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Journal of Biomaterials Science, Polymer Edition Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tbsp20

A functional drug delivery platform for targeting and imaging cancer cells based on Pluronic F127 a

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Denghao Zhang , Liang Tao , Hongli Zhao , Huihui Yuan & Minbo Lan

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Shanghai Key Laboratory of Functional Materials Chemistry, and Research Centre of Analysis and Test, East China University of Science and Technology, Shanghai 200237, P.R. China b

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State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, P.R. China Accepted author version posted online: 17 Mar 2015.Published online: 08 Apr 2015.

To cite this article: Denghao Zhang, Liang Tao, Hongli Zhao, Huihui Yuan & Minbo Lan (2015) A functional drug delivery platform for targeting and imaging cancer cells based on Pluronic F127, Journal of Biomaterials Science, Polymer Edition, 26:8, 468-482, DOI: 10.1080/09205063.2015.1030136 To link to this article: http://dx.doi.org/10.1080/09205063.2015.1030136

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Journal of Biomaterials Science, Polymer Edition, 2015 Vol. 26, No. 8, 468–482, http://dx.doi.org/10.1080/09205063.2015.1030136

A functional drug delivery platform for targeting and imaging cancer cells based on Pluronic F127 Denghao Zhanga, Liang Taoa, Hongli Zhaoa*, Huihui Yuana and Minbo Lana,b*

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a Shanghai Key Laboratory of Functional Materials Chemistry, and Research Centre of Analysis and Test, East China University of Science and Technology, Shanghai 200237, P.R. China; bState Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, P.R. China

(Received 18 December 2014; accepted 13 March 2015) Functional polymeric micelles play an important role in the efficient delivery of therapeutic drugs into tumours. In this study, a functional drug delivery platform with ligands for targeting and fluorescent imaging was designed based on Pluronic F127 (PF127). Using folic acid (FA) and fluorescein isothiocyanate (FITC) to chemically conjugate with PF127, two functional polymers, Pluronic F127-FA (PF127-FA) and Pluronic F127-FITC (PF127-FITC), were synthesized. Solasodine-loaded micelles were then prepared via the thin-film hydration method. By employing A549 and HeLa cells, the results of in vitro cell assays performed using confocal laser scanning microscopy and flow cytometry suggested that the proposed micelles could provide the desired specific targeting and fluorescent imaging functions. In addition, the results of in vitro cytotoxicity experiments showed that the growth inhibition rates of A549 and HeLa cells treated with solasodine-loaded micelles were remarkably higher than those of cells treated with free solasodine. Solasodine-loaded micelles exhibited a more distinct inhibitory effect against HeLa cells than against A549 cells. Thus, an effective drug delivery system for targeting and imaging cancer cells was developed. Keywords: Pluronic F127; fluorescent imaging; targeted delivery; functional micelles; solasodine

1. Introduction The past decades have witnessed distinguished developments, improvements and breakthroughs in drug research and drug delivery system (DDS) for cancer therapies.[1–5] To date, many classical and successful nanomaterials used to transport anticancer drugs into tumours have been established and implemented, the most prominent among which include polymeric nanomaterials [6–8] (nanoparticles, nanogels, nanocapsules, and polymeric micelles), liposomes [9,10] and dendrimers.[11–13] Among these materials, polymeric micelles with a core-shell structure, typically formed by the self-assembly of amphiphilic polymers, are garnering increasing attention.[14,15] The core-shell configuration is indispensible to increasing drug stability and drug solubility as well as providing a prolonged circulation period and preferential accumulation in tumours via the enhanced permeability and retention effect after administration.[16–19] *Corresponding authors. Email: [email protected] (H. Zhao); [email protected] (M. Lan) © 2015 Taylor & Francis

