N-Boc-Histidine-Capped PLGA-PEG-PLGA as a Smart Polymer for Drug Delivery Sensitive to Tumor Extracellular ph

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1 Full Paper N-Boc-Histidine-Capped PLGA-PEG-PLGA as a Smart Polymer for Drug Delivery Sensitive to Tumor Extracellular ph Guangtao Chang, Chong Li, Weiyue Lu, Jiandong Ding* A ph-sensitive polymer was synthesized by introducing the N-Boc-histidine to the ends of a PLGA-PEG-PLGA block copolymer. The synthesized polymer was confirmed to be biodegradable and biocompatible, well dissolved in water and forming micelles above the CMC. DOX was employed as a model anticancer drug. In vitro drug release from micelles of N-Boc-histidinecapped PLGA-PEG-PLGA exhibited significant difference between ph ¼ 6.2 and ph ¼ 7.4, whereas DOX release from micelles composed of un-capped virgin polymers was not significantly sensitive to medium ph. Uptake of DOX from micelles of the new polymer into MDA-MB- 435 solid tumor cells was also observed, and ph sensitivity was confirmed. Hence, the N-Boc-histidine capped PLGA-PEG-PLGA might be a promising material for tumor targeting. Introduction In the anticancer field, nano-sized drug carriers, especially micelles, have become a research focus and can significantly improve the drug efficacy and reduce the side effects. [1 7] Besides the effect of enhanced permeability and retention (EPR) in tumor, [8,9] the slight acidic environment of solid tumors has recently been thought as another cue for designing tumor-targeting materials. [10] The micelles G. T. Chang, J. D. Ding Key Laboratory of Molecular Engineering of Polymers of Ministry of Education, Department of Macromolecular Science, Laboratory of Advanced Materials, Fudan University, Shanghai , China Fax: þ ; jdding1@fudan.edu.cn C. Li, W. Y. Lu Department of Pharmaceutics, School of Pharmacy, Fudan University, Shanghai , China sensitive to tumor extracellular ph (ph e ) could switch the release kinetics of drugs out of nano-carriers, from a relatively slow release at circulation to a fast release at tumor sites. The ph e is around , lower than physiological ph ¼ 7.4, which is caused by the high rate of glycolysis at tumor sites under either aerobic or anaerobic conditions. [11] One has designed polymers of some groups neutral at physiological ph but ionizable at ph e. For instance, Bae and his coworkers [12 15] developed a series of nano-particles containing the moiety of polyhistidine to make the anticancer drug delivery system sensitive to ph e. Lee and his coworkers [16,17] synthesized block copolymers containing a poly(beta-amino ester) block to achieve the micellization/demicellization transition between physiological ph and ph e. It seems worthy to mention other ph-sensitive micelles of some polymers containing an acid-cleavable linkage with an acid-labile bond such as hydrazone, [18 22] acetal [23 25] or 1248 ß 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim wileyonlinelibrary.com DOI: /mabi

2 N-Boc-Histidine-Capped PLGA-PEG-PLGA as... benzoic-imine, [26,27] etc. The pertinent carriers could physically encapsulate drugs or more frequently chemically conjugate with drugs. The drugs enter into cells together with polymers by endocytosis, and are then released in the late endosomes at ph ¼ or further in lysosomes at ph ¼ ApH e -targeting polymer with sufficient sensitivity to the weak acidic ph e mixes with a drug, and the drug should be released outside tumor cells and then diffuse into the cells. Since we prefer not to introduce any chemical reaction to drugs for convenience of potential approval of a new medicament, we select physical encapsulation of anticancer drugs into micelles. In this case, an acid-cleavable linkage seems not absolutely necessary and a preestablished biodegradable block with many acid-labile bonds might be an alternative choice. Hence, it might be helpful to try an amphiphilic polymer, which contains ph e -sensitive groups and meanwhile include a biodegradable block with accelerated hydrolysis in the acidic environment, as shown in Scheme 1. Then the underlying micelles composed of the polymers