Different Insight into Amphiphilic PEG-PLA Copolymers: Influence of

Article pubs.acs.org/Biomac

Different Insight into Amphiphilic PEG-PLA Copolymers: Influence of Macromolecular Architecture on the Micelle Formation and Cellular Uptake Cinzia Garofalo,†,‡ Giovanna Capuano,†,§ Rosa Sottile,‡ Rossana Tallerico,‡ Renata Adami,∥ Ernesto Reverchon,∥ Ennio Carbone,*,‡ Lorella Izzo,*,§,⊥ and Daniela Pappalardo*,# ‡

Dipartimento di Medicina Sperimentale e Clinica, Università degli Studi “Magna Graecia” di Catanzaro, viale Europa, Catanzaro 88100, Italy § Dipartimento di Chimica e Biologia, Università degli Studi di Salerno, and ⊥NANOMATES, Research Centre for NANOMAterials and nanoTEchnology via Giovanni Paolo II 132, Fisciano 84084 (SA), Italy ∥ Dipartimento di Ingegneria Industriale, Università degli Studi di Salerno, via Giovanni Paolo II 132, Fisciano 84084 (SA), Italy # Dipartimento di Scienze e Tecnologie, Università del Sannio, via dei Mulini 59/A, 82100 Benevento, Italy S Supporting Information *

ABSTRACT: One constrain in the use of micellar carriers as drug delivery systems (DDSs) is their low stability in aqueous solution. In this study “tree-shaped” copolymers of general formula mPEG-(PLA)n (n = 1, 2 or 4; mPEG = poly(ethylene glycol) monomethylether 2K or 5K Da; PLA = atactic or isotactic poly(lactide)) were synthesized to evaluate the architecture and chemical composition effect on the micelles formation and stability. Copolymers with mPEG/PLA ratio of about 1:1 wt/wt were obtained using a “core-first” synthetic route. Dynamic Light Scattering (DLS), Field Emission Scanning Electron Microscopy (FESEM), and Zeta Potential measurements showed that mPEG2K-(PD,LLA)2 copolymer, characterized by mPEG chain of 2000 Da and two blocks of atactic PLA, was able to form monodisperse and stable micelles. To analyze the interaction among micelles and tumor cells, FITC conjugated mPEG-(PLA)n were synthesized. The derived micelles were tested on two, histological different, tumor cell lines: HEK293t and HeLa cells. Fluorescence Activated Cells Sorter (FACS) analysis showed that the FITC conjugated mPEG2K(PD,LLA)2 copolymer stain tumor cells with high efficiency. Our data demonstrate that both PEG size and PLA structure control the biological interaction between the micelles and biological systems. Moreover, using confocal microscopy analysis, the staining of tumor cells obtained after incubation with mPEG2K-(PD,LLA)2 was shown to be localized inside the tumor cells. Indeed, the mPEG2K-(PD,LLA)2 paclitaxel-loaded micelles mediate a potent antitumor cytotoxicity effect.

■

INTRODUCTION

can be achieved. The most promising assemblies are constituted by block copolymers made of hydrophobic polyesters, such as poly(lactide) (PLA) and hydrophilic poly(ethylene glycol) (PEG).5−7 As matter of fact, PLA and PEG nicely fulfills the strict requirements for a biomedical application. PLA has high biocompatibility, nontoxicity, and moreover biodegradability, which is important to maintain clearance of the nanoconstruct after injection and delivery of the cargo and to minimize the risk of toxic buildup of the polymer carrier in tissue. On the other hand, PEG not only is hydrophilic and soluble in both water and organic solvents, but also exhibits excellent biocompatibility, lack of toxicity, and absence of immunogenicity. Thus, the interest in the study of PEG-PLA based copolymers as drug delivery systems (DDSs) greatly increased in the last decades. A huge number of

Pharmacological treatment of solid tumor has been hampered by the fast drug clearance and degradation. Therefore, a clever drug delivery approach relies on the preservation of the antitumor drug and their specific transport inside the tumor cell target. The enhanced permeability and retention effect (EPR), well documented in solid tumor, open a new opportunity window for the tumor treatment based on polymeric nanoparticles delivery.1 Amphiphilic block copolymers can readily undergo microphase separation in selective solvents to form micelles, thus affording discrete nanostructures through a spontaneous process. As a result of the capability to load lipophilic molecules into the hydrophobic core, polymer micelles have been widely studied to solubilize and deliver poorly watersoluble drugs.2−4 By tuning copolymer properties such as the chemical structure of the monomers, the blocks molecular weight, and the architecture, a collection of micellar structures © 2013 American Chemical Society

Received: November 12, 2013 Published: December 12, 2013 403

dx.doi.org/10.1021/bm401812r | Biomacromolecules 2014, 15, 403−415

Biomacromolecules

Article

Table 1. Microstructural Details, Molecular Weight, and Molecular Weight Distribution of mPEG-(PLA)n Copolymers run

sample

PLA/mPEga (w/w)

Mn,NMRa(KDa)

Mn,GPb (KDa)

PDIb

Mol of lactidea (%)

LA units/armc

1 2 3 4 5 6 7 8 9

mPEG5K-(PD,LLA) mPEG5K-(PD,LLA)2 mPEG5K-(PD,LLA)4 mPEG5K-(PLLA) mPEG5K-(PLLA)2 mPEG5K-(PLLA)4 mPEG2K-(PLLA) mPEG2K-(PD,LLA) mPEG2K-(PD,LLA)2

1.0 0.55 1.5 1.0 1.1 1.4 1.1 1.0 1.4

10.4 7.8 12.3 10.1 10.3 11.9 4.2 4.0 4.9

18.2 9.9 11.8 10.8 10.8 11.6 3.7 3.4 5.9

1.12 1.06 1.03 1.02 1.02 1.16 1.16 1.13 1.22

25 14 31 24 24 30 26 24 31

37 10 13 36 19 12 16 14 10

Determined by NMR. bObtained from GPC. cLA units/arm have been calculated according to the equation: (Mn,NMR − Mn,PEG)/(144× number of arms).

a

To address these issues, a collection of mPEG-(PLA)n copolymers have been designed, by varying several factors, such as the length of hydrophobic and hydrophilic blocks, the architecture, the tacticity of the poly(lactide) segments. The formation of aggregates of the obtained “tree-shaped” copolymers in aqueous medium was detected by evaluating the critical micelle concentration (CMC). The micelles dimension and polydispersity, and the zeta potential were evaluated in order to understand the architecture and chemical composition effect on autoassembly process and micelle stability. The fate of the synthesized materials, conjugated with the fluorescent label fluorescein isothiocyanate (FITC), was evaluated and localized on two different tumor histologically distinct tumor cell lines, HEK293t and HeLa, respectively, derived from kidney and uterine cancer. The interactions with tumor cells were analyzed by two techniques, the flow cytometry (FACS) and the confocal microscopy; while to assess the drug delivery efficiency, cell viability assays have been used. While the matter has been largely debated in the case of linear amphiphilic block copolymers,2 it was not investigated in the case of nonlinear mPEG-(PLA)n copolymers.

