Article pubs.acs.org/Biomac
HPMA Copolymers as Surfactants in the Preparation of Biocompatible Nanoparticles for Biomedical Application Annette Kelsch,†,‡ Stephanie Tomcin,‡ Kristin Rausch,§ Matthias Barz,† Volker Mailan̈ der,‡,∥ Manfred Schmidt,§ Katharina Landfester,*,‡ and Rudolf Zentel*,† †
Institute of Organic Chemistry, Johannes Gutenberg-University Mainz, Duesbergweg 10-14, 55128 Mainz, Germany Max Planck Institute for Polymer Research, Ackermannweg 10, 55128 Mainz, Germany § Institute of Physical Chemistry, Johannes Gutenberg-University Mainz, Jakob-Welder-Weg 11, 55128 Mainz, Germany ∥ III. Medical Clinic, University Medicine of the Johannes Gutenberg University, Langenbeckstr. 1, 55131 Mainz, Germany ‡
S Supporting Information *
ABSTRACT: In this work we describe the application of amphiphilic N-(2-hydroxypropyl)methacrylamide (HPMA)based copolymers as polymeric surfactants in miniemulsion techniques. HPMA-based copolymers with different ratios of HPMA (hydrophilic) to laurylmethacrylate (LMA; hydrophobic) units were synthesized by RAFT polymerization and postpolymerization modification. The amphiphilic polymers can act as detergents in both the miniemulsion polymerization of styrene and the miniemulsion process in combination with solvent evaporation, which was applied to polystyrene and polylactide. Under optimized conditions, monodisperse colloids can be prepared. The most promising results could be obtained by using the block copolymer with a ratio of 90/10. Preliminary cell uptake studies showed that polymer-stabilized nanoparticles have only minor unspecific cellular internalization in HeLa cells. Furthermore, cytotoxicity assays showed no particle-attributed toxicity. In addition, the copolymer-stabilized particles preserved the shape and size in human blood serum as demonstrated by dynamic light scattering. fragmentation chain transfer (RAFT) polymerization.19,20 Additionally, techniques for selective end group functionalization have been developed, broadening the chemical toolbox21,22 available for the next generation of polymers used as nanomedicines. Despite their potential, water-soluble polymers have, of course, some drawbacks, which are related to the flexible conformation of the polymer chain itself. As a result, they do not possess a well-defined shape (only an average size). There is no defined inside, in which one can safely encapsulate a sensitive compound and no defined outside, at which a targeting moiety can be presented. One may overcome these problems by using polymer colloids (size 10−100 nm), which consist of a water-insoluble hydrophobic polymer core stabilized by detergents at the outside. Polymer colloids have a stable shape and they offer a defined interface to which targeting moieties can be attached to, while a sensitive cargo can be encapsulated in the inner core. Such colloids can be made by various methods, from which the miniemulsion technique is most flexible for the encapsulation of the cargo.23,24 One variant of this method is the miniemulsion
1. INTRODUCTION Over the last decades, “nanomedicines”,1 a term describing nanoparticles, for example, polymer drug conjugates, polymer protein conjugates, and organic or inorganic nanoparticles used for medical application, gained widespread attention in medicine and biology.2 All these nanometer-sized carrier systems have in common that they modify the body distribution or presentation of the biologically active compound linked to or incorporated into them in comparison to the free compound. Most prominent are -in this context- an increased plasma half-life time -due to size related slower renal clearanceand a passive accumulation in well vascularized solid tumors due to the EPR (enhanced permeability and retention) effect.3,4 The most prominent polymers used for the synthesis of nanomedicines are so far poly(ethylene glycol) (PEG), poly[N(2-hydroxypropyl)methacrylamide] (PHPMA), poly(glutamic acid) (PGA), or dextran.5−7 Up to now, PHPMA-based drug conjugates are among the most carefully studied polymer therapeutics.8,9 HPMA copolymers are polymerized by radical polymerization and thus offer the advantage that a multitude of reactive groups can be copolymerized, which can be used to attach even sensitive functionalities.10−17 Furthermore, advances in controlled radical polymerization have enabled the synthesis of defined HPMA-based copolymers by atom transfer radical polymerization (ATRP)18 or reversible addition− © XXXX American Chemical Society
