 and 37 °C. The
medium was then discarded and 100 µL of serumꢀfree RPMI media, pH 6.6, containing folic
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acid (10 µg/mL) and Ce6 or Ce6@VNS (0.1 µg/mL) were added to each cell and further
incubated for 1 h at 37 °C. Intracellular Ce6 uptake or PDT was done as described above.
ROS assay. After irradiation, ROS generation induced by Ce6 was measured using the
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DCFHꢀDA method. In brief, the fluorogenic substrate DCFHꢀDA, a cell permeable dye,
oxidized to the highly fluorescent DCF when exposed to ROS, which was used to monitor
intracellular ROS generation. The cells were treated with Ce6 or Ce6ꢀapsulated VNS (0.1
µg/mL) containing DCFHꢀDA at a final concentration of 20 µM in RPMI media (free of phenol
red) at different pH (6.6 or 7.4) and incubated at 25 or 37 °C for 3 h. Next, the medium was
removed and the cells were washed twice with PBS, and 100 µL of RPMI media (free of phenol
2
red) was added to each well. After irradiation at 1.0 J/cm (664 nm), ROS generation was based
on fluorescence intensity measurements at an excitation of 485 nm and emission of 535 nm using
an Infinite M200 pro microplate reader (Tecan).
Flow cytometry analysis. To evaluate apoptotic and necrotic cells, FITCꢀannexin V and
PI were used, respectively. Following PDT, the uptake of these two reagents was assessed using
a FACScan flow cytometer. KB cells were treated with Ce6 or Ce6@VNS (0.1 µg/mL) in serumꢀ
free RPMI medium at pH 6.6. The cells were incubated at 25 or 37 °C for 3 h, the medium
discarded, the cells washed twice with PBS, and 100 µL of fresh serumꢀfree medium added to
2
each well. The cells were then exposed to a dose of 1.0 J/cm of radiation. Cells were harvested
by trypsinization and washed with PBS. The cells were resuspended in binding buffer (10 mM 4ꢀ
(
2ꢀhydroxyethyl)ꢀ1ꢀpiperazineethanesulfonic acid [HEPES] at pH 7.4, 150 mM NaCl, 5 mM
KCl, 1 mM MgCl , and 1.8 mM CaCl ) containing FITCꢀannexin V (1 µg/mL) and PI (10
2
2
µg/mL), and further incubated for 15 min at room temperature (25 °C) in the dark. The cells
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were immediately analyzed using a FACScan flow cytometer (Becton Dickinson Biosciences,
Franklin Lakes, NJ, USA).
Statistical data analysis. Statistical analyses were performed using analysis of variance
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(ANOVA). All experiments were performed at least three times, and data are presented as mean
± standard error of the mean (SEM).
RESULTS AND DISCUSSIONS
Synthesis of PE−p(His) and PE−p(NIPAM)40−FA conjugates. The synthetic procedures
40
of PE−p(His)40 and PE–p(NIPAM) –FA conjugates are summarized in Scheme 1. A high
40
composition of PC and PE was simultaneously extracted from soybean lecithin by using a mixed
2
2
solvent consisting of ethanol and chloroform. For the synthesis of PE−p(His) the ringꢀopening
4
0
10
polymerization (ROP) of BnꢀHisꢀNCA was performed using PE
of the resulting PE p(BnꢀHis) conjugate by using NMR and gel permeation chromatograph
showed that the block size of BnꢀHis was about 40 units. The benzyl side chains were then
deprotected by treating PE p(BnꢀHis) with HBr/AcOH in trifluoroacetic acid, and the polymer
−NH as an initiator. Analyses
2
−
−
4
0
was characterized by NMR (Figure S1 of supporting information (SI)). The peak integral ratio of
the protons of PE (δ = 0.9–1.6 ppm) and imidazole (a+b+c+d) in the peptide block (δ 7.2 to 8.5
ppm) was used to calculate the number average molecular weight (M ), confirming that there
n
was no loss of the repeating histidine units during the deprotection.