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The Pluronic (also known as Poloxamer or Synperonic) series is composed of ethylene oxide and propylene oxide in the following triblock copolymer structure: PEOx-PPOy-PEOx.[20–22] When dissolved in aqueous media, Pluronic unimers form micelles by self-assembly. The core of the micelles consists of hydrophobic PPO blocks and the shell consists of hydrophilic PEO chains. Micelles can solubilize poorly soluble drugs and impart better stability and drug pharmacokinetics and biodistribution, allowing for enhanced antineoplastic efficacy. Regarding the safety of Pluronics micells for humans, intensive studies have been performed on the micelles as a DDS via different routes, such as intravenous, subcutaneous, intraperitoneal, and intramuscular routes.[23,24] Specifically, five Pluronic copolymers have been approved by the FDA and are listed in the US Pharmacopoeia, namely Pluronic F127, F108, F87, F68, and L44.[25,26] Because of their poor solubility in aqueous media, many anticancer drugs, such as Paclitaxel and Doxorubicin, can’t exhibit a satisfactory anticancer effect, despite their excellent therapeutic properties in inhibiting and killing cancer cells. Thus, it is urgent that an operative method for overcoming these deficiencies be established. Several formulations based on Pluronic micells have been developed for clinical uses, such as SP1049C (Canada),[27,28] SP1017 (Canada),[29,30] Fluosol-DA (Japan) [31] and RheothRx (Canada).[32,33] Solasodine, a type of steroidal alkaloid, is extracted from members of the solanum genus, such as potatoes and tomatoes, and exhibits excellent bioactivities against fungi, viruses, and especially tumours.[34–36] However, free solasodine shows no remarkable anticancer efficacy against tumour cells because of its poor solubility and passive endocytosis. For example, it has been reported that the rates of cell growth inhibition of solasodine against human cervical cancer cells (HeLa) and human breast adenocarcinoma cells (MCF-7) at a concentration of 4.1 μg mL−1 were 7 and 33%, respectively while 9 and 24% at a concentration of 41 μg mL−1, respectively.[36] Considering the above-described advantages, the use of Pluronic micelles may be an effective method for improving the antitumour efficacy of solasodine by fabricating solasodine-loaded Pluronic micelles. In this study, a functional solasodine loaded delivery system based on PF127 was developed. The system exhibited the dual-functions of targeting and fluorescent imaging tumour cells by chemically conjugating FA and fluorescein isothiocyanate (FITC) with PF127. A series of in vitro cell assays were further performed to evaluate the biological activities of solasodine-loaded micelles.

2. Materials and methods 2.1. Materials 2.1.1. Reagents Solasodine was kindly provided by Shanghai Pharmaceutical Technology Co. Ltd (Shanghai, China). Pluronic F127 (PF127), 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) and 6-diamidino-2-phenylindole (DAPI) were purchased from Sigma-Aldrich (USA). Penicillin–streptomycin, Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum and a 0.25% (w:v) trypsin–0.03% (w:v) EDTA solution were purchased from Gibco (Gaithersberg, MD, USA). Folic acid (FA), FITC, 1-ethyl-3[3dimethylaminopropyl] carbodiimide hydrochloride (EDC), dimethyl sulfoxide (DMSO), tetrahydrofuran (THF) N,N′-carbonyldiimidazole (CDI) and N-hydroxysuccinimide (NHS)

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were obtained from Sinopharm Chemical Reagent Co. Ltd. All other reagents employed were of analytical grade and used without further purification. 2.1.2. Cell lines Human non-small lung cancer cells (A549), human cervical cancer cells (HeLa) and human normal bronchial epithelial cells (16HBE) were purchased from the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (Shanghai, China).