polymeric architectures have been explored as DDSs, ranging from di- to multiblock copolymers,8 from linear to star,9 to branched,10 or by changing the stereochemistry of the PLA blocks.11 Some of these DDSs have already reached the stage of clinical trial, such as the Genexol-PM, studied as a carrier of the antitumor drug paclitaxel.12 The micelles stability prevent drug cargo release before reaching the target cells. Indeed, one of the limits in the use of micellar carriers as DDSs is their low stability in aqueous solution.3 The latest literature reported the use of linear diblock copolymers made of PEG and aliphatic polyesters of various composition for the fabrication of stable micelles by controlling the crystallinity of the core.13 Recently we reported the synthesis of “tree-shaped” copolymers of general formula mPEG-(PLA)n, where the mPEG (poly(ethylene glycol) monomethylether) is the trunk, from which one, two, or four atactic or isotactic polyester arms start.14 It was envisaged that such nonlinear amphiphilic block copolymer should also lead to discrete micelles formation through a spontaneous process and that variation in the architecture could affect the solution nanostructure stability through enhanced hydrophobic interaction between the PLA arms. Micronization by supercritical assisted atomization was performed on linear and branched mPEG-(PLA)n copolymers, with the branched ones affording well-defined microparticles.15 Recent scientific literature underlined the potential value of analogous nonlinear “Y-shaped” or “four-arm star-like” PEGPLA based copolymers compared with the linear ones in forming self-assembly particles.16 On the other hand, the actual usefulness of these systems as DDSs was not assessed. In fact, the abnormal self-assembly behavior observed would hamper their use as DDSs, provoking the formation of microscale assemblies,16 too big for intravenous injection use.1 However, the copolymers described in this latter work were restricted to those having stereoregular poly(L-lactide) blocks and mPEG with Mn = 2000 Da.16 Other recent works also debated the influence of the copolymers architecture, and the synthesis of “linear dendritic”17 or “Y-shaped”18 copolymers made of PEG and poly(ε-caprolactone) was reported. Prompted by these considerations, we investigated the stability of a panel of “tree-shaped” mPEG-(PLA)n copolymers to select those able to form stable micelles having dimension suitable for DDSs use. Moreover, to establish whether the PEG dimensions or the PLA shape play a role in the biological interaction of the micelles, and to identify, among the micelles set, whose better deserve as antitumor drug delivery carrier, their interaction with tumor cell lines were analyzed in vitro.

■

EXPERIMENTAL SECTION

Materials. All manipulations involving air-sensitive compounds were carried out under nitrogen atmosphere using Schlenk or drybox techniques. Poly(ethylene glycol) monomethylether (mPEG2K, Mn =2000 Da; mPEG5K, Mn =5000 Da), purchased from Aldrich, was dried in vacuo over phosphorus pentoxide for 72 h at 25 °C prior to use. D,L-Lactide and L-lactide, purchased from Aldrich, were crystallized from dry toluene, then dried in vacuo with phosphorus pentoxide for 72 h at 25 °C. Toluene, purchased from Aldrich, was dried over sodium and distilled before use. Al(CH3)3, N,N′-carbonyldiimidazole, fluorescein isothiocyanate isomer I (FITC), phosphorus pentoxide, triethylamine, and pyrene were supplied from Aldrich and used as received. Dialysis of the copolymers was performed with membrane Spectra/Por Dialysis MWCO 6−8000, supplied from Aldrich. Instruments and Measurements. The NMR spectra were recorded on a Bruker Avance 400 spectrometer (1H, 400.00 MHz; 13 C, 100.62 MHz) at 25 °C. Deuterated solvents were purchased from Cambridge Isotope Laboratories, Inc. degassed and dried over activated 3 Å molecular sieves prior to use. Chemical shifts (δ) are listed as parts per million. 1H NMR spectra are referenced using the residual solvent peak at δ 7.26 ppm for CDCl3. The molecular weights (Mn) and the polydispersity index (PDI = Mw/Mn) of polymer samples were measured by GPC at 30 °C, using THF as solvent, flow rate of eluant 1.0 mL/min, and narrow polystyrene standards as reference. The measurements were performed on a Waters 1525 binary system equipped with a Waters 2414 RI detector using four 404


Biomacromolecules

Article

Scheme 1

Styragel columns (range 1000−1000000 Å). Every value was the average of two independent measurements. Synthesis of the mPEG-(PLA)n Copolymers. The copolymers were synthesized according to the procedures developed by some of us;14 their properties are reported in Table 1 and their architectures are described in Scheme 1. A typical procedure is described below for the copolymer mPEG2KPD,LLA (run 8, Table 1). A magnetically stirred reactor vessel (50 mL) was charged sequentially with a solution of mPEG2k (Mn = 2000 Da; 0.701 g; 0.35 mmol) and AlMe3 (25 mg; 0.35 mmol) in toluene (10 mL). The mixture was stirred for 1 h. Subsequently, D,L-lactide (1.00 g, 6.94 mmol) was added, and the mixture was stirred at 70 °C for 48 h. Conversions were monitored by integration of the monomer versus polymer methine resonances in the 1H NMR spectrum of crude product (in CDCl3). At complete monomer conversion the mixture was poured into hexane (200 mL), the precipitated polymer was recovered by filtration, washed with methanol, and dried at 40 °C in a vacuum oven. The polymer was characterized by NMR spectroscopy and GPC analysis. 1 H NMR (CDCl3, 25 °C): δ 1.54 (m, −CHCH3−), 3.64 (s, −CH2−), 5.15 (m, −CHCH3−). 13C NMR (CDCl3, 25 °C): δ 16.8 [−C(O)OCHCH3−], 69.2 [C(O)OCHCH3], 70.2 (−CH2−), 169.8 (−COO−). GPC: Mn = 3.4 KDa, PDI = 1.13. Mn,NMR = 4.0 KDa. Micelles Preparation and Pyrene Fluorescence Spectroscopy. mPEG-(PLA)n micelles were prepared by dissolving the copolymers (6 mg) in 2 mL of acetone at room temperature and dialyzing the mixture against 5 L of water for 48 h. The critical micelle concentration (CMC) was determined using pyrene as a fluorescence probe. Samples for fluorescence spectroscopy were prepared by diluting the micelles solutions to 10 different concentrations in the range 1.0 × 10−4 mol/L to 1.0 × 10−7 mol/L. Each sample was then obtained by dropping a pyrene solution (5.0 × 10−6 mol/L in acetone) into an empty vial, adding one of the copolymer solutions previously prepared, and evaporating the acetone by gentle heating. The volume of the needed copolymer solution was calculated to have a final pyrene concentration in water of 6.0 × 10−7 mol/L, which is slightly below the pyrene saturation concentration at 22 °C. Fluorescence spectra were recorded using a Varian luminescence spectometer at an excitation wavelenght of 335 nm and at 22 °C. The intensities of the bands I1 at 372 nm and I3 at 383