The FA conjugated PE−p(NIPAM)40 was synthesized by combining the RAFT
polymerization with the azideꢀalkyne and thiolꢀene click reactions (Scheme 1). Firstly alkyne
2
7
terminated RAFT chain transfer agent (CTA) was used for the polymerization of NIPAM to
yield p(NIPAM) terminated with alkyne moiety. The resulting compound was subject to click
4
0
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Scheme 1. Synthesis routes of (i) PE–p(His) and (ii) PE–p(NIPAM) –FA.
4
0
40
reaction with PE modified with azide group. The aminolysis of RAFT CTA fragment and the
subsequent removal of the catalyst by column filtration and thiocarbonyl moieties yielded
PE−p(NIPAM) –SH hybrids. Figure 1(A) shows the FT−IR spectra of alkyneꢀterminated
4
0
p(NIPAM) , azideꢀfunctionalized PE, and PE−p(NIPAM) hybrids. In the synthesis of
4
0
40
−1
PE−p(NIPAM)40 hybrids, the azide stretching of PE−N at 2230 cm disappeared in the
3
3
1
PE−p(NIPAM)40 spectra. Additionally, the single P NMR chemical shift at δ = −1.3 ppm
2
8
confirms the successful conjugation of the lipids in p(NIPAM) . Hay et al. reported a strong
4
0
31
isotropic resonance at 1.33 ppm in the
P NMR of dimyristoylphosphatidylꢀ
ethanolamine−p(NIPAM) hybrids obtained by carbodiimide coupling strategy. The removal of
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thiocarbonyl moiety was confirmed by comparing the UV−visible spectra of CTA, p(NIPAM) ,
4
0
PE−p(NIPAM) , and PE−p(NIPAM) −SH, shown in Figure 1(B). The RAFT entity (typical
40
40
absorbance at 308 nm) completely disappeared after aminolysis.
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Figure 1. (A) FT−IR spectra of alkyneꢀterminated p(NIPAM) , azideꢀfunctionalized PE and
4
0
PE−p(NIPAM)40 hybrids, and (B) UV−visible spectra of RAFT CTA, p(NIPAM)40,
PE−p(NIPAM) and PE−p(NIPAM) −SH.
4
0
40
The resulting ω−functional thiol group was utilized for the conjugation of allyl folate as a
targeting ligand by thiol−ene click reaction. The folic acidꢀtethered PE−p(NIPAM)40
lipopolymers were purified by precipitation and lyophilization to remove the nonconjugated allyl
1
folate. H NMR (Figure S2 of SI) spectroscopy was used to confirm the folic acid conjugation in
the PE−p(NIPAM) −SH lipopolymer. The chemical shift at δ = 8.67 ppm shows the presence of
4
0
25
a pteridine ring of the allylꢀfolate in the PE−p(NIPAM) −FA conjugate. The allyl proton of
4
0
allyl folate at δ = 5.29 ppm and 5.64 ppm (–CH=CH ) disappeared after the thiol−ene reaction,
2
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and the proton signals near δ = 6–7.5 ppm of folate merged with the N−H proton of the
p(NIPAM) repeating units. The spectroscopic data reveal the successful attachment of the folic
40
acid ligand to the lipopolymer hybrids. Note that the removal of CTA endꢀgroups is one of the
major issues in the RAFT polymerization, since their decomposition produces malodorous
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sulfurꢀcontaining materials. In this work, we converted the CTA fragment to thiol group to
attach folate for enhance the targetivity of NCs.
Fabrication of NCs. New generation liposomal formulations like PEGylated liposomes may
extend blood circulation time, vary drug distribution in the body, and hence reduce the possible
side effects related to the drugs; however, they displayed severe and harmful toxic effects on
normal organs and tissues in clinical trials, most probably relating to PEGylation. Moreover,
most of the liposomes and liposomal formulations were developed for drug delivery vehicles. As
a means of developing a new pH/temperature responsive and targeted lipopolymer PDT
formulation that may overcome the drawbacks of the conventional liposomal formulations, we
have chosen a system consisting of lecithin oriented PC, PE
−p(His) conjugate where p(His)
block shows pH response, and FA conjugated PE
temperature response.