Received: September 15, 2012 Revised: November 12, 2012
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technique in combination with solvent evaporation.25 For this purpose, polymer and drug (both hydrophobic) are dissolved in a volatile water-immiscible organic solvent. Then emulsification takes place under high shear force to disperse the oil-phase in the continuous phase consisting of water and a predissolved surfactant, mostly sodium dodecyl sulfate (SDS). Subsequently, the solvent gets evaporated to obtain the final nanoparticles.26 Such negatively charged nanoparticles made from, for example, polystyrene, poly(methylmethacrylate), and poly(L-lactide) (PLLA) were taken up by HeLa cells, whereby the uptake depends, to some extent, on the polymer core, but much more on the surfactant, which was used during colloid preparation.25,27 One drawback of this preparation method is the use of classical detergents, for example, SDS. Assuming adsorption is mainly driven by the hydrophobic effect, detergents are not permanently fixed to the surface of the colloids. Due to their high critical micelle concentration (CMC) values (cmc of SDS is about 0.01 M), the detergent will diffuse away from the surface into solution causing cellular toxicity by denaturation of proteins and changes in cell membrane fluidity. Therefore, time-consuming dialysis is required to purify the colloids after preparation. Due to the fact that detergents are not permanently attached to the surface they can hardly be used for a reliable surface functionalization, for example, by binding targeting moieties (e.g., antibodies) to the surface. To overcome this problem, the use of detergent-like systems (e.g., amphiphilic polymers) with a low CMC seems attractive. They will stick well to a hydrophobic core, while their hydrophilic corona can be functionalized and used to shape the colloidal surface. Up to now, however, only a small number of polymeric surfactants has been introduced to disperse systems,28 more precisely to miniemulsion polymerization.29 According to those needs we describe in the following article, for the first time, the use of various amphiphilic HPMA/LMA copolymers in miniemulsion polymerization and miniemulsion approaches. The polymer colloids, prepared in this way, are permanently sterically stabilized with copolymers, for which routes for further functionalization are well established. Now, colloids can be synthesized being highly biocompatible, multifunctional, and based on clinically investigated materials2,30 such as PLA and PHPMA.
2.2. Synthesis of Copolymers. The polymers P1−P6 were prepared in analogy to the literature.13 Details are added as Supporting Information. 2.3. Characterization of the Copolymers. All 1H and 13C NMR spectra were recorded on a Bruker 300 MHz FT-NMR spectrometer. Chemical shifts (δ) are given in parts per million (ppm) relative to TMS. All 19F NMR spectra were recorded on a Bruker 400 MHz FTNMR spectrometer. Chemical shifts (δ) are given in ppm relative to CCl3F. Polymers were dried at 40 °C overnight under vacuum and subsequently characterized by size exclusion chromatography (SEC). SEC was performed in tetrahydrofurane (THF) as solvent using the following system: pump PU 1580, auto sampler AS 1555, UV-detector UV 1575, RI-detector RI 1530 from Jasco, and miniDAWN Tristar light scattering detector from Wyatt. Columns were used from MZ Analysentechnik: MZ-Gel SDplus 102 Å, MZ-Gel SDplus 104 Å, and MZ-Gel SDplus 106 Å. The elution diagrams were analyzed using the ASTRA 4.73.04 software from Wyatt Technology. Calibration was done using polystyrene standards. The flow rate was 1 mL/min at a temperature of 25 °C and the salt content was 0.1 mmol/mL. 2.4. Preparation of the Nanoparticles. Polystyrene-based nanoparticles were synthesized via miniemulsion polymerization. A mixture of 400 mg styrene, 8 mg initiator (V59), and 5 mg ultrahydrophobe (hexadecane) was added to the aqueous phase containing 10 mg of P1−P6 and 10 g demineralized water. After 1 h of pre-emulsification, the mixture was sonicated, while it was cooled in an ice bath for 180 s at 70% amplitude (Branson sonifier W450 digital, 1/4” tip). The polymerization was performed for 16 h at 72 °C with the stirring rate fixed at 500 rpm. For polymer particles, obtained by the solvent evaporation method combined with the miniemulsion technique, 100 mg polymer was dissolved in 2.1 g toluene. The macroemulsion was prepared by adding the aqueous phase consisting of 5 to 50 mg of dissolved P1−P6 in 10 g water to the