nm were evaluated and their ratio plotted versus the copolymer concentration. Paclitaxel-Loaded Micelles. Paclitaxel-loaded micelles were prepared by dissolving 0.5 mg of paclitaxel in 0.5 mL of methylene chloride and adding this solution to 1 mL of methylene chloride containing 2.5 mg of the copolymer mPEG2K-(PD,LLA)2. Water was subsequently added in order to obtain an aqueous copolymer concentration ranging from 1.5 × 10−4 mol/L to 1.5 × 10−9 mol/L and methylene chloride was evaporated at room temperature under stirring overnight. To remove the free paclitaxel, the drug-loaded micelles were centrifuged for 30 min at 3000 rpm. The solid, unloaded paclitaxel was collected and dissolved in methanol for determining the concentration by UV−vis measurements. Loaded paclitaxel was then determined by the difference of the starting drug concentration and the unloaded drug. The micelles drug loading was evaluated using the following equation: drug content(%) = (weight of PTX in the micelles /weight of micelles) × 100 = 11% Micelles Size. Size (MS) and the size distribution (MSD) of the micelles were measured by dynamic laser scattering (DLS) using a Nanosizer (NanoZS Malvern Instrument, U.K.) equipped with a He− Ne laser operating at 4.0 mW and 633 nm, that afford the hydrodynamic diameter of the micelles. To prepare the micellar solutions, the samples were dissolved in acetone, loaded in a dialysis membrane and placed in water for two days, in order to obtain stable micelles in aqueous solution.21 DLS analysis were performed setting the temperature at 5 °C, since at this temperature the dynamic exchanges among the micelles are slower, and at pH 7 and pH 5.22 For the analysis at pH 7, the samples were diluted with distilled water up to 2 × 10−5 mol/L and stabilized for two days; for the analysis at pH 5, the samples were diluted with monobasic/dibasic buffer phosphate and soon analyzed in order to avoid degradation of the polylactide. All the analyses were repeated 6 times per each sample. Size (MS) and the size distribution (MSD) of the micelles were also measured by FESEM photomicrographs using the Sigma Scan Pro 405


Biomacromolecules

Article

Triton X-100 stock, final dilution 1×), with the first wash containing DAPI (1 mg/mL stock, final dilution 1:1000 Molecular Probes). Cells were recovered in mounting medium (ProLong Antifade Molecular Probes) and mounted on a glass coverslip. The images were collected on a Leica TCS SP2 confocal microscope (Leica Mycrosystems, Wetzlar, Germany) with a 63× Apo PLA oil immersion objective (numerical aperture, 1.4) and 60 μm aperture. Cells were scanned from the bottom to the top (usually 7−10 horizontal scan) to identify the central plane for the evaluation of nanoparticles distribution; Zstacks of images were collected using a step increment of 0.2 μm between planes; at least 30 independent fields were scanned for each experimental point. HeLa cells were seeded on a cover glass placed into 6 well plates (1 × 105) and allowed to settle for 24 h, then treated for 2 and 24 h with mPEG5K-(PLLA), mPEG2K-(PD,LLA)2, mPEG2K(PLLA), mPEG5K-(PLLA)2 copolymers. Then were processed like other cell line. Cell Viability Assay. The cytotoxicity of paclitaxel-loaded micelles was evaluated using the MTT (3-(4,5-dimethiltiazol-2-yl)-2,5diphenyl-tetrazolium bromide) (Sigma Aldrich, Milan, Italy) cell viability assay for 72 h of incubation with HeLa cells. Cells were seeded into sterile 96-well plates (2 × 103). Cells were allowed to settle for 24 h before the polymers were added. Ranging from stock solution, MTT solution was prepared at a concentration of 5 mg/mL in RPMI-1640. Solution was filtered with a 0.2 μm filter, and aliquots were stored at −20 °C. Cells were counted and allowed to settle for 24 h using cells treated with Paclitaxel as positive control (Sandoz 6 mg/ mL 853, 91 g/mol) and cells treated with empty nanoparticles as negative control. After 24 h, cells were treated with Paclitaxel at 1 × 10−6 mol/L, 1 × 10−7 mol/L, 1 × 10−10 mol/L concentration, corresponding to mPEG2K-(PD,LLA)2 concentration (1.56 × 10−6 mol/L, 1.56 × 10−7 mol/L, 1.56 × 10−10 mol/L). MTT assay was done as described elsewhere.23 After a 68 h incubation, MTT was added to each well (20 μL of 5 mg/mL solution in PBS) and the cells were incubated for another 4 h. The precipitated formazan crystals were dissolved in absolute isopropanol 0.04 mol/L HCl. The plates were read spectrophotometrically at 490 and 655 nm using a Bio Rad iMark Microplate Reader. The absorbance values were represented as the percentage of cell viability using the control cells value as 100% cell viability.

Software (release 5.0, Aspire Software International Ashburn, VA). Approximately 500 micelles were measured for each particle size distribution calculation. Histograms representing the particle size distribution were fitted using Microcal Origin Software (release 8.0, Microcal Software, Inc., Northampton, MA). Zeta Potential. The zeta-potential was measured using Nanosizer (NanoZS Malvern Instrument, U.K.) by electrophoretic light scattering. This technique allowed measuring the zeta potential by the calculation of the electrophoretic mobility of the micelles suspended in the liquid. Morphological Analysis. The morphology of the micelles was observed by a field emission-scanning electron microscope (FESEM, mod. LEO 1525, Carl Zeiss SMT AG, Oberkochen, Germany). The samples were prepared using the solvent evaporation method. The polymer was dissolved in dichloromethane, then water was dropped in the solution up to a concentration of 2 × 10−5 mol/L. The formed emulsion was vigorously stirred overnight at room temperature in order to evaporate the dichloromethane and obtain the micellar solution. A drop of this solution was put on a carbon tab previously stuck to an aluminum stub (Agar Scientific, Stansted, U.K.) and kept overnight in a vacuum oven at 37 °C. Then, samples were coated with gold (layer thickness 250 Å) using a sputter coater (mod. 108 Å, Agar Scientific). For the micelles loaded with the paclitaxel, the unloaded active principle was separated in the solution using a centrifuge at 3000 rpm for 30 min. To better evaluate the morphology of the micelles, two detectors provided by the FESEM were used: in-lens and SE2 (secondary electron). Synthesis of FITC-Labeled Copolymers. The procedure of labeling with fluorescein isothiocyanate (FITC) is described herein for the mPEG2K-(PDLLA)2 copolymer (Table 1, run 9). The preparation of all the other fluorescent-labeled samples was performed by analogous procedures. In a 25 mL reaction flask, equipped with a magnetic stir bar, and under a nitrogen atmosphere, fluorescein isothiocyanate (0.295 mmol) and N,N′-carbonyldiimidazole (0,290 mmol) were dissolved in 6 mL of tetrahydrofuran and stirred at room temperature for 4 h. Subsequently, the reaction mixture was added to a solution of the mPEG2K-(PDLLA)2 (0.2003 g, 4.0 KDa, 0.0498 mmol) copolymer and 0.2 equiv of triethylamine (1.38 mL, 0.00995 mmol) in 5 mL of THF, with stirring and under nitrogen. The mixture was left under stirring for 24 h. The product was purified by dialysis against water using a cellulose membrane (cutoff = 6000−8000 g/mol). The solution of the purified product was lyophilized. An orange solid was recovered (0.1603 g, 0.0362 mmol, yield = 73%). The FITC-labeled copolymers were characterized by 1H NMR in CDCl3 and by IR analysis (see Supporting Information). 1 H NMR (300 MHz, CDCl3) δ 1.54 (m, 288), 3.64 (s, 454), 5.17 (m, 96), 6.61 (m, 4), 6.75 (m, 3), 7.45 (m, 2), 7.76 (m, 2). Cell Lines. HEK293t cell line derived from human embryonic kidney cells and HeLa cell line derived from human cervix adenocarcinoma were growth in complete DMEM (Invitrogen, Milan, Italy) supplemented with 10% FBS and 1% penicillin/ streptomycin (Invitrogen). All cell lines were kept at 37 °C in a 5% CO2 humidified incubator. Flow Cytometry Analysis. HEK293t cell line was analyzed by flow cytometry after polymers treatment between 2 and 24 h. Cells were seeded into sterile 6-well plates (1 × 105) and allowed to settle for 24 h. Then cells were treated with polymers for 2 and 24 h and were washed with PBS 1X (Sigma Aldrich, Milan, Italy) twice. Cells were acquired by flow cytometry using a BD Facs Canto II Flow Cytometer (San Jose, Ca, U.S.A.) and were analyzed with the software FlowJo 7.6.4/9.3. version. The statistical analysis was determined using GraphPad Prism 4 software. Confocal Microscopy Analysis. HEK293t cells were seeded on a cover glass placed into six-well plates (1 × 105) and allowed to settle for 24 h. Then, linear and branched copolymers were added for 2 and 24 h. After the incubation, the medium was removed, cells were washed 2 times with PBS 1×. Cells were fixed with fixation solution (Cytofix kit; BD Biosciences San Diego California) for 15 min at 4 °C and washed twice with permeabilization solution (5× PBS, 5% BSA,5%