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2.2. Synthesis of functional copolymer PF127-FA and PF127-FITC 2.2.1. Synthesis of Pluronic F127-NH2 (PF127-NH2) PF127-NH2 was synthesized as an intermediate for the preparation of PF127-FA and PF127-FITC according to a method reported in a previous study.[37] PF127 was purified by dissolving in acetone and pouring the solution into excessively cooled hexane to obtain a purified white powder for the reactions. PF127 (1.2604 g, 1.00 × 10−4 mol) was dissolved in dry THF (20 mL), resulting in a clear solution. Excess CDI (0.1644 g, 1.00 × 10−3 mol) in dry THF (25 mL) was dropped into the PF127 solution under nitrogen with a peristaltic pump at room temperature. After the reaction, the solution was treated with dialysis (a molecular weight cut-off of 8000–12,000 g mol−1) to remove the compounds with low molecular weight, such as CDI. The dialysis was performed against ultrapure water for 72 h, during which the water was replaced every 6 h for the first 18 h and then changed every 18 h, followed by lyophilization. Pluronic F127-CDI (PF127-CDI) was obtained as a white powder. Furthermore, PF127-CDI (0.6350 g, 5.00 × 10−5 mol) dissolved in dry THF (20 mL) was added dropwise into THF containing 1,2-ethylenediamine (4.500 g, 7.50 × 10−2 mol) with a peristaltic pump. After the reaction, the solution was treated by dialysis and lyophilization. 2.2.2. Synthesis of PF127-FA PF127-FA was prepared according to a method described in a previous study, with modifications.[37,38] Briefly, FA (0.2636 g, 6.00 × 10−6 mol) was slowly added to DMSO (25 mL) under sonication to facilitate dissolution, then, PF127-NH2 (0.2525 g, 2.00 × 10−5 mol) in DMSO (10 mL), EDC (0.2750 g, 1.34 × 10−4 mol), NHS (0.0160 g, 1.39 × 10−4 mol), and triethylamine (0.3650 g, 3.60 × 10−3 mol) were added sequentially into DMSO containing FA under magnetic stirring in the dark for 24 h. The mixture was treated by dialysis and lyophilization. 2.2.3. Synthesis of PF127-FITC PF127-NH2 (0.2519 g, 2.00 × 10−5 mol) was dissolved in dry THF (10 mL), FITC (0.0143 g, 4.00 × 10−5 mol) in dry THF (10 mL) was then added dropwise. The mixture was maintained under magnetic stirring for 12 h in the dark. PF127-FITC was obtained by dialysis and lyophilization. 2.3. Preparation of solasodine-loaded micelles The classic thin-film hydration method was utilized to prepare polymeric drug loaded micelles. Typically, PF127 (0.0950 g, 7.54 × 10−6 mol), PF127-FA (0.0025 g,

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1.90 × 10−7 mol), PF127-FITC (0.0025 g, 1.90 × 10−7 mol) and solasodine (0.001 g, 2.42 × 10−6 mol) were dissolved completely in 20 mL CHCl3 in a round-bottom flask, followed by rotary evaporation to remove CHCl3 to form a thin drug-polymer film. Residual CHCl3 was further removed under vacuum at room temperature overnight. The thin film was hydrated with 5 mL ultrapure water, and the solution was sonicated to prepare solasodine loaded-micelles. Finally, the micelles were filtrated through a hydrophilic filtration membrane (0.45 μm) to remove the unloaded solasodine, followed by lyophilization. The blank micelles were prepared by the same process. The content of solasodine in the micelles was determined by the high-performance liquid chromatography (HPLC) method on a C18 Eclipse-XDB column (5 μm, 250 × 4.6 mm) with a mobile phase consisting of acetonitrile and water (89:11, v:v) at a flow rate of 1.0 mL min−1. The detection wavelength was selected to be 210 nm, and solasodine content was quantified according to the standard curve obtained previously. The drug loading content (DLC) and drug loading efficiency (DLE) were calculated by the following equations: Weight of drug in micelle  100% Weight of drug loaded micelle

(1)

Weight of drug in micelle  100% Weight of drug added into micelle

(2)