−p(NIPAM) where p(NIPAM) block shows
NCs consisting of PC, PE
simultaneous heatingꢀcooling followed by dialysis. The dried hybrid films fabricated by
controlling the weight ratio of PC/PE p(His) /PE p(NIPAM)40 FA to 50/25/25, 60/20/20,
0/15/15, 80/10/10, 90/5/5 were dissolved in mixed solvent of ethanol and DMSO, then water
was added into the solution. The turbid solutions were heated to 50 °C for 1 h to obtain clear
−p(His) , and PE−p(NIPAM)40−FA were fabricated by
40
−
−
−
4
0
7
−
1
solution mixtures, then were allowed to cool slowly at a rate of 1 °C min . The resulting
solutions were subjected to dialysis and lyophilization to yield VNS powders. The size and
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morphology of the fabricated hybrid vesicles were characterized by TEM (Figure 2). The
average particle size is around 100 nm. The beneficial properties of liposomes as drug carriers
led to the development of fabrication methods such as extrusion, highꢀenergy sonic
fragmentation, highꢀpressure homogenization, solvent injection, freezeꢀthaw cycling, deꢀ
/rehydration, reverse evaporation, electroꢀformation, solid film dehydration, detergent dialysis,
etc. Different preparation techniques result in different types of liposomes, categorized by
lamellarity (multiꢀ/unilamellar vesicles) and size range [small (≤100 nm), intermediate (100–250
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nm), large (≤250 nm), or giant (> 1 ꢀm)]). Surface charge of vesicles is classified into anionic,
cationic, or neutral base on the chemical structure of lipid used. The lecithin PC is one of the
typical charge neutral phosphatidylcholines.
Figure 2. TEM images of nanocarriers fabricated by PC, PE–p(HIS) , and PE–p(NIPAM) –FA
4
0
40
mixtures composed of different weight ratios: (a) 50/25/25, (b) 60/20/20, (c) 70/15/15, (d)
0/10/10 and (e) 90/5/5.
8
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According to the sizes of the NCs consisting of PC, PE−p(His) and PE−p(NIPAM)40−FA,
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the vesicles obtained here can be categorized as small range. The size of the hybrid VNSs in
different formulations was also confirmed by DLS (Table 1). As the relative composition of PC
increases from 50% to 90%, the average particle size decreases from 125 nm to 97 nm. Note that
the particle size distribution is also reasonably narrow. In fact the undesirable and irreproducible
distribution of liposomes fabricated by pure phospholipids is one of the critical factors of
importance in industrial applications. The lamellarity of the NCs is ambiguous, because of the
participation of the long p(His) and p(NIPAM) chains in forming vesicles. In fact, the vesicles
are quite different from those fabricated by pure PC, so that they are much like micelles (vide
infra).
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Table 1. Compositions, size and physical properties of cationic dual responsive nanocarriers.
Composition
Ce6
Ce6
DLE
(%)
45
Average particle ζ potential (mV)
[
b]
Sample
PC/ PE–p(His) /PE–
DLC
(%)
4
0
[a]
size (nm)
at pH 6.6
p(NIPAM) –FA
4
0
A
B
C
D
5.0/2.5/2.5
6.0/2.0/2.0
7.0/1.5/1.5
8.0/1.0/1.0
125±8
119±5
108±10
100±6
97±10
+24.3
+18.1
+12.2
+10.3
+3.9
8.67
7.20
5.91
3.21
2.86
38
32
25
E
9.0/0.05/0.05
19
[
a]
[b]
Determined by DLS. Drug (Ce6) loading content and drug loading efficiency.
Careful investigation of the TEM images of the NCs in Figure 2 shows an interesting
transformation of the lipid hybrid NCs from VNSs to liposome vesicles. Conjugating polymer
chains into the hydrophilic head leads to the creation of vesicles different from uniꢀ and
multilamellar phospholipid vesicles (liposomes) that are spherical softꢀmatter particles consisting
of one or more bilayer membrane(s), encapsulating a volume of aqueous medium. The VNSs
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obtained by PC, PE–p(His) , and PE–p(NIPAM) –FA with 50/25/25 ratio are semisolid
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spheres, wherein the aqueous lipid phase is replaced by macromolecular constituents. Figure 3
illustrates the transformation of the VNSs according to the variation of composition. Considering
both inside and outside of the phospholipid membranes are covered by hydrophobic p(His) and
hydrophilic p(NIPAM) constituents, the aqueous volume is reduced to a minimum, giving rise to
steric interactions between neighboring vesicles and thus to a semisolid consistency. As a result
the VNSs in this condition are much like liposome gels, wherein hydrophobic polymer forms
embedding and hydrophilic polymer form encapsulating matrices.