could exhibit tumor targeting with accelerated drug release outside cells in a tumor site, and the drugs still remaining in the nanocarriers could possibly be internalized into cells and further released after polymer degradation in endosomes. By these two ways, the efficacy of a drug delivery system might be quite good, while the material technique is not complicated. We thus design a polymer of the above two characteristics, namely, containing ph e -sensitive groups and biodegradable blocks, via histidine end-capping of a biodegradable amphiphilic polymer PLGA-PEG-PLGA consisting of poly[(d,l-lactic acid)-co-(glycolic acid)] (PLGA) and poly(ethylene glycol) (PEG) blocks. Our group has recently performed a series of research on PLGA-PEG-PLGA as an injectable hydrogel, [28 34] following the pioneering work of hydrogels of block copolymers composed of PEG and biodegradable aliphatic polyester. [35] Since the accelerated hydrolysis of PLGA under acidic environment is well known in the field of biomaterials, [36 39] the present paper pays attention to examination of the availability of ph e sensitivity of the new polymer containing only two histidine groups at the ends of the synthesized linear macromolecules. Figure 1 shows the synthesis route of this polymer abbreviated as his-plga-peg-plga-his. Micelles might be formed in water with a PLGA-rich core and a PEG-rich corona, and micelle sizes might be tuned by ionization degree of the imidazole between the circulation ph and the tumor ph e. So the release rate of the loaded drug might be switched, as shown in Scheme 1. The micelles are expected to accelerate drug release at tumor ph e compared with normal physiological condition. Since a cationic micelle is more probably endocytosed by cells, [40] the positive charge of the imidazole of the present micelles might also facilitate Scheme 1. (a) Schematic presentation of the design of the micelles composed of amphiphilic polymers with end capped by N-Bochistidine and the sensitivity of micelles to tumor extracellular ph as a circulated nano-carrier of anti-tumor drugs. (b) Presentation of the routes of drugs into nucleus of tumor cells after injection. The PEG corona could prolong the circulation time of the micelles in blood, and the micelles with appropriate sizes could enter into tumor sites due to the EPR effect. Part of DOX could be released from the ph-sensitive micelles outside tumor cells in an accelerated way due to the weak acidic extracellular ph, and then diffuse into the cytoplasm. Some micelles might be internalized by the tumor cell, and then the biodegradable polymer could be hydrolyzed in the acidic endosomes or lysosomes, which also result in release of DOX into the cytoplasm. The DOX in the cytoplasm could partially enter into cell nucleus. internalization by the tumor cell, although the present manuscript is more focused on accelerated drug release out of micelles due to tumor ph e. The present paper will report the synthesis of the polymer, the determination of pk a of the his groups [N- Boc-histidine groups; Boc ¼ tert-butoxycarbonyl] at the ends of the amphiphilic block copolymer, the measurement of critical micelle concentration (CMC) of the end-capped amphiphiles, the confirmation of biodegradation and biocompatibility of the synthesized copolymer, and the examination of in vitro release of an anticancer drug doxorubicin (DOX) from polymeric micelles at typical ph values. ß 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

3 G. T. Chang, C. Li, W. Y. Lu, J. D. Ding Gel Permeation Chromatography (GPC) The molecular weights (MWs) and MW distribution of polymers were measured by GPC in an Agilent 1100 apparatus with a differential refractometer as a detector. Tetrahydrofuran was used as an eluting solvent at a flow rate of 1.0 ml min 1 at 35 8C, and polystyrene (PS) was used as MW standard. NMR Spectrometry 1 H NMR measurements were performed in a Bruker DMX500 spectrometer at resonance frequency of 500 MHz. The ph in the NMR experiments (pd) was adjusted by NaOD or DCl solution in D 2 O. The relation between pd and ph is pd ¼ ph þ 0.4. [41,42] Figure 