DLC ð%Þ ¼ DLE ð%Þ ¼

2.4. Cellular uptake in vitro Confocal laser scanning microscopy (CLSM) and flow cytometry were employed to evaluate the efficacy of cellular uptake for the micelles composed of equimolar amounts of PF127-FA and PF127-FITC. In detail, for the CLSM study, A549 and HeLa cells were seeded onto aseptic glass coverslips in a six-well culture plate at a density of 5.0 × 104 well−1 and incubated for 24 h for thorough attachment. Then, the cells were treated with micelles containing equimolar amounts of PF127-FA and PF127-FITC (200 μg mL−1) for 4 and 24 h, respectively. The cells were washed three times with cold PBS and fixed with 4% paraformaldehyde in PBS for 30 min, following treatment with 1% Triton X-100 and DAPI (5 μg mL−1) for 10 min each at room temperature. Finally, the cells were mounted and observed using CLSM on the DAPI channel (blue) and on the FITC channel (green) with an excitation wavelength of 488 nm and an emission wavelength of 520 nm. For the flow cytometry study, A549 and HeLa cells were seeded in six-well culture plates at a density of 1.0 × 106 well−1 in 1.5 mL medium and cultured for 4 or 24 h. The cells were then treated with micelles containing equimolar amounts of PF127-FA and PF127-FITC (200 μg mL−1) for 4 or 24 h, respectively. In addition, cells treated with pure medium served as the control. The culture medium was discarded, and the cells were washed with PBS three times. The cells were subsequently trypsinized and obtained by centrifugation. Finally, the cell pellets were resuspended in PBS for the subsequent measurement of fluorescence intensity on a flow cytometer with an excitation wavelength of 488 nm and an emission wavelength of 520 nm.

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2.5. FA competition assays FA competition assays were performed to confirm FR-mediated endocytosis. CLSM and flow cytometry were used to analyse the samples. For the CLSM assay, HeLa cells were seeded onto aseptic glass coverslips in a sixwell culture plate at a density of 5.0 × 104 well−1 and incubated for 24 h for thorough attachment. HeLa cells without any treatment were set as the control. Sample S1 was treated with micelles without PF127-FA. S2 and S3 were treated with micelles containing equimolar amounts of PF127-FA and PF127-FITC (200 μg mL−1, respectively) as well as various concentrations of free FA (100 and 1 μg mL−1, respectively). S4 was treated with micelles containing equimolar amounts of PF127-FA and PF127-FITC (200 μg mL−1, respectively) for 24 h. Finally, the cells were washed three times with cold PBS and fixed with 4% paraformaldehyde in PBS for 30 min, following treatment with 1% Triton X-100 and DAPI (5 μg mL−1) for 10 min each at room temperature. Finally, the cells were mounted and using a CLSM on the DAPI channel (blue) and on the FITC channel (green) with an excitation wavelength of 488 nm and an emission wavelength of 520 nm. For the flow cytometry assay, HeLa cells were seeded in six-well culture plates at a density of 1.0 × 106 well−1 in 1.5 mL medium and cultured for 24 h. The five samples were treated by the same procedure as CLSM assay. Then the cells were trypsinized and obtained by centrifugation. Finally, the cell pellets were resuspended in PBS for the subsequent measurement of fluorescence intensity on flow cytometer with an excitation wavelength of 488 nm and an emission wavelength of 520 nm. 2.6. Cytotoxicity assay The 16HBE and A549 cells were seeded in a 96-well cell culture plate at a density of 8 × 103 well−1 in 200 μL and cultured for 24 h under a 5% CO2 atmosphere at 37 °C. The cells were subsequently incubated with PF127, PF127-FA, and PF127-FITC at the same concentrations ranging from 62.5 to 1000 μg mL−1. Cell viability was measured using an MTT assay after 24 or 48 h. A microplate reader was employed to measure the absorbance at 492 and 630 nm. 2.7. Characterization of the materials The content of solasodine loaded in the micelles was measured by HPLC (Agilent 1260, USA). 1H NMR analysis was conducted on a nuclear magnetic resonance spectrometer (Bruker, Avance 400 MHz, Germany). The infrared adsorption spectrum of each polymer was measured using Fourier transform infrared spectroscopy (FT-IR, Thermo Fisher Scientific, Nicolet 6700, USA). The diameter and zeta potential were measured via dynamic light scattering (DLS, Beckman Coulter, DelsaTM Nano analyzer, USA). The micelle morphology was further observed using a high-resolution transmission electron microscope (HR-TEM, JEOL, JEM-2100, Japan), with a beam energy of 200 kV and equipped with a Gatan camera. The micelle solution was deposited in a dropwise manner onto a copper grid, dyed with 2 wt% phosphotungstic acid aqueous solution three times and air-dried for observation. The relative colour intensity of the formazan crystals was measured on a microplate reader (ThermoFisher Scientific Multiskan MK3, USA). Flow cytometry analysis was performed using a flow cytometer (BD, FACSAria, USA). Confocal analysis was performed on a confocal laser scanning microscope (CLSM, Nikon A1R, Japan).