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The macromolecular constituents originated packing density decreases accordingly as the
amount of them decreases. As expected, minimizing the amount of PE–p(His) , and PE–
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p(NIPAM) –FA conjugates during the micellization, say PC, PE–p(His) , and PE–
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40
p(NIPAM) –FA in 90/5/5 ratio, resulted in predominantly hollow vesicles (Figure 2(e)). On the
4
0
other hand stable VNS could not be achieved by increasing the amount of PE–p(His) , and PE–
40
p(NIPAM) –FA conjugates higher than 50%, due to severe steric interactions preventing
4
0
phospholipids and their fragments from spontaneous selfꢀassembly.
Electrostatic interaction is an important secondary force affecting the structure, stability,
and function of liposomes. In addition to particle size and its distribution and shape surface
charge plays a key role in the cellular uptake of liposomes, since the uptake of them by cells is
proceeded by a twoꢀstep process: first, a binding step on the cell membrane and second, the
internalization step. Furthermore, surface charge affects not only the stability of dosage forms
and their release rate but also their circulation in the blood value stream and absorption into body
membranes. Table 1 shows zeta potential (ξ) values of the VNSs that are associated with a
realistic magnitude of surface charge. The ξ value decreases monotonously from +24.3 (PC/PE–
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Figure 3. (A) Diagrammatic representation of VNS bearing Ce6 fabricated by PC, PE–p(His) ,
4
0
and PE–p(NIPAM) –FA mixtures; (B) morphological transition of VNS from semisolid VNSs
4
0
to liposomes by modulating relative composition of PC, PE–p(His) , and PE–p(NIPAM) –FA.
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p(His) /PE–p(NIPAM) –FA = 50/25/25) to +3.9 (PC/PE–p(His) /PE–p(NIPAM) –FA =
4
0
40
40
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90/5/5), as the PC ratio increases, demonstrating the nanoꢀsuspension stability of VNSs
decreases as the PC ratio increases. It is interesting to note that all the vascular structures are
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characterized by cationic surface charge. Positive ξ value is important for drug delivery since it
can facilitate effective adhesion to cancer cell surfaces, which are usually charged negatively due
to the translocation of negatively charged constituents of the inner layer of the cell membrane
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(
e.g., phosphatidylserine, anionic phospholipids, glycoproteinsand proteoglycans) , prolonging
the drug release and enhancing the drug bioavalibility in the internal tissue due to the
electrostatic interaction.
Conventional liposomes prepared by the selfꢀassembly of neutral phospholipids are
structurally unstable, have low drug loading capabilities and tend to leak the internalized drugs
through their boundary. Sterically stabilized or PEGꢀylated liposomes, so called stealth
liposomes, were reported to provides stabilization enhancement and additional protection of the
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encapsulated substances.
Likewise, the VNSs fabricated from the mixture of PC, PE–
p(His) , and PE–p(NIPAM) –FA are expected to show stealth structure and peculiar
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functionality, since the pHꢀsensitive and hydrophobic p(His) chains are embedded inside of the
structure and temperatureꢀsensitive p(NIPAM) chains are located outside of the structure.
Considering the attractive PDT drug candidate Ce6 is hydrophobic and big molecule, the VNSs
are expected to show different DLC and DLE values in encapsulating Ce6. The surface charge of
the particles and binding type between the drug and nanoparticles are the important parameters
that determine the rate of desorption of the drug in the nanoparticles and drug loading
3
3, 34
efficiency.
Table 1 summarizes those values of VNSs obtained from different compositions. The DLC
value of VNSs fabricated by PC/PE–p(His) /PE–p(NIPAM) –FA mixtures in 50/25/25 and in
40
40
90/5/5 ratios is 8.67% and 2.96%, respectively, showing nearly 3ꢀfold difference. The value
decreases monotonously as the PC ratio increases. The increase of p(His) chains inside of VNS
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may increase the van der Waals interaction and πꢀπ stacking between p(His) fragments and Ce6
molecules. Note that PSs loaded cationic liposomes have high affinity for angiogenic endothelial
1
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and tumor cells.