1. Synthesis route of PLGA-PEG-PLGA and its N-Boc-histidine capped derivative, his-plga-peg-plga-his. Experimental Part Materials PEG (1 500 Da) was purchased from Fluka, stannous 2-ethylhexanoate (stannous octoate), 4-dimethylaminopridine (DMAP) and DOX HCl were from Sigma. D,L-lactide (LA) and glycolide (GA) were obtained from Purac and used without further treatment. N-Boc-L-histidine, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC HCl) were purchased from GL Biochem (Shanghai, China) Ltd. All other chemicals were of reagent grade and used as purchased without further purification. For cell culture experiments, medium, serumandotherrelated reagents werepurchasedfromgibcoco., USA. Potentiometric Titration After 10 ml of 1 wt.-% N-Boc-histidine-capped copolymer in 0.9 wt.-% NaCl solution were prepared, the solution was titrated with 0.1 M HCl at C under argon atmosphere. The physiological saline solution with 0.9 wt.-% NaCl was used as a blank. The volume of added HCl solution and ph were recorded to obtain the titration profile. The corrected curve was fitted using the Henderson-Hasselbalch (HH) Equation to obtain the apparent pk a of the group his at the end of the block copolymer. The degree of ionization (a) of the imidazole group is defined as ½his þ Š aðphþ ½his þ Šþ½hisŠ where [his] and [his þ ] are concentrations of deionized and ionized N-Boc-histidine groups at the ends of the amphiphilic copolymer, respectively. The pk a of the weak base in the his-plga-peg-plgahis is defined as (1) Synthesis of N-Boc-Histidine-Capped PLGA-PEG-PLGA First, the virgin PLGA-PEG-PLGA triblock copolymer was synthesized using stannous octoate as catalyst. The ring-opening polymerization of LA and GA was performed in the presence of PEG. The copolymer was collected and freeze-dried after precipitated in water at 80 8C. PLGA-PEG-PLGA (15 g), N-Boc-histidine (3.3 g), EDC HCl (3.5 g), DMAP (2.6 g) and dichloromethane (120 ml) were added to a dry three-necked round bottom flask, and the reaction was carried out at room temperature for 48 h under argon atmosphere. Then the solvent was partially evaporated. Polymers in the residue solution were precipitated in diethyl ether, further purified through dissolving in diluted hydrochloric acid, and reprecipitated at 80 8C in water to wipe off the remaining EDC HCl, DMAP and N-Boc-histidine residues. The precipitate was freezedried for 48 h. pk a 1gK a ; K a ðhisþðhþ Þ ðhis þ Þ where K a is the equilibrium dissociation constant and (H þ ), (his) and (his þ ) are the activities of medium proton, deionized and ionized imidazoles, respectively. Neglecting the difference of activity and concentration, a could be related to pk a as CMC 1 a ¼ (3) 1 þ 10 ph pka CMC values were determined by the method of hydrophobic dye solubilization. 150 ml of M 1,6-diphenyl-1,3,5-hexatriene (2) 1250 ß 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim DOI: /mabi

4 N-Boc-Histidine-Capped PLGA-PEG-PLGA as... (DPH) methanol solution was injected into 150 ml phosphatebufferedsaline (PBS) solution, and then stirredatroom temperature for 4 h to evaporate the methanol. The PBS solution containing dye was divided into three parts with ph adjusted to the scheduled value. A 1.0 wt.-% polymer solution was also adjusted to the corresponding ph, and then diluted into three series of concentrations with the corresponding PBS. Partitioning of DPH into a hydrophobic domain such as the core of a micelle results in a stronger absorbance at 337, 356 and 376 nm. The CMC was estimated as the crossing point when the extrapolation of the absorbance at 376 nm with respect to that at 400 nm was plotted against the logarithmic polymer concentration. series of time, and the DOX release profiles were measured via UV absorbance at 479 nm. Cell Culture The human breast cancer cell line MDA-MB-435 and human normal cell line L02 were purchased from the cell bank of Chinese Academy of Science. Cells were maintained in RPMI1640 cell culture medium with 10% heat-inactivated bovine calf serum (BCS) under 5% CO 2 at 37 8C. Dynamic Light Scattering (DLS) DLS measurements were