■

RESULTS AND DISCUSSION Rationale for the Design of PEG-PLA Copolymers with Enhanced Micellar Stability. A first parameter, strongly influencing the micellar properties, is the hydrophobic/ hydrophilic weight ratio. In amphiphilic copolymers the hydrophobic chain length parallels with increased stability and reduced CMC of the micelles.24,25 In the case of linear poly(ethylene glycol)-poly(D,L-lactide) block copolymers, a clear decrease of the size distribution of the micelles was observed by increasing the molecular weight of the PLA segment, and unimodal micelles were formed for the block copolymers having PLA/PEG weight ratio in the range 0.50− 1.28.6 As a consequence, all the copolymer samples of this work were prepared by setting a PLA/PEG weight ratio in the same range (see Table 1). The PLA/PEG weight ratio was in the range 1.0−1.5 for all the copolymers, apart from the sample mPEG5K-(PD,LLA)2 (Table 1, run 2), prepared with a PLA/ PEG weight ratio of 0.55 for comparison with the sample mPEG2K-(PD,LLA)2 with which it shares the architecture and the same PD,LLA blocks lengths, but differs for the PEG molecular weight (Table 1, run 9). Furthermore, intermolecular interactions in the micelle core could also influence stability. In this respect, the architecture of the copolymers could have a strong influence.7−9 The copolymers described in this work have been designed with a “tree shaped” structure, that is, a linear polymer chain made of mPEG having from one to four PLA arms (Scheme 1). The 406


Biomacromolecules

Article

ratio between PLA/PEG weight ratio, were characterized by 1H NMR and gel permeation chromatography (GPC; Table 1). These measurements showed that the copolymers were obtained as the exclusive polymerization product. As a matter of fact, Mn for all the samples was higher than that of the starting mPEG core. GPC analysis showed monomodal curves with narrow molecular weight distribution for all the copolymers (PDI = 1.02−1.22), which indicates that the products were not mixtures of different copolymers and homopolymers. However, star or hyperbranched copolymers generally show lower hydrodymanic volume in solution compared to linear analogs of similar molar mass.30,31 As a result, the values obtained by GPC should be regarded with special care. Therefore, the actual molecular weight of the copolymers was evaluated by NMR (Mn,NMR), being known the molecular weight of the mPEG trunk, as previously reported.14 A good agreement with the Mn,NMR and the theoretical molecular weight Mnth was noticed (see Table S1 in the Supporting Information). The PLA/mPEG weight ratio, and the number of lactide units in each arm were also determined by NMR.14 The used synthetic pathways allowed a perfect tuning of these features, with a perfect control of the macromolecular architecture. Micellization of mPEG-(PLA)n Copolymers Monitored by Fluorescence Spectroscopy. The aggregation behavior of branched amphiphilic copolymers with respect to the corresponding linear ones is of crucial importance for the evaluation of aggregates stability in solution and consequently for their applicability as drug carriers. On the other side, it is reported that even unimolecular micelles correlate with the biodistribution, potential toxicity, and antitumor activity.32−34 The formation of aggregates of amphiphilic copolymers in aqueous medium was detected by evaluating the critical micelle concentrations (CMC). CMC is correlated to the nature and characteristics of the hydrophobic part of an amphiphilic copolymer. Intramolecular and intermolecular aggregate forces leading to the formation of micelles are proportional to hydrophobicity. A decrease in the hydrophobic interactive forces may affect the micelles stability, leading to higher amount of copolymer needed to form the micelles and consequently to an increase of CMC value.11 CMCs of the synthesized copolymers were determined by means of a fluorescence spectroscopy method using pyrene as a probe. The micelle solutions were prepared by the dialysis method with the solubilization of copolymers in a watermiscible organic solvent such as acetone. Micelles were formed adding water to the copolymer solutions, and dialyzing against water to remove the solvent. The copolymers aqueous solutions at different concentrations were then used for the preparation of solutions containing pyrene. The CMC values of mPEG-(PLA)n copolymers with different compositions are listed in Table 2 and are in the range 3.8 × 10−6 to 2.5 × 10−5 mol/L. The CMC of mPEG(PD,LLA) are lower than that of mPEG-(PLLA) (e.g., 3.8 × 10−6 for mPEG5K-(PD,LLA), run 1 vs 1.1 × 10−5 for mPEG5K(PLLA), run 4). Given that they share the same mPEG (5000 Da) moiety and the linear architecture, the tacticity of PLA blocks is a major determining factor for their CMCs. This behavior is nicely in agreement with previous literature results by Shaver et al.11 and Wang et al.,35 where a decrease of CMC by decreasing the PLA block stereoregularity was reported. The tacticity of PLA, on the contrary, is irrelevant for the branched copolymers. In these latter the CMCs seem more affected by

presence of the PLA segments could influence the micellar stability favoring intermolecular interactions in the core. For comparison purpose, linear block copolymer of comparable molecular weight were also prepared. Stereochemistry, moreover, plays an important role in determining the physicochemical properties of the polymer. Not only the physical properties (melting point, glass transition temperature, crystallinity) but also the biodegradation of these polymers relate directly to their microstructure. For instance, the in vitro degradation of poly(L-lactide) (PLLA) is much slower than that of poly(D,L-lactide) (PD,LLA).26 It is expected also that changes in the tacticity of the hydrophobic block may influence the kinetic stability, encapsulation efficiency, and transport of an entrapped therapeutic agent in drug delivery applications. Previous works on polymethacrylic acids27 and on P(HPMA)-block-P(LA) copolymers23 underlined this aspect. We also predicted that the tacticity of the PLA chains could influence the self-assembly of the mPEG-(PLA)n copolymers in solution, and their potential use as drug delivery systems.14 Very recently, variation in micellization and degradation properties of linear diblock PEG-PLA copolymers featuring varying tacticities in the PLA blocks have been reported.11 The stereochemistry of the hydrophobic segments was therefore taken into account: copolymers bearing stereoregular isotactic PLLA branches, or stereoirregular, atactic PD,LLA have been prepared. Finally, it is known from the literature that the molecular weight distribution of the constituent copolymers could affect the size distribution of the micelles. Providing that the micelle has a core−shell structure, due to the phase separation, PLA segments should be uniformly distributed in the core. This condition obviously requires that the PLA segments have a narrow distribution in their molecular weight.6 Consequently, a controlled synthetic pathway to obtain copolymers with narrow polydispersities was selected.14 Synthesis of the mPEG-(PLA)n Copolymers. The copolymers were obtained by using a simple, fast, and versatile chemical approach that allowed a perfect tuning of molecular weight, PDI, and number of branching. The synthesis was achieved through a “core-first” approach on a linear mPEG initiator-core, modified in order to bear two or four hydroxyl end groups as the starting point for the ring-opening polymerization (ROP) of L- or D,L-lactide. For the synthesis, a hydrophilic linear core based on mPEG (Mn 2000 and 5000 Da) was chosen. The starting core, with one terminal hydroxyl group, n = 1, was dendronized to obtain a first generation core bearing two hydroxyl groups. A novel macromolecular generation was then grown from these branching points to achieve the second generation core bearing four hydroxyl groups.28,29 The ring-opening polymerization (ROP) of L- or D,L-lactides were carried out using the so prepared macroinitiators in the presence of trimethylaluminum in toluene solution at 70 °C (Scheme 1). The ring-opening occurred with retention of configuration. Polymerizations performed in the presence of Llactide afforded isotactic polymer, while in the presence of the racemic mixture (D,L-lactide), atactic blocks were obtained. This approach, finally, resulted in “tree-shaped” polymers, with one (AB structure), two (AB2 structure), and four (AB4 structure) atactic or isotactic polyester arms.14 The use of AlMe3 as initiator for the ROP of lactide allowed a perfect control of the PDI of the obtained copolymer samples, minimizing deleterious transesterification reactions. The obtained copolymers with different architectures, and fixed 407