In addition Figure S3 shows the absorption and fluorescence spectra of the
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Ce6 loaded PC/PE–p(His) /PE–p(NIPAM) –FA (50/25/25) VNS. The peak at 663 nm in both
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spectra shows the drug loading ca of the system. The fluorescence spectra also demonstrate the
o
pH responsive release of Ce6 from the VNS at 37 C. The enhanced release of Ce6 may occur
not only by the pHꢀinduced destabilization but also by the thermoresponsive behavior of our
o
VNS at 37 C.
Time dependent release of Ce6. The time dependent releases of Ce6 were evaluated at
different temperatures and pHs in phosphate buffered saline (PBS, pH 7.4) by using UVꢀVis
spectroscopy. Note that the absorption maximum of Ce6 appears at 663 nm in solution The Ce6ꢀ
loaded VNS fabricated by PC/PE–p(His) /PE–p(NIPAM) –FA in 5.0/2.5/2.5 ratio was chosen
4
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40
as a representative sample because it showed the highest DLE and DLC values. The release
performance was observed for 50 h at different temperatures and the resulting profiles are shown
in Figure 4. Comparing the release of Ce6 at 37 °C (∼55% release in 50 h) to that of Ce6 at 25
°
C (~ 15% release in 50 h), it is evident that the release is accelerated around the transition
temperature of the p(NIPAM) block. Accordingly the drug release obtained at 41 °C showed a
o
conspicuous initial bursting of Ce6 (~35%) that is comparable to that (~11%) obtained at 37 C.
Thus, the drug release tests at far above LCST of the VNSs may not show any beneficial effects
o
and detailed investigations were not performed at higher temperature than 37 C. The
temperature sensitivity of the VNSs is reflected by variations of the p(NIPAM) block that shows
a LCST around our body temperature from the hydrophilic to the hydrophobic state in response
to temperature. The hydrophobicity of the p(NIPAM) block induces low solubility of the
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polymer in water by breaking the hydrogen bond of the amide functionality in p(NIPAM) chain,
destabilizing the VNSs. Therefore, the LCST of p(NIPAM) chains clearly affect the Ce6 release
from VNS owing to the thermal transition of p(NIPAM) chain.
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Since tumor tissues contain large, acidic outside plasma pH gradients, while normal tissues
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generally have alkaline outside pH gradients , the extracellular pH of tumors is significantly
3
6
lower than that of normal tissues under physiological conditions . Thus, the pHꢀresponsive
vesicles should show improved fusogenic potential to an endosome mimicking anionic
membrane at acidic pH. Considering the ξ value of the VNSs of this study is positive under
acidic pH conditions and the size of the particle is around 100 nm, the cationic liposomes should
adhere to the negatively charged tumor cell membrane and accelerate the intracellular uptake by
37
epithelial cells. For the effective PDT, vesicles bearing Ce6 should preferably release Ce6 in
the targeted tumor cells. In vitro release rates of Ce6 were investigated by a set of timeꢀ
dependent release experiments at different pHs 37 °C (Figure 4(B)). About 52% of the
incorporated Ce6 is released within 50 h at pH 7.4. The release rate is accelerated at acidic pH
conditions, thus, the value increases to 58, 73, and 82% at pH 7, 6.6, and 6.0, respectively. The
accelerated rate, especially at pH 6.0, might be related to pKa (∼6.0) of the imidazole side chain
of the p(His) block. These data clearly demonstrate the release rate is sensitive to pH due to the
pHꢀinduced destabilization of the p(His) block within the VNSs. Because the p(His) block is
ionizable under acidic conditions, its swelling behavior greatly depends on the pH due to the
ionizationꢀdeionization of the imidazole ring of the p(His) block. Under acidic conditions, the
p(His) block is ionized and the charged imidazole groups repel each other leading to high
swelling.