organic phase and subsequent magnetic stirring of the mixture at high speed for 60 min. Afterward, the macroemulsion was subjected to ultrasonication under ice cooling for 180 s at 70% amplitude in a pulse regime (10 s sonication, 5 s pause) using the same sonifier as mentioned above. The obtained miniemulsion was transferred to a reaction flask with a large size neck and stirred for 1 h at 86 °C for complete azeotropic evaporation of the organic solvent.31 To avoid a reduction of the continuous phase, water was added from time to time. 2.5. Characterization of the Nanoparticles. The average size of the final polymer particles was determined by dynamic light scattering (DLS). All light scattering experiments were done using an instrument consisting of a HeNe laser (632.8 nm, 25 mW output power), an ALVCGS 8F SLS/DLS 5022F goniometer equipped with eight simultaneously working ALV 7004 correlators, and eight QEAPD Avalanche photodiode detectors. Scanning electron microscopy (SEM) was used to study the morphology of the polymer particles, dried on a silica wafer. The images were recorded by using a field emission microscope (LEO 1530 Gemini) working at an acceleration voltage of 0.74 V. 2.6. Aggregation Behavior in Human Blood Serum and Cell Culture Medium. Particle size of D6 and D8 in water and analysis of potential interaction of D6 and D8 with blood serum as well as the under section 2.7 described cell culture medium were determined by DLS analysis as described.32 In brief, solutions of these particles for light scattering experiments were prepared in a dust free flow box. Cylindrical quartz cuvettes (20 mm diameter, Hellma, Müllheim) were cleaned by dust-free distilled acetone. The particle solutions were filtered through Millex-AA syringe filter with 800 nm pore size (Merck Millipore) before use. DLS experiments were performed with the instrument described above. 2.7. Cell Culture. Human cervix adenocarcinoma cells, established from the epitheloid cervix carcinoma of a 31-year-old black woman, Henrietta Lacks (HeLa), in 1951 (Leibniz-Institute, DSMZ-German Collection of Microorganisms and Cell Cultures), were kept in Dulbecco's Modified Eagle Medium (DMEM) without phenol red supplemented with 10 vol % fetal bovine serum (FBS), 100 units penicillin, and 1 vol % GlutaMAX (all from Invitrogen, Germany).
2. EXPERIMENTAL SECTION 2.1. Materials. All chemicals were reagent grade, obtained from Aldrich and used without further purification, unless indicated otherwise. Oregon green cadaverin was purchased from Invitrogen. All solvents were of analytical grade. Pentafluorophenol was obtained from Fluorochem (Great Britain, U.K.) and distilled prior to use. Dioxane and dimethylsulfoxide (DMSO) used in the syntheses were freshly distilled from a sodium/potassium mixture. 2,2′-Azobis(isobutyronitrile) (AIBN) was recrystallized from diethyl ether and stored at −7 °C. Lauryl methacrylate was distilled at 93 °C and 6 × 10−3 mbar prior to use. Styrene (Sigma-Aldrich, 99%) was passed through a basic alumina column to remove inhibitor. Hexadecane (Merck, 99%) and N-(2,6-diisopropylphenyl)perylene-3,4-dicarboximide (PMI, BASF) were used as received. 2,2′-Azobis(2-methylbutyronitrile) (V-59) was purchased from Wako Chemicals. Polystyrene (Mw = 120000 g/mol), PDLLA_1 (Mw = 28000 g/ mol), and PDLLA_2 (Mw = 120000 g/mol) were purchased from Sigma-Aldrich. Deuterated chloroform-d1 was purchased from Deutero GmbH, dried, and stored over molecular sieves. Dialyses were performed with Cellu SepH1 membranes (Membrane Filtration Products, Inc.) with a nominal molecular weight cutoff of 1000 g/ mol and Spectra/Por membranes (Roth) with a nominal molecular weight cutoff of 3500 g/mol. B
dx.doi.org/10.1021/bm301453g | Biomacromolecules XXXX, XXX, XXX−XXX
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Scheme 1. Synthetic Pathway to Random and Block Copolymers Based on PHPMA Using the Active Ester Approach