Biomacromolecules

Article

Table 2. Microstructural Details, CMCs, and ΔG Micelles Formation of mPEG-(PLA)n Copolymers run

sample

1

mPEG5K(PD,LLA) mPEG5K(PD,LLA)2 mPEG5K(PD,LLA)4 mPEG5K(PLLA) mPEG5K(PLLA)2 mPEG5K(PLLA)4 mPEG2K(PLLA) mPEG2K(PD,LLA) mPEG2K(PD,LLA)2

2 3 4 5 6 7 8 9 a

LA units/ arm

Mn,NMR KDa

Mn,GPC KDa

CMC (mol/L)

ΔGa (KJ/ mol)

37

10.4

18.2

3.8 × 10−6

−30.94

10

7.8

9.9

4.5 × 10−6

−30.52

13

12.3

11.8

1.0 × 10−5

−28.54

36

10.1

10.8

1.1 × 10−5

−28.30

19

10.3

10.8

4.5 × 10−6

−30.52

12

11.9

11.6

1.0 × 10−5

−28.54

16

4.2

3.7

2.5 × 10−5

−26.27

14

4.0

3.4

4.5 × 10−6

−30.52

10

4.9

5.9

3.8 × 10−6

−30.94

probably more copolymers will self-assembly into one micelle, to reduce the surface tension in the hydrophobic core−solvent interface, and thus increasing the free energy of micellization. Micellar Size Distribution and Zeta Potential. The micellar size distribution was evaluated both at pH 7 (Figure 1) and pH 5 (Figure 2) to simulate the cytoplasm and lysosomial environment respectively (see also Tables S2 and S3 in the Supporting Information). Data reported in Figure 1 show the presence of a bimodal size distribution at pH 7. It was detected the presence of two peaks indicating polymer aggregations with two different diameters, the first of which was in the range 52−300 nm and the second in the range 730−1073 nm. The smallest diameter was attributed to the dimension of a single polymeric micelle while the second, that was also the most abundant, would derive from the aggregation of a few micelles. It is worth noting that the diameter relative to the higher aggregation was generally 4-fold higher that the lower one, indicating that the aggregation of four micelles was particularly stable. Interestingly, the only sample able to give a monomodal distribution characterized by the presence of the lower aggregation was the branched mPEG2K-(PD,LLA)2 (PLA/mPEG 1.4 w/w, LA units/arm ∼ 10). The corresponding copolymer mPEG5K(PD,LLA)2, characterized by the same architecture, similar number of LA units (∼10 per arms) but mPEG 5K and PLA/ mPEG 0.55 w/w, gave exclusively micelles aggregation, confirming the importance of the hydrophobic/hydrophilic weight ratio for the formation of stable micelles. However, such a ratio is not the only parameter to influence the micelles stability. In fact, the copolymer mPEG5K-(PLLA)2, with

ΔG = RT ln CMC.

the degree of branching: the two arms mPEG-(PLA) 2 copolymers show CMC values 1 order of magnitude lower than that of four arms mPEG-(PLA)4 copolymers and comparable with that of the corresponding linear copolymers. This unexpected behavior suggests that the major steric hindrance of the hydrophobic part in the four arms copolymers affects the intermolecular aggregate forces. In this case,

Figure 1. Micelles size distribution obtained at pH 7. 408


Biomacromolecules

Article

Figure 2. Micelles size distribution obtained at pH 5.

indicated that the samples exist as a dynamic system. In fact, the presence of aggregates, likely formed by 2−4 micelles that dynamically aggregate and disaggregate, could cause the variability of the zeta potential values. The micelles formed by mPEG2K-(PD,LLA)2, on the contrary, showed a zeta potential of −41 mV, meaning that such a copolymer tends to form stable micelles, thus, confirming the DLS data that indicated the presence of only one diameter size and no higher aggregation. Furthermore, the values of the zeta potential were all negative, meaning that the micelle surfaces are negative, well explaining the increasing of aggregation showed by DLS at pH 5. Morphological Analysis. The FESEM photomicrograph in Figure 3 shows that micelles obtained from mPEG2K(PD,LLA)2 had a spherical shape.

isotactic PLA blocks, still gave micelles aggregation even with PLA/mPEG weight ratio 1.1. Apparently, the thermodynamic and kinetic stability derive from a delicate hydrophobic/ hydrophilic balance determined both by the relative length and spatial distribution of the two components. From the micellar size distribution it can be concluded that the presence of branches, of the appropriate hydrophobic/hydrophilic balance and of atactic PLA blocks can contribute to enhance hydrophobic interactions influencing micelles stability. The DLS analysis performed at pH 5 (Figure 2) gave similar results, i.e., a bimodal micellar size distributions, albeit with an increase in the percentage of aggregates. The sample mPEG2K(PD,LLA)2 showed now two peaks, indicating that once inside the lysosomes, micelles might aggregate. We also performed zeta potential measurements, in order to evaluate the micelles stability with a different method. Data reported in Table 3 showed that, apart for the sample mPEG2K(PD,LLA)2, all the other samples had a value between −20 and −30 mV, typical of instable systems. These data explained the attitude of the micellar suspension to aggregate, in accordance with the DLS analysis. The broad standard deviation values Table 3. Zeta Potential of the Micelles sample

zeta potential (mV)

std dev (mV)

mPEG5K-(PD,LLA) mPEG5K-(PD,LLA)2 mPEG5K-(PD,LLA)4 mPEG5K-(PLLA) mPEG5K-(PLLA)2 mPEG5K-(PLLA)4 mPEG2K-(PLLA) mPEG2K-(PD,LLA) mPEG2K-(PD,LLA)2

−27 −20 −26 −31 −24 −24 −29 −33 −41

16 7 12 8 6 7 10 7 11

Figure 3. FESEM photomicrograph of the micelles obtained from the sample mPEG2K-(PD,LLA)2 (SE2 detector). 409