performed in a light scattering spectrophotometer (Autosizer 4700, Malvern) with a vertically polarized incident beam at 532 nm supplied by an argon-ion laser. A scattering angel of 908 was detected in this study. Before measurements, all samples were treated with a 0.45-mm filter (Millipore). The Stokes-Einstein Equation was employed to calculate the hydrodynamic radius of the micelles. Polymers were dissolved in PBS, and medium ph was adjusted by addition of 4 M NaOH or 4 M HCl. Each sample was equilibrated for 10 min at 37 8C. Cytotoxicity of his-plga-peg-plga-his to Normal Cells The cytotoxicity of polymers was assessed using the MTT assay [MTT ¼ 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]. The human normal liver cells L02 were cultured as described above cells per well were seeded in 96 well microtiter plates and incubated for 24 h in the above condition. A series of polymer concentrations were prepared and added to each well and incubated at 37 8C for 72 h. The MTT experiments were conducted in triplicate. Uptake of DOX by Tumor Cells In vitro Degradation The polymer solution was prepared in PBS solution. Every vial containing 4 ml of polymer solution (1 wt.-%) was immersed in a shaking water bath at 37 8C, 35 strokes per min. A vial was collected at a predetermined time, and the hydrodynamic radius hr h i was measured by DLS. MDA-MB-435 cells were cultured onto six-well plates. After 24 h, the medium was replaced by fresh medium at ph ¼ 6.2 or 7.4, followed by addition of DOX-loaded micelles (10 mg ml 1 DOX equiv.) or DOX HCl with the equivalent DOX concentration. At a given time, the cells were washed with PBS (ph ¼ 7.4) and fixed using 4% (w/v) formaldehyde at 4 8C for 30 min. Then the cellular uptake of free DOX or DOX from micelles at different ph values was observed by fluorescence microscopy. Drug Loading and in vitro Release DOX-loaded micelles were prepared by a solvent evaporation method similarly to the blank micelles. Briefly, DOX HCl (1 mg, 1 equiv.) was stirred with triethylamine (1.5 equiv.) in chloroform/ methanol (1:1, v/v), followed by dropwise addition to chloroform/ methanol (1:1, v/v) solution of polymers (2 ml, 5 mg ml 1 ). After removing organic solvent using a rotary evaporator, the formed thin film was dispersed by addition of 1 ml of distilled water (ph ¼ 7.4) under gentle shaking for 2 h and treated in a bath sonicator for 15 min. Then the micelles were passed through syringe filters (0.45 mm, Millipore) and transferred into a dialysis bag (cut-off MW 3 500) to dialyze against pure water for further removal of free DOX overnight. The measurement of drug loading amount was performed by dissolving lyophilized DOX-loaded micelles in chloroform/methanol (1:1, v/v), followed by quantification of DOX based on UV absorbance at 479 nm. In vitro DOX release was performed by the following steps: firstly, dispersion of the lyophilized drug-loaded micelles were prepared and transferred into a dialysis bag (cut-off MW 3 500), then the bag was incubated in 30 ml PBS (ph ¼ 6.2 or 7.4) at 37 8Cin a water bath under shaking. The solution was collected after a given Results and Discussion Synthesis and Characterization of his-plga-peg- PLGA-his PLGA-PEG-PLGA triblock copolymers were synthesized via ring-opening polymerization of D,L-lactide and glycolide with PEG as macroinitiators. The virgin triblock copolymer was then conjugated with the N-Boc-histidine in the presence of EDC HCl and DMAP (Figure 1). The Boc group linked to histidine was kept in order to avoid complex due to the otherwise exposed amine group with pk a around Figure 2 shows the representative 1 H NMR spectrum of his- PLGA-PEG-PLGA-his. All proton signals of the N-Bochistidine capped copolymers were assigned in Figure 2. The MW and composition were determined from 1 H NMR using PEG (1 500 Da) as standard, the MW distribution was measured from GPC relative to PS standard. The calculated N-Boc-histidine substitution rate and MW of the synthesized polymers are given in Table 1. ß 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