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Scheme 1. Schematic illustration of the synthesis of PF127-CDI, PF127-NH2, PF127-FA and PF127-FITC.

3. Results and discussion 3.1. Synthesis and characterization of PF127-FITC and PF127-FA Two functional copolymers, PF127-FITC and PF127-FA, with the capacities of fluorescence imaging and targeting capacities, respectively, were synthesized by conjugating FA and FITC with PF127. The synthetic processes are shown in Scheme 1. The chemical structures of the copolymers were characterized by 1H NMR (400 MHz, DMSO-d6, ppm) and FT-IR, respectively. Figure 1 shows that, compared with the 1H NMR

Figure 1. 1H NMR spectra (400 MHz, DMSO-d6) of PF127 (A), PF127-FITC (B) and PF127FA (C). The insert in the upper left corner shows partially magnified spectra for PF127 (A1), PF127-FITC (B1) and PF127-FA (C1).

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FT-IR spectra of PF127 (A), PF127-FITC (B) and PF127-FA (C).

spectrum of PF127 (Figure 1(A)), new peaks can be found in the spectra of PF127NH2, PF127-FITC and PF127-FA (inset of Figure 1). A new peak at δ 4.51 for Ho belonging to –NH- in PF127-NH2 appears (B1 of the inset of Figure 1). As shown in the 1H NMR spectrum of PF127-FITC (C1 in the inset of Figure 1), new peaks near δ 6.50–7.00 ppm are attributed to the Hi and Hj aromatic protons of two phenyl groups in FITC, and the peak at δ 7.00–7.50 ppm for Hh belongs to aromatic protons of the phenyl group connected to –NH2 in FITC. Similarly, in the 1H NMR spectrum of PF127-FA (D1 in the inset of Figure 1), new characteristic peaks emerged from protons in the FA structure, primarily at δ 4.45 ppm for Hq, δ 6.60 ppm for Hp, δ 7.59 ppm for Hn and δ 8.62 ppm for Hm. In the study, PF127-FA and PF127-FITC were prepared successfully. The FT-IR spectra of PF127, PF127-NH2, PF127-FITC and PF127-FA are shown in Figure 2. Compared with curve A for PF127, the new absorption peak observed at 1717.49 cm−1 belongs to the carbonyl group (–C=O) in curve B for PF127-NH2. Other new absorption peaks formed at 1753.5 and 1720.0 cm−1, corresponding to the carbonyl group (–C=O) in curve C for PF127-FITC. The absorptions peaks occurring between 1577.2 and 1701.1 cm−1 in curve D are associated with the amide group (–CO–NH–) in the PF127-FA structure. Overall, the FT-IR and 1H-NMR spectra are consistent with the desired copolymer products. 3.2. Characterization of solasodine-loaded Pluronic micelles The size, distribution, zeta potential and morphology of the micelles were determined by DLS and HR-TEM. The average size and polydispersity index (PDI) of a blank micelle were approximately 21.7 nm and 0.084. The encapsulation of solasodine resulted in an increase in diameter and in PDI to 26.8 nm and 0.139 (Figure 3(A)), respectively. The zeta potential of the micelles was −3.05 ± 0.10 mV. The DLC and

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Figure 3. DLS image of solasodine-loaded micelles (A). HR-TEM image of solasodine- loaded micelles stained with phosphotungstic acid aqueous solution three timed and air-dried for observation (B).