Biomacromolecules

Article

FITC-labeled micelles. Then, the cells were analyzed by fluorescence activated flow cytometry for their fluorescence intensity signals. In the first experimental set, the HEK293t cell line was treated for 2 and 24 h with the following copolymers: mPEG5K-(PLLA), mPEG2K-(PD,LLA)2, mPEG5K-(PLLA)2, and mPEG5K-(PLLA)4 (Figure 5A,B). Among the tested copolymers, the strongest fluorescent intensity signal was observed using mPEG2K-(PD,LLA)2. The data suggested that the interactions pattern of mPEG2K-(PD,LLA)2 polymer with HEK293t might be influenced by the molecular weight of PEG (2000 Da). Therefore, it was conceivable that the intrinsic characteristic of copolymers structure might regulate the ability of nanoparticles to form micelles able to bind cells. To further study the relevance of the PEG molecular weight, the copolymers sharing the same PEG moiety of 2000 Da (see Table 1, runs 1−7) were tested again on the HEK293t cell line with 2 and 24 h treatments (Figure 5C-D). The micelles composed by mPEG2K-(PD,LLA)2 gave the most reproducible and highest fluorescence intensity signal reaching meaningfully statistic values (Figure 5E-F). However, in some experiments also the linear mPEG2K-(PD,LLA) showed a high fluorescence intensity (Figure 5E). The fluorescence intensity increased over the time as demonstrated comparing the micelles median of fluorescence intensity (MIFs) at 2 and 24 h. It is worth noting that the MIF gave data on the capability of different micelles to stain tumor cells at single cell level. On the contrary, to obtain information at cell population level, we analyzed the percentage of cells that are stained by the different micelles (Figure 5G,H) after 2 and 24 h of incubation. To validate and extend the previous data, a panel of linear and AB2 copolymers, bearing mPEG2K and mPEG5K (see Table 1, runs 4, 5, 7, and 9), was screened at micellar concentration using 2 and 24 h incubation times with HeLa tumor cells (Figure 6A,B). These data showed that mPEG2K-(PLLA) polymer was able to stain more than mPEG2K-(PD,LLA)2 after 2 and 24 h. The micelles staining capability of the tumor cells increase over the time; compare Figure 4C with Figure 4D. The micelles composed by mPEG5K-(PLLA) surprisingly in this cell system showed a nice staining capability. As described before, the micelles staining capability was analyzed also as percentage of stained cells within the tumor population (Figure 6E,F). On the HeLa cell lines the mPEG2K-(PD,LLA)2, mPEG2K-(PLLA), and mPEG5K-(PD,LLA) micelles gave the highest rate of stained cells. The data strength was evaluated by statistical analysis performed using the MFI for each polymer tested (Figure 6C,D). In both tumor cell lines studied (293t and Hela), the cell population viability percentage did not change over the incubation times used (data not shown). We concluded from this two experimental sets on both the tumor cell lines that the highest efficiency and reproducibility in staining tumor cells is confined to the polymer with a PEG of 2000 Da, and bearing two PD,LLA arms. Therefore, the mPEG2K-(PD,LLA)2 is the only polymer that generate micelles able to stain efficiently (MFI) both the kidney (HEK293t) and uterine (HeLa) derived tumor cell lines. To further investigate the nature of the micelles interaction with tumor cells, we set to dissect the micelles/cells interaction in confocal microscopy experiments. Cell Distribution Analysis of mPEG5K-(PLLA), mPEG5K(PLLA)2, mPEG2K-(PD,LLA)2, and mPEG2K(PLLA) Copolymers in Human Cancer Cells HEK293t and HeLa by Confocal Microscopy. To understand if the staining signals

The micellar size distribution measured from the analysis of the FESEM photomicrographs showed smaller diameters compared to the one given by the DLS analysis. This is due to the preparation of the sample that for the FESEM consists in dehydrating the micelles on the sample holder, that reduces the diameters of micelles compared to the one evaluated in suspension by DLS.36 In fact, the hydrophilic mPEG shell is probably highly hydrated when the micelles are in suspension and the reduction of micelles size dimension is probably due to a lack of water from mPEG. In Figure 4, FESEM photomicrograph acquired using the inlens detector allowed obtaining images differences in the

Figure 4. FESEM photomicrograph of the micelles obtained from the sample mPEG2K-(PD,LLA)2 (in-lens detector).

electronic variations on the sample with high lateral resolution and confirmed the spherical micelles shape and the smaller dimensions with respect to the ones obtained by DLS. We also evaluated the size of micelles loaded with the hydrophobic antitumor drug paclitaxel (PTX) by FESEM. As expected, the corresponding diameter of micelles increased with respect to the ones unloaded for the presence of the drug molecules (Table 4). Table 4. Micellar Size Distribution of mPEG2K-(PD,LLA)2 Measured by FESEM Photomicrographs and DLS sample

diameter (nm; FESEM)

diameter (nm; DLS)

mPEG2K-(PD,LLA)2 mPEG2K-(PD,LLA)2 + PTX

143 ± 28 171 ± 40

267 ± 77

Cytofluorimetric Analysis of the Cellular Interactions of PEG-PLA Copolymers. To monitor the interaction between the polymers and tumor cells, the fluorescent dye fluorescein isothiocyanate isomer I (FITC) was conjugated at the terminal chain of the PLA. All the copolymers were used at the critical micellar concentration (CMC). Since FITC is a hydrophobic molecule, it will tend to reduce the CMC of the conjugated copolymers with respect to the free ones. As a consequence, the use of the CMC of free copolymers surely allowed the performance of cytofluorimetric analysis in the presence of micelles. As negative control the cells were incubated with free FITC dye, at the same concentration used for copolymers, as previously described. 19,20 To investigate the interactions among different micelles on a biological system, two tumor cell lines were incubated with 410


Biomacromolecules

Article

Figure 5. Flow cytometry analysis of mPEG-(PLA)n copolymer interaction with HEK293t cell line. (A, B) mPEG5K-(PLLA)4, mPEG5K-(PLLA)2, mPEG5K- (PLLA), and mPEG2K-(PD,LLA)2; polymer-derived micelles (represented by different color lines) have been added to the tumor cell culture and kept either at 2 or 24 h. Fluorescein was used as negative control. (C, D) mPEG2K-(PLLA), mPEG2K-(PD,LLA), and mPEG2K(PD,LLA)2; polymer-derived micelles (represented by different color lines) have been added to the tumor cell culture and kept either at 2 or 24 h. Fluorescein was used as negative control. (E, F) Statistical analysis of mean of fluorescent intensity (MFI) data obtained from five independent experiments. Statistical analysis with pairwise, two-tailed Student t test; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. (G, H) Percentage of positive cells after 2 and 24 h treatment with PEG-PLA polymers where fluorescein and polymers were compared by the same statistical analysis used for panels E and F. Statistical analysis was accomplished by Student t test, pairwise, two-tailed. Symbols: *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001.