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Figure 4. Timeꢀdependent releases of Ce6 from the VNS fabricated by PC/PE–p(His) /PE–
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0
p(NIPAM) –FA (50/25/25) estimated (A) at different temperatures (pH = 7.4) and (B) at
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0
different pH values (temperature = 37 °C).
Cytotoxicity of VNSs fabricated by PC/PE–p(His) /PE–p(NIPAM) –FA hybrids.
40
40
Above results showed the VNSs fabricated by PC/PE–p(His) /PE–p(NIPAM) –FA are
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characterized by i) stable and small particle size (~ 100 nm) and narrow particle size distribution,
ii) positive surface charge, iii) high hydrophobic drug (Ce6) loading efficiency, iv) covalently
linked targeting moiety, and v) smartness, in which the drug release is responsive to temperature
as well as pH. Accordingly the VNSs of this study have some pros in targeted drug delivery.
3
8
However, positively charged lipids are not approved by FDA for clinical use and in some cases
3
9
cationic charge is claimed to enhance cytotoxicity . Positive charge shows a membrane
destabilization and concomitantly destructive effect resulting from an interaction of positive
4
0
charge and negative charge of membrane. The biocompatibility of the newly formulated VNSs
was tested against human fibroblast CCDꢀ986Sk cells and human oral squamous carcinoma KB
cells. As shown in Figure 5, the viability of the two kinds of cells was higher than 80% in a
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polymer concentration range from 0 to 10 µg/mL over 24 h of cultivation, even though
considerable cell cytotoxicity was observed at very high concentrations like 100 µg/mL that is
not practical for in vitro and/or in vivo study. The result reveals that the VNSs show substantial
biocompatibility in cell culture and have no acute and intrinsic cytotoxicity against carcinorma
(KB) and normal (CCDꢀ986Sk) cells.
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The dark toxicity of free Ce6 and Ce6@VNS was also studied using KB cells. As shown in
Figure 6, no significant dark toxicity is observed in the presence of less than 1.0 µg/mL Ce6 or
Ce6@VNS. Additionally, pH has no effect on cell viability. These results indicate that drug,
polymer, and culture conditions have no effect on cell survival.
Figure 5. Intrinsic cytotoxicity of VNSs fabricated by PC/PE–p(His) /PE–p(NIPAM) –FA
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0
40
mixture in different formulations against (A) CCD986ꢀSk and (B) KB cells. After starvation for
8 h, the cells were treated with various concentration of polymer micelle. Cell viability was
revealed as percent of control. In each concentration PC/PEꢀp(His) /PEꢀp(NIPAM) ꢀFA ratio is
1
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corresponding to 50/25/25, 60/20/20, 70/15/15, 80/10/10 and 90/5/5 in order from the left.
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Figure 6. Dark toxicity of (A) free Ce6 and (B) Ce6@VNS (PC/PE–p(His) /PE–p(NIPAM) –
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FA = 50/25/25) against KB cells; KB cells were incubated with 0ꢀ1 µg/mL Ce6 or Ce6@VNS in
the dark at different pH conditions for 3 h. Cell survival was expressed as the percentage of the
control (not exposed to Ce6 or Ce6@VNS) and was determined using the MTT assay.
Intracellular uptake of Ce6 or Ce6@VNS. The uptakes of free Ce6 and encapsulated
Ce6 inside of Ce6@VNS in KB cells were also investigated. As shown in Figure 7, the two
systems show a dramatic difference in the degree of intracellular uptake of Ce6. Incubating KB
cells with 0.1 µg/mL of Ce6 or Ce6@VNS for 3 h at different pH (pH 6.6 or 7.4) and culture
temperatures (25 or 37 °C), the amount of free Ce6 inside of the cell is almost negligible;
however, considerable amount of encapsulated Ce6 inside of Ce6@VNS is observed inside of
the cell. In addition Ce6 molecules are more efficiently delivered inside of the cells at low pH
and 37 °C. Note that the KB cell membrane is most probably in acidic condition. Evidently the
dramatic increase of intracellular Ce6 delivery must be related with the features of Ce6@VNS:
i.e. suitable size (~ 100 nm) and size distribution, stability, cationic surface charge, specificity
(or targetability), and smartness responding to pH and temperature. The efficient delivery of Ce6
is a prerequisite condition to perform an efficient PDT.
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