DLE were calculated to be 0.8 and 65.7%, respectively, which were relatively low compared with the values observed for PTX, DOX and other model drugs because solasodine is poorly soluble in aqueous media.[38,39] The HR-TEM image in Figure 3(B) shows spherical micelles with a homogeneous size distribution. The micelle size measured by DLS was generally larger than that measured by TEM because the hydrophilic shell size of the micelles slightly shrank when the TEM samples were prepared by dripping micelle solution onto the copper grid and allowing it to dry. 3.3. Cellular uptake in vitro In this study, CLSM and flow cytometry were used to evaluate the targeting property of FA on the in vitro cellular uptake behaviour of the micelles against 16HBE, A549 and HeLa cells, which show no, low and high positive expression of the folate receptor (FR), respectively.[40,41] Micelles composed of equimolar amounts of PF127-FA and PF127-FITC were incubated with 16HBE, A549 and HeLa cells. The 16HBE cells did not show any uptake because they did not express FR.[41,42] For the A549 and HeLa cells, as shown in Figure 4, the strong green fluorescence indicated that a large amount of FITC was absorbed into the cells via endocytosis. The intensity of green fluorescence increased with time. At the same incubation time, i.e. 4 or 24 h, the fluorescence intensity for HeLa cells was much stronger than the intensity for A549 cells. The results were consistent with our hypothesis that the expression level of FR on the surface of HeLa cells was higher than that on A549 cells. To quantitatively investigate FR mediated endocytosis, flow cytometry analysis was conducted. A minimum of 100,000 events were acquired for each sample. By treatment with micelles containing equimolar amounts of PF127-FA and PF127-FITC for 4 or

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Figure 4. Confocal microscopy images of A549 and HeLa cells treated with micelles composed of PF127-FA and PF127-FITC (200 μg mL−1) for 4 or 24 h. Green fluorescence arose from FITC in PF127-FITC, and blue fluorescence arose from DAPI stained on the nuclei.

24 h, A549 and HeLa cells were acquired for fluorescence intensity determination. From Figure 5, we can conclude that a longer incubation time resulted in a stronger fluorescence intensity. For the control group, the fluorescence intensity was nearly equal, whereas after incubation for 4 or 24 h, the fluorescence intensity of PF127-FITC exhibited by HeLa cells was significantly stronger than that exhibited by A549 cells. All of the results were consistent with our hypothesis that micelles were internalized via an FR-mediated pathway. Thus, the micelles can be considered suitable nanoscale vehicles for delivering hydrophobic drugs with higher efficacy. With respect to the assay of different expressions of FR on the surface of A549 and HeLa cells, Figures 4 and 5 indicate that the expression level of FR on the surface of HeLa was higher than that on A549. Specifically, compared with the A549 cellular uptake of micelles containing PF127-FA and PF127-FITC, HeLa cells appeared to absorb more PF127-FITC, as evidenced by the fluorescence intensity at 4 or 24 h, respectively. This result is consistent with previous reports.[41,42]

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Figure 5. Fluorescence activated cell sorter (FACS) analysis of FITC intensity of cells (A549 and HeLa) treated with micelles composed of PF127-FA and PF127-FITC (200 μg mL−1) for 4 or 24 h (excitation: 488 nm, emission: 520 nm).

3.4. FA competition assays FA competition assays were employed to ensure endocytosis was mediated by FR on the surface of the HeLa cells. As shown in Figure 7, the fluorescence intensity of sample S4 (HeLa cells incubated with micelles containing PF127-FA and PF127-FITC) was much stronger than that of S1 (HeLa cells incubated with micelles without PF127FA). This phenomenon could be explained by the fact that more micelles were absorbed via the specific uptake pathway mediated by FR than by non-specific uptake. Furthermore, due to the competition between free FA and PF127-FA to specifically bind with FR, the fluorescence intensities of sample S2 (HeLa cells treated with micelles containing 200 μg mL−1 PF127-FA and PF127-FITC, as well as 100 μg mL−1 free FA) and sample S3 (HeLa cells treated with micelles containing 200 μg mL−1 PF127-FA and PF127-FITC, as well as 1 μg mL−1 free FA) were stronger than that of S1, whereas the intensities were weaker than the intensity of S4 (HeLa cells treated with micelles containing 200 μg mL−1 PF127-FA and PF127-FITC). The features shown in the result of CLSM image in Figure 6 are consistent with the results of flow cytometry shown in Figure 7. 3.5. Cytotoxicity assay in vitro It is necessary to evaluate the cytotoxicity of polymeric materials designed for drug delivery applications. In the study, the cytotoxicity of various polymers was determined by an MTT assay. The cell viability of drug resistant A549 and 16HBE cells against PF127, PF127-FA and PF127-FITC is shown in Figure 8. All of cell lines showed an inhibition rate of less than 80%, despite the concentration reaching 1 mg mL−1. An identical MTT assay was used to evaluate the efficacy of free solasodine and solasodine-loaded micelles in inhibiting the growth of A549 and HeLa cells. The tested concentrations of solasodine in each case were 0.630, 1.25, 2.50, 5.00 and 10.0 μg mL−1. As illustrated in Figure 9, the solasodine-loaded micelles led to lower cell viability than that measured for A549 and HeLa cells incubated with free solasodine because the micelles could hold more drug molecules for administration and enforce FR-mediated endocytosis. Treated with free solasodine and solasodine-loaded micelles at different concentrations, the A549 and HeLa cells showed different survival rates.