The nuclei were stained with Dapi, nanoparticles were characterized by the presence of FITC to the terminal chain of poly(lactide). We observed that the fluorescence signals belonging to mPEG2K-(PD,LLA)2 micelles incubation with HEK293t clearly belong to cytoplasm internalized nanoparticles. Indeed, after 2 h we observed an average of 1 nanoparticle per cell, while after 24 h of the treatment, several nanoparticles were found in the tumor cell cytoplasm (Figure 5A). mPEG2K(PLLA) micelles enter in the tumor cell cytoplasm in less efficient way since they can be found outside the cells as well. In contrast mPEG5K-(PLLA) copolymers

collected by FACS analysis derive from an endocellular or cell surface localization of the micelles, confocal microscopy experiments were performed on selected copolymer samples. HEK293t were incubated with mPEG5K-(PLLA), mPEG5K(PLLA)2, mPEG2K(PLLA), and mPEG2K-(PD,LLA)2 copolymers (Table 1, runs 4, 6, and 9) and fluorescein for 2 and 24 h (Figure 7A,B). Images were analyzed by using confocal microscopy Leica-TCS-SP2. To evaluate the cell distribution of polymeric micelles, cells were analyzed using at least three fields for each experimental point. 411


Biomacromolecules

Article

Figure 6. Flow cytometry analysis on HeLa cell line and interaction of mPEG-(PLA)n copolymer. (A, B) mPEG2K-(PD,PLLA)2, mPEG2K-(PLLA), mPEG5K- (PLLA), and mPEG5K-(PLLA); polymer-derived micelles (represented by different color lines) have been added to the tumor cell culture and kept either at 2 or 24 h. Fluorescein was used as negative control. (C, D) Statistic analysis of mean of fluorescent intensity (MFI) data obtained from five independent experiments. Statistical analysis with pairwise, two-tailed Student t test; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001. (E, F) Percentage of positive cells after 2 and 24 h treatment with PEG-PLA polymers where Fluorescein and polymers were compared by the same statistical analysis used for panels E and F. Statistical analysis was accomplished by Student t test, pairwise, two-tailed. Symbols: *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001.

(Figure 6B). As observed with HEK293t cells experiments, the mPEG2K-(PD,LLA)2 micelles enter inside the cells. On the contrary, the linear copolymer mPEG2K-(PLLA) gave less reproducible results and showed the presence of fluorescinated particles even outside the cells. It should be noted that the mPEG 2K -(PD,LLA) 2 micelles localize in the HEK293t cytoplasm, while in HeLa cells they can be found in nuclei and cytoplasm. Therefore, we concluded that mPEG2K(PD,LLA)2 micelles are specifically entering in both tumor cell type. Cell Toxicity Assay of mPEG2K-(PD,LLA)2 Copolymer in HeLa Cell Line. The drug delivery capability of mPEG2K(PD,LLA)2 was evaluated by the MTT (3-(4,5-dimethiltiazol-2yl)-2,5-diphenyl-tetrazolium bromide) cell viability assay. HeLa cells were used as cell targets (Figure 8A−C). Cells were treated with paclitaxel (PTX) as a positive control, empty

generated aggregate outside the cells, too big to enter in the cell cytoplasm, mPEG5K-(PLLA)2 does not enter in the cells and is quickly washed away during the sample preparation, while free fluorescein presented a diffuse pattern on the cells (Figure 5A,B). This kind of polymer did not penetrate the cells. As matter of fact the DLS analysis disclosed for these nanoparticles bimodal distribution and formation of large aggregates. Thus, mPEG2K-(PD,LLA)2 polymer-derived micelles staining is due to their capability to enter in the tumors cytoplasm compartment most probably by endocytosis. It is noteworthy that their localization is outside the nuclei as demonstrated by the nuclear staining with DAPI and micelles FITC reciprocal localization. To extend our observations this experimental setting was repeated using mPEG2K-(PD,LLA)2 and mPEG2K-(PLLA) copolymers (Table 1, runs 7 and 8) on the HeLa cell line 412


Biomacromolecules

Article

Figure 7. Confocal microscopy analysis of cellular distribution of mPEG-(PLA)n copolymers in human cancer cells HEK293t and HeLa. (A) The distribution of mPEG-(PLA)n copolymers (green) on HEK293t cell line after 2 and 24 h incubation time. (B) The distribution of mPEG-(PLA)n copolymers (green) on HEK293t cell line after 2 and 24 h incubation time. The cell nuclei were stained with DAPI (blue). For each panel, a single plane confocal image shows the central section of the cell. Scale bars: 26.5 μm.

tacticity. The two-arm copolymers gave more stable micelles showing CMC values comparable with that of the linear ones and 1 order of magnitude lower than that of the corresponding four arms copolymers. In the latter case, the major steric hindrance of the four hydrophobic arms influences the intermolecular aggregate forces most probably reducing the surface tension in the core−solvent interface. The two-arm mPEG2K-(PDLLA)2 characterized by mPEG 2000 Da and atactic PLA appeared the best copolymer in giving a discrete formation of monodispersed micelle, as shown by DLS and zeta potential measurements. On the contrary, the other structures showed higher tendency to aggregate and consequently very low stability both at pH 7.4 and 5.0. Indeed, we demonstrated that the tumor uptake of the micelles mPEG-(PLA)n depend on the chemical moieties and the copolymer architecture as well. Our functional data in biological systems demonstrated that the mPEG2K-(PD,LLA)2 enter in two different tumor cell lines with high efficiency. Its intracellular distribution is cell-type dependent. In the HEK293t cell line, the mPEG2K-(PD,LLA)2 distributed in the cytoplasm, while in HeLa cells they can reach the nuclei. Moreover, the MTT test showed that mPEG2K-(PD,LLA)2 copolymers, once loaded with PTX, are able to kill tumor cells. In contrast, the copolymers having mPEG5K, such as mPEG5K-(PLLA)2, do not penetrate the cells. As matter of fact, the DLS analysis disclosed for these nanoparticles bimodal distribution and formation of large aggregates.

mPEG2K-(PD,LLA)2 copolymer as a negative control, and paclitaxel-loaded copolymer mPEG2K-(PD,LLA)2PTX. The concentrations of paclitaxel and PTX-copolymer used in dose response titration experiments ranged from 0.001 to 1000 nm (Figure 8A−C). The mPEG2K-(PD,LLA)2PTX loaded micelles gave a considerable high antitumor cytotoxic effect at 1.56 × 10−6 mol/L (Figure 8A). These results indicate that micelles generated using mPEG2K-(PD,LLA)2 copolymer are able to deliver the drug inside the HeLa cells, inducing tumor cells death.

■

CONCLUSIONS

“Tree-shaped” copolymers mPEG-(PLA)n, where n = 2 or 4, mPEG = 2000 or 5000 Da and PEG/PLA ratio ∼1:1 w/w have been successfully synthesized. Parameters such as the length of hydrophobic and hydrophilic blocks, the architecture and the tacticity of the PLA blocks were properly modulated with a good control over the final architecture, as proved by the 1H NMR and GPC characterization. The critical micelle concentrations, the micelles dimension and polydispersity, and the zeta potential were evaluated in order to understand the architecture and chemical composition effect on autoassembly process and micelle stability. Our data demonstrated that the PLA tacticity influence the CMC of linear copolymers. However, this stabilizing effect was not observed for branched architecture characterized by shortest PLA arms. Apparently, CMCs of branched copolymers were influenced more by the degree of branching rather than PLA 413


Biomacromolecules

Article

terial is available free of charge via the Internet at http://pubs. acs.org.

■

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]; [email protected]; pappalardo@ unisannio.it. Author Contributions †

These authors equally contributed to the paper (C.G. and G.C.). Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS L.I. is grateful to MIUR (FARB 2011) for financial support. D.P. is grateful to Università del Sannio (FAR 2012) for financial support. E.C. is grateful to Associazione Italiana per la Ricerca sul Cancro (AIRC) (AIRC IG 10189), Axel WennerGren Foundation. E.C. work has been supported by a UICC International Cancer Technology Transfer Fellowship.