Cells were grown in a humidified incubator at 37 °C and 5% CO2. One day before the experiment started, adherent cells were detached using 0.5% trypsin (Gibco, Germany). For the MTS assay, cells were seeded out in 96-well plates (Becton Dickinson, U.S.A.) at a density of 0.8 × 105 cells per well. For CLSM imaging, 8500 HeLas per cm2 were seeded out in ibidi μ-dish35 mm,low (IBIDI, Germany). After readhesion overnight cells were washed once with Dulbecco's phosphate buffered saline (PBS, Invitrogen, Germany) before treated with nanoparticles. Incubation times and concentration of nanoparticles differ with the kind of experiment (see below). 2.8. MTS Assay. To show that the synthesized nanoparticles have no toxic influence on HeLa cells, a MTS assay of all particles was performed. MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt) is reduced NADH-dependent by cells to a colored formazan product, which is soluble in culture medium. The concentration of formazan is photometrically detectable with a maximum absorption at 490 nm. To detect the absorption in each well a Plate Reader Infinite M1000 (Tecan, Germany) was used. Cells were incubated with nanoparticles at concentrations of 150, 300, 600, 1000, and 1200 μg/mL for 72 h in a humidified incubator at 37 °C and 5% CO2. After incubation time, the MTS assay was done in medium following the manufacturer's protocol of CellTiter 96 Aqueous One Solution Cell Proliferation Assay (Promega, U.S.A.). The absorption signal of formazan is directly proportional to the number of living cells. The cell viability can be determined according to literature.33 2.9. CLSM. Confocal laser scanning microscopy (CLSM) was performed to demonstrate the intracellular uptake of nontoxic nanoparticles tested via MTS assay. Therefore, cells were incubated with nanoparticles at a concentration of 150 μg/mL for 2, 6, 12, and 24 h. After incubation time, HeLas were washed three times with medium to remove nanoparticles that were not taken up, covered with medium, and stained with 0.2 μL of CellMask Deep Red (Invitrogen, Germany) to detect cell membranes as well. Images were taken with Leica LAS AF Software on a Leica TCS SP5 II microscope equipped
with five lasers (multiline argon laser with 458, 476, 488, 496, and 514 nm, a DPSS 561 nm, a HeNe laser with 494 and 633 nm lasers, and a 592 nm CW STED laser) with a HCX PL APO CS 63x/1.4−0.6 oilimmersion objective. Oregon Green labeled nanoparticles were excited with a 488 nm argon laser line and detected with a Leica HyD (hybrid detector) at 500−560 nm. The detection of the CellMask Deep Red labeled cell membranes occurred at 650−730 nm when excited at 633 nm with a HeNe laser. Images were taken with a pinhole size of 1 AE and a line average of 4. To avoid crosstalk, a serial mode was used for imaging.
3. RESULTS AND DISCUSSION 3.1. Synthetic Concept of HPMA-Based Block and Random Copolymers. At first, various amphiphilic HPMAbased block and random copolymers were synthesized (see Scheme 1). By applying the RAFT polymerization technique in combination with the reactive ester approach,10 it is possible to obtain well-defined HPMA-based polymers with very narrow molecular weight distributions and variable functionalization (such as dyes) by polymer analogous reaction. The polymer characteristics are displayed in Table 1. It is well-known that polymers of such kind self-assemble in water into complex aggregates10,34 starting with a ratio of about 10 mol % of hydrophobic LMA segments, leaving the HPMA segments immersed in the aqueous solution. Another advantage of these detergent-like systems is their very low critical micelle concentration (cmc) of around 5 × 10−4 mg/mL for the random copolymers and around 4.1 × 10−6 mg/mL for the block copolymers.13 Based on earlier work,10,13 these HPMAbased copolymers are well-known to be nontoxic up to a concentration of 2 mg/mL, which emphasizes their usage as polymeric surfactants in miniemulsion techniques for the C
dx.doi.org/10.1021/bm301453g | Biomacromolecules XXXX, XXX, XXX−XXX
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Table 1. Characterizations of Reactive Copolymers (P1-R− P6-R) and HPMA-Based Copolymers (P1−P6) Prepared Thereof code P1-R P2-R P3-R P4-R P5-R P6-R P1 P1OG P2 P3 P4 P5 P6
copolymers P(PFPMA)-bP(LMA) P(PFPMA)-bP(LMA) P(PFPMA)-bP(LMA) P(PFPMA)-ranP(LMA) P(PFPMA)-ranP(LMA) P(PFPMA)-ranP(LMA) P(HPMA)-b-P(LMA) P(HPMA)/OG-bP(LMA) P(HPMA)-b-P(LMA) P(HPMA)-b-P(LMA) P(HPMA)-ranP(LMA) P(HPMA)-ranP(LMA) P(HPMA)-ranP(LMA)
monomer ratio
yield (%)
Mn in g/mol
D
90:10a
87
15500b
1.21
80:20a
85
18500b
1.18
60:40a
89
24700b
1.13
90:10a
68
17600b
1.16
80:20a
72
22000b
1.19
60:40a
75
20000b
1.19
88:12c 88:12c,e
81 80
9500d 9500d
1.21 1.21
79:21c 61:39c 89:11c
83 79 82
12500d 19000d 10800d
1.18 1.13 1.16
75:25c
77
15000d
1.19
50:50c
78
16000d
1.19
Table 2. PS-Particles Stabilized by Block and Random Copolymers via Miniemulsion Polymerization
b
code
core
water to monomer ratio
D1 D2 D3 D4
PS PS PS PS
10:0.4 10:0.4 10:0.4 10:0.4
a
polymeric surfactant
initiator
⟨1/Rh⟩z−1a (nm)
μ2a
P4 P1 P5 P2
V59 V59 V59 V59
103 100 91 107