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Figure 6. Confocal microscopy images of FA competition assays. HeLa cells treated with only DMEM (control); HeLa cells treated with micelles without PF127-FA (S1); HeLa cells treated with micelles containing 200 μg mL−1 PF127-FA and PF127-FITC, as well as 100 μg mL−1 free FA (S2); HeLa cells treated with micelles containing 200 μg mL−1 PF127-FA and PF127-FITC, as well as 1 μg mL−1 free FA (S3); and HeLa cells treated with micelles containing 200 μg mL−1 PF127-FA and PF127-FITC (S4).

Using 10.0 μg mL−1 drugs against the A549 and HeLa cells as an example, the cell survival rates were 90 and 75% for free solasodine, respectively, whereas the cell survival rates were 40 and 20% for solasodine-loaded micelles, respectively. It could be hypothesized that the micelles played an important role in delivering the drugs into

Figure 7. Flow cytometry images of FA competition assays. HeLa cells treated with only DMEM (control); HeLa cells treated with micelles without PF127-FA (S1); HeLa cells treated with micelles containing 200 μg mL−1 PF127-FA and PF127-FITC, respectively, as well as 100 μg mL−1 free FA (S2); HeLa cells treated with micelles containing 200 μg mL−1 PF127-FA and PF127-FITC, as well as 1 μg mL−1 free FA (S3); and HeLa cells treated with micelles containing 200 μg mL−1 PF127-FA and PF127-FITC (S4).

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Figure 8. Cytotoxicity of PF127, PF127-FA and PF127-FITC against 16-HBE (A and C) and A549 (B and D) for 24 or 48 h at different concentrations.

Figure 9. Efficacy of cell growth inhibition of free solasodine (A) and solasodine- loaded micelles (B) against A549 and HeLa cells for 24 or 48 h at different concentrations.

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cancer cells, and the different survival rates between the A549 and HeLa cells resulted from the high specific binding of FR and FA in the HeLa cells.

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4. Conclusions In summary, an efficient drug delivery micelle platform based on FA- and FITCconjugated PF127 was developed in this study. Two copolymers, PF127-FA and PF127-FITC, were synthesized by chemically conjugating FA and FITC with PF127. Solasodine-loaded micelles were fabricated based on the two functional copolymers. The results of in vitro cell assays indicated that the functional micelles enhanced cellular uptake performance via FR-mediated endocytosis in FR positive HeLa cells compared with A549 cells with low FR expression. The solasodine-loaded micelles may offer enhanced efficacy for the inhibition of cancerous cell growth, approximately 4-fold that provided by free solasodine. Thus, the functional drug delivery platform may have excellent potential for applications in lipophilic drug delivery. Disclosure statement No potential conflict of interest was reported by the authors.

Funding This work was supported by the Science and Technology Commission of Shanghai Municipality (STCSM, contract Nos. 11nm0505700 and 13142201001).

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A functional drug delivery platform for targeting and imaging cancer cells based on Pluronic F127.

Functional polymeric micelles play an important role in the efficient delivery of therapeutic drugs into tumours. In this study, a functional drug del...
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