■

(1) Fang, J.; Nakamura, H.; Maeda, H. Adv. Drug Delivery Rev. 2011, 63, 130−151. (2) Kataoka, K.; Kwon, G. S.; Yokoyama, M.; Okano, T.; Sakurai, Y. J. Controlled Release 1993, 24, 119−132. (3) Owen, S. C.; Chan, D. P. Y.; Shoichet, M. S. Nano Today 2012, 7, 53−65. (4) Gaucher, G.; Dufresne, M.-H.; P. Sant, V. P.; Kang, N.; Maysinger, D.; Leroux, J.-C. J. Controlled Release 2005, 109, 169−188. (5) Jain, R. A. Biomaterials 2000, 21, 2475−2490. (6) Yasugi, K.; Nagasaki, Y.; Kato, M.; Kataoka, K. J. Controlled Release 1999, 62, 89−100. (7) Tanodekaew, S.; Pannu, R.; Heatley, F.; Attwood, D.; Booth, C. Macromol. Chem. Phys. 1997, 198, 927−944. (8) Venkatraman, S. S.; Jie, P.; Min, F.; Freddy, B. Y. C.; Leong-Huat, G. Int. J. Pharm. 2005, 298, 219−232. (9) Sallam, L. E.; Dean, D.; Bray, T. L. Polymer 2006, 47, 310. (10) Joo, M. K.; Sohn, Y. S.; Jeong, B. Macromolecules 2007, 40, 5111−5115. (11) Agatemor, C.; Shaver, M. P. Biomacromolecules 2013, 14, 699. (12) Kim, D. W.; Kim, S. Y.; Kim, H. K.; Kim, S. W.; Shin, S. W.; Kim, J. S.; Park, K.; Lee, M. Y.; Heo, D. S. Ann. Oncol. 2007, 18, 2009− 2014. (13) Glavas, L.; Olsén, P.; Odelius, K.; Albertsson, A.-C. Biomacromolecules 2013, 14, 4150−4156. (14) Izzo, L.; Pappalardo, D. Macromol. Chem. Phys. 2010, 211, 2171−2178. (15) Adami, R.; Liparoti, S.; Izzo, L.; Pappalardo, D.; Reverchon, E. J. Supercrit. Fluids 2012, 72, 15−21. (16) Li, T.; Han, R.; Wang, M.; Liu, C.; Jing, X.; Huang, Y. Macromol. Biosci. 2011, 11, 1570−1578. (17) Lundberg, P.; Walter, M. V.; Montaňez, D.; Hult, A.; Nyström, A.; Malkoch, M. Polym. Chem. 2011, 2, 394−402. (18) Maglio, G.; Nicodemi, F.; Conte, C.; Palumbo, R.; Tirino, P.; Panza, E.; Ianaro, A.; Ungaro, F.; Quaglia, F. Biomacromolecules 2011, 12, 4221−4229. (19) Chen, H.; Kim, S.; L., L.; Wang, S.; Park, K.; Cheng, J.-X Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 6596−60. (20) Savić, R.; Luo, L.; Eisenberg, A.; Maysinger, D. Science 2003, 300, 615−618. (21) Li, Y.; Rong, Q. X.; Maitani, Y.; Nagay, T. Nanotechnology 2009, 20, 055106. (22) Hemlinger, G.; Yuan, F.; Dellian, M.; Jain, R. K. Nat. Med. 1997, 3470−477.

Figure 8. mPEG2K-(PD,LLA)2 copolymer drug delivery analysis in HeLa cell line by cell toxicity assay. (A−C) Cells were treated with paclitaxel as positive control; empty mPEG2K-(PD,LLA)2 polymer and paclitaxel-loaded mPEG2K-(PD,LLA)2 micelles. The concentrations used were 1.56 × 10−6 mol/L, 1.56 × 10−7 mol/L, 1.56 ×10−10 mol/L. (A) Untreated and paclitaxel, empty polymer, and paclitaxel-loaded micelles treated cells were compared. Statistical analysis was accomplished by Student t test, pairwise, two-tailed. Symbols: *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001.

In conclusion, we found that a complex and fine balance of several parameters such as the hydrophobic/hydrophilic ratio, the mPEG block length, and the presence of branches influenced the micelles stability and its capability to target tumor cells in an unpredictable way. Here, we proposed that the copolymer mPEG2K-(PD,LLA)2 should be used to generate a new micelle class able to target efficiently the tumor cell and delivery antitumor cytotoxic effect.

■

REFERENCES

ASSOCIATED CONTENT

S Supporting Information *

Experimental conditions and molecular weights of the mPEG(PLA)n copolymers, micelles DLS analysis data, 1H NMR spectra of copolymers, and 1H NMR and IR spectra of the FITC conjugated mPEG2K-(PD,LLA)2 copolymer. This ma414


Biomacromolecules

Article

(23) Barz, M.; Armiñań , A.; Canal, F.; Wolf, F.; Koynov, K.; Frey, H.; Zentel, R.; Vicent, M. J. J. Controlled Release 2012, 163, 63−74. (24) Kang, N.; Leroux, J.-C Polymer 2004, 45, 8967−8980. (25) Jette, K. K.; Law, D.; Schmitt, E. A.; Kwon, G. S. Pharm. Res. 2004, 21, 1184−1191. (26) Schindler, A.; Jeffcoat, R.; Kimmel, G. L.; Pitt, C. G.; Wall, M. E.; Zweidinger, R. In Contemporary Topics in Polymer Science; Pearce, E. M., Schaefgen, J. R., Eds.; Plenum Press: New York, 1977; Vol. 2, p 251. (27) Izzo, L.; Griffths, P. C.; Nilmini, R.; King, S. M.; Wallom, K. L.; Ferguson, E. L.; Duncan, R. Int. J. Pharm. 2011, 408, 213−222.24. (28) Sun, X.; Zhang, H.; Huang, X.; Wang, X.; Zhou, Q.-F. Polymer 2005, 46, 5251. (29) Ihre, H.; Padilla de Jesús, O. L.; Fréchet, M. J. J. Am. Chem. Soc. 2001, 123, 5908. (30) Stradman, S.; Tenhu, H. Polymer 2007, 48, 3938. (31) Kowalczuk-Bleja, A.; Sierocka, B.; Muzynski, J.; Trzebicka, B.; Dworak, A. Polymer 2005, 46, 8555. (32) Searle, F.; Gac-Breton, S.; Keane, R.; Dimitrijevic, S.; Brocchini, S.; Duncan, R. Bioconjugate Chem. 2001, 12, 711. (33) Keane, R.; Gac-Breton, S.; Searle, F.; Duncan, R. J. Pharm. Pharmacol. 2000, 52 (Suppl), 52. (34) Mella, M.; Izzo, L. Polymer 2010, 51, 3582. (35) Zhang, J.; Wang, L.-Q.; Wang, H.; Tu, K. Biomacromolecules 2006, 7, 2492. (36) Guo, X. D.; Tandiono, F.; Wiradharma, N.; Khor, D.; Tan, C. G.; Khan, M.; Qian, Y.; Yang, Y.-Y. Biomaterials 2008, 29, 4838−4846.

415


Different Insight into Amphiphilic PEG-PLA Copolymers: Influence of

Recommend Documents