5 G. T. Chang, C. Li, W. Y. Lu, J. D. Ding between circulation ph and tumor ph e. The ionization degree (a) is obtained from aðphþ ¼ VðpHÞ V 0 V end V 0 (5) As shown in Figure 3b, a varies with ph. Micellization of his-plga-peg-plga-his in Water Figure 2. 1 H NMR spectrum of the N-Boc-histidine capped PLGA- PEG-PLGA. A potentiometric titration was performed to determine the pk a of the N-Boc-histidine-capped polymers. The pk a of the imidazole group is usually 7.23 (25 8C) in small molecules such as histidine methyl ester. [43] The value might be altered after linkage to an amphiphilic block copolymer. It is non-trivial to accurately determine the pk a of a weak acid or alkaline group in a colloid. [32,44] The pk a values in amphiphilic block copolymers in water might change with the ionization degree a, as shown in Equation (3). So we use its value at a ¼ 0.5, namely, pk a (0.5) to describe the ionization ability of the polymers. The titration curve was corrected with 0.9 wt.-% NaCl as a blank, as shown in Figure 3a. The corrected curve was fitted with the HH equation. The Equation in this case could be written as V end V 0 V ¼ V 0 þ (4) 1 þ 10 n½pkað0:5þ phš Here, V is the volume of added HCl solution at a certain ph, V 0 and V end denote the volume of HCl solution at the start and end points in the titration, respectively, and n is an adjustable constant. The resultant pk a (0.5) after correcting with the physiological saline solution was That pk a (0.5) is between the circulation ph and the tumor tissue ph e. An ionization transition might happen As a potential drug carrier, CMC is an important factor for an amphiphile. [5,13,45,46] In this paper, CMC was determined via the method of hydrophobic dye solubilization. The CMC values shown in Figure 4 are around 0.03 wt.-%. The ionization state of the end groups influences micelle sizes significantly in higher concentrations of polymers. The DLS results at different ph values are shown in Figure 5a. The hydrodynamic radius ( hr h i) increase with the increase of ph (Figure 5b). The apparent volume of micelles at ph ¼ 8.00 is 5.3 fold of that at ph ¼ H NMR was employed to further examine the ph effects on the self-assembly behaviors of the polymers in water. Figure 6 shows the signals assigned to the lactyl protons CH 3 and the Boc CH 3 with increasing ph. At ph ¼ 5.30, the micelles were loose, the PLGA segment and the Boc group had high mobility, thus the signals showed narrow and strong resonance peaks in the 1 H NMR spectrum. At ph ¼ 7.54, the PLGA segment and the Boc- group were buried in the micelles and had low mobility, so the signals of the end were relatively less intensive. With decrease of ph, the imidazole of the histidine was protonated and acted as a more hydrophilic group. The aggregation number might also be decreased. After rearrangement due to self assembly of less chains of relatively more hydrophilic chains, the micelles became smaller and looser. The decrease of hr h i at the slight acidic ph may be useful for the anticancer drug release and for the enhanced EPR effect at the tumor site. If we define the transition ph of the micelle sizes as ph, the value could also be obtained via fitting with HH equation, resulting in ph ¼ 6.39, while the corresponding a was The ph is not equal to the pk a (0.5), because the abrupt change of micelle sizes happens unnecessarily when half of weak alkaline groups are ionized. Table 1. Basic characterization of the synthesized PLGA-PEG-PLGA and its N-Boc-histidine capped derivative. a) Copolymer, M n LA/GA a) N-Boc-Histidine substitution a) mol mol 1 b) M w =M n PLGA-PEG-PLGA / his-plga-peg-lga-his /1 92% 1.16 a) M n and LA/GA ratio were obtained from 1 H NMR; b) The MW distribution described by M w =M n was determined from GPC ß 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim DOI: /mabi

6 N-Boc-Histidine-Capped PLGA-PEG-PLGA as... Figure 4. Absorbance difference of M DPH at 376 and 400 nm as a function of polymer concentration. The calculated CMC values are 0.39, 0.30, and 0.26 mg ml 1 at ph ¼ 4.00, 6.30, and 8.00, respectively. The potential toxicity or biocompatibility of this polymeric material was also preliminarily evaluated by MTT assay. No serious reduction of viability of human normal liver cells L02 was observed after addition of the synthesized polymers of concentration up to 2.5 mg ml 1 (Figure 8). Figure 3. (a) Suspension ph versus V, volume of 0.1 M HCl solution added into 10 ml of water or polymer aqueous solutions at C. The hollow circles (*) represent the data of the 1 wt.- % N-Boc-histidine-capped PLGA-PEG-PLGA in 0.9 wt.-% NaCl solution; the hollow upper triangles (D) represent the data of the 0.9 wt.-% NaCl solution. The filled circles (*) show the titration curve of the aqueous solution of N-Boc-histidine capped PLGA-PEG-PLGA after correction using 0.9 wt.-% NaCl solution as a blank. The line corresponding to the filled circles is a fit by HH equation, resulting in pk a (0.5) ¼ , n ¼ , V 0 ¼ ml, V end ¼ ml; the other two lines are just for guidance of eyes. (b) Degree of ionization (a) of 1 wt.-% Boc-histidine-capped PLGA-PEG-PLGA in 0.9 wt.-% NaCl solution as a function of ph. The filled circles (*) represent the experimental data obtained by Equation (5); the solid line is a fit with HH Equation [similar to Equation (4)] resulting in pk a (0.5) ¼ Biodegradability and Biocompatibility Biodegradability is important for a potential drug carrier. We examined the micelle degradation by DLS, as shown in Figure 7. While the micellar state maintained at least in one week, both sizes and the scattering intensity declined during degradation. The degradation of the hydrophobic PLGA block makes the copolymer chains relatively more hydrophilic. The amphiphilic chains are probably rearranged into smaller micelles in order to maintain stability of the nanoparticles in water. Figure 5. (a) Hydrodynamic radius of copolymer micelles in PBS (1 wt.-%) as a function of ph at 37 8C. The solid line is a fit from HH Equation resulting in ph ¼ , n ¼ (b) Size distribution at three typical ph values at 37 8C. ß 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

7 G. T. Chang, C. Li, W. Y. Lu, J. D. Ding In vitro DOX Release and Cellular Uptake of DOX from Micelles Figure 6. 1 H NMR spectra of N-Boc-histidine capped PLGA-PEG- PLGA solution (1 wt.-%) in D 2 O at indicated ph values at 25 8C. The solution ph was adjusted by addition of DCl and NaOD. Figure 7. Hydrodynamic radius R h and scattering intensity I as a function of degradation time t for an aqueous suspension of micelles of N-Boc-histidine capped PLGA-PEG-PLGA at 37 8C and ph ¼ 7.4. Every sample was measured three times. The lines are just for guidance of eyes. The drug loading efficiency and capacity were assessed and calculated as % and %, respectively. It seems worthy of noting that the hydrophilic DOX HCl was treated by triethylamine in an organic solvent in our loading experiment, which converted the DOX HCl into DOX free base. The DOX was thus able to be loaded into the hydrophobic PLGA core of micelles. The in vitro release of DOX from the micelles was investigated by dialysis. As shown in Figure 9, DOX was relatively slowly released from micelles of his-plga-peg-plga-his under ph ¼ 7.4, as compared to ph ¼ 6.2, indicating that the modified micelles respond to change from normal neutral ph ¼ 7.4 to acidic tumor ph e 6.2. It should be indicated that the ionization of the amine of DOX and thus the solubility of DOX in water are also ph dependent. The pk a of DOX is about 8.4 at 25 8C. [47] An estimation under an ideal condition and upon neglecting the influence of the ionic strength reads that the ionization degrees of DOX are theoretically 91% at ph ¼ 7.4 and 99% at ph ¼ 6.2. So, the change of ionization degree of drug itself is less significant between ph ¼ 7.4 and ph ¼ 6.2 than that of our end-capped copolymer his-plga-peg-plga-his, which pk a was determined as 6.72 (Figure 3). Our in vitro release experiments illustrate that DOX release did not exhibit significant difference at the two ph values in the case of virgin PLGA-PEG-PLGA polymers (Figure 9). So, the ph dependent DOX release from micelles of his-plga- PEG-PLGA-his in our examined ph range (Figure 9) might mainly come from the intelligence of our copolymer derivative designed by introducing histidine end groups as shown in Figure 1 and 2, instead of from the ph dependent solubility of DOX. Figure 8. Viability of human normal liver cells L02 incubated in cell culture medium with 10% serum at indicated concentrations of N-Boc-histidine capped PLGA-PEG-PLGA for 72 h. The viability in the absence of copolymer is set as 100%. MTT experiments were performed in triplicate. Figure 9. In vitro release profiles of DOX from micelles at indicated ph values. The release amount was evaluated by UV absorbance at 479 nm. Micelles of N-Boc-histidine capped PLGA- PEG-PLGA and those of virgin PLGA-PEG-PLGA were examined. Three independent samples were detected for each condition ß 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim DOI: /mabi

8 N-Boc-Histidine-Capped PLGA-PEG-PLGA as... Conclusion By introducing the ph-sensitive moiety N-Boc-histidine into the end of PLGA- PEG-PLGA, we synthesized a ph-sensitive polymer with appropriate pk a and constructed micelles ready to accelerate the release of drugs under the slightly acidic, tumor extracellular micro-environments. The basic properties of the new material and characteristic parameters of the formed micelles in water were evaluated. The in vitro release assays using DOX as a model drug validated the mode of drug release. The N-Boc-histidine capped PLGA-PEG-PLGA polymer might be a potential candidate as an anticancer release carrier in view of its biodegradability, biocompatibility, and sensitivity to tumor extracellular ph. Figure 10. Cellular uptake of DOX at indicated time and ph values. Human breast cancer cells MDA-MB-435 were observed. The upper part refers to micelles of his-plga-peg- PLGA-his with DOX loaded. The lower part shows images in the cases of free DOX and DOX-load micelles composed of non-capped virgin polymers. Shown are merged images of fluorescent micrographs and corresponding phase-contrast micrographs. The red fluorescence of DOX is shown as white in the black and white image. Bars: 150 mm. The same ph-sensitive trend was also observed in cellular uptake of DOX from micelles investigated by microscopy (Figure 10). DOX emits red florescence and targets cell nucleus. The efficacy of free DOX illustrates that DOX can diffuse into the human breast cancer cells MDA-MB-435 rather fast. More DOX uptake was seen in the group of ph-sensitive micelles at ph ¼ 6.2 than the physiological ph, while no significant difference wasfoundbetweenph¼ 6.2 and 7.4 for either the group of free DOX or the group of micelles composed of virgin block copolymer without N-Boc-histidine capping. These results suggest that the N-Boc-histidine capped PLGA-PEG-PLGA micelles may be suitable for tumor targeting based on the acidic tumor extracellular microenvironment. Acknowledgements: The authors gratefully acknowledge financial support from the Chinese Ministry of Science and Technology (973 Programs No. 2009CB and No. 2010CB934000), the NSF of China (Grant No ), the National Science and Technology Major Project (No. 2009ZX ), the Science and Technology Developing Foundation of Shanghai (Grant No ), and the Shanghai Education Committee (Project No. B112). Received: March 15, 2010; Revised: April 28, 2010; Published online: June 30, 2010; DOI: /mabi Keywords: block copolymers; drug delivery systems; micelles [1] J. S. Park, T. H. Han, K. Y. Lee, S. S. Han, J. J. Hwang, D. H. Moon, S. Y. Kim, Y. W. Cho, J. Controlled Release 2006, 115, 37. [2] C. K. Huang, C. L. Lo, H. H. Chen, G. H. Hsiue, Adv. Funct. Mater. 2007, 17, [3] K. T. Oh, H. Q. Yin, E. S. Lee, Y. H. Bae, J. Mater. Chem. 2007, 17, [4] M. Licciardi, E. F. Craparo, G. Giammona, S. P. Armes, Y. Tang, A. L. Lewis, Macromol. Biosci. 2008, 8, 615. [5] H. Lee, C. H. Ahn, T. G. Park, Macromol. Biosci. 2009, 9, 336. [6] X. Han, J. Liu, M. Liu, C. Xie, C. Y. Zhan, B. Gu, Y. Liu, L. L. Feng, W. Y. Lu, Int. J. Pharm. 2009, 372, 125. [7] C. Y. Zhan, B. Gu, C. Xie, J. Li, Y. Lin, W. Y. Lu, J. Controlled Release 2010, 143, 136. ß 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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