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De Novo Synthesis and Cellular Uptake of Organic Nanocapsules with Tunable Surface Chemistry Kun Huang,† Amy Jacobs,‡ and Javid Rzayev*,† † ‡
Department of Chemistry, University at Buffalo, The State University of New York, Buffalo, New York 14260, United States Department of Microbiology and Immunology, University at Buffalo, The State University of New York, Buffalo, New York 14214, United States
bS Supporting Information ABSTRACT: Water-soluble organic nanocapsules were prepared from bottlebrush copolymers with triblock terpolymer side chains composed of a degradable inner block (polylactide), a cross-linkable middle block (poly(4-butenylstyrene)), and a functional outer block (poly(styrene-co-maleic anhydride)). Bottlebrush copolymers are macromolecules with a long linear backbone and shorter polymeric side chains densely grafted onto the backbone. Hollow cylindrical nanoparticles were prepared by peripheral cross-linking of the bottlebrush copolymers and subsequent selective removal of the core. Reactive anhydride groups of the outer functional layer allowed for the preparation of nanocapsules with tunable surface characteristics. Cellular uptake of negatively charged organic nanocapsules showed strong surface chemistry dependence. The presence of hydrophobic groups on the nanocapsule surface was necessary for their nonspecific association with the cell membrane and subsequent internalization by endocytosis. The length of surface grafted oligoethylene glycol chains also had a dramatic influence on the intracellular accumulation of nanocapsules. Macropinocytosis was shown to be the predominant pathway for the cellular uptake of organic nanocapsules.
’ INTRODUCTION Intracellular delivery of therapeutic and diagnostic agents often requires the use of encapsulating constructs and delivery vehicles to circumvent the limited solubility and poor stability of drug molecules and molecular probes under biological conditions as well as to achieve cellular internalization and cell-specific targeting.13 A variety of nonviral delivery systems based on organic and inorganic nanomaterials are being developed, such as polymers,46 liposomes,7 dendrimers,8 and nanoparticles.911 Functionalized carbon nanotubes have also been shown to act as efficient intracellular transporters of proteins and DNA.1219 The multifaceted nature of the drug delivery process poses stringent requirements on the structure and function of the delivery vehicles in terms of drug binding and release, cellular uptake, intracellular trafficking, and overall biocompatibility. The transport of nanocarriers through the cellular membrane represents one of the challenging steps in effective delivery of therapeutics. The surface chemistry of synthetic constructs plays an important role in determining nanocarriercell interactions and directing the intracellular trafficking.2024 Therefore, the availability of organic nanomaterials with precisely tuned dimensions and chemical functionalities is not only required for the fabrications of robust delivery vehicles but also allows for the preparation of model systems to gain better understanding of the r 2011 American Chemical Society
underlying interactions between synthetic nanomaterials and biological structures. We recently developed a new methodology for the preparation of organic nanotubes with controlled structural parameters, such as pore size and length as well as functional composition.25 This approach is based on single-molecule templating of core shell bottlebrush copolymers, which take on a cylindrical shape in solution. Bottlebrush copolymers are a class of branched macromolecules with a long polymeric backbone and shorter polymeric side chains densely grafted along the backbone.26,27 The steric repulsion between side chains causes the backbone to adopt an extended conformation.28,29 Recent progress in controlled polymerization techniques opened a way for the synthesis of bottlebrush copolymers with finely tuned chemical compositions.3037 The single-molecule templating method allows one to convert coreshell bottlebrush copolymers to standalone tubular nanoobjects while preserving overall molecular dimensions. Such organic nano-objects bring distinct advantages over the existing nanomaterial delivery systems: (1) controlled dimensions (including length and pore diameter), (2) chemical versatility and tunability, and (3) accessible cavity for binding small and Received: March 25, 2011 Revised: May 4, 2011 Published: May 12, 2011 2327
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Biomacromolecules large guest molecules. The degree of structural control that can be achieved by this method has been demonstrated by the fabrication of organic nanotubes with open/closed ends38 as well as conjugated and amphiphilic polymer shells.25,39 The development of synthetic virus-mimics requires access to organic nano-objects not only with controlled dimensions but also with regulated surface properties that can dictate their interactions with biological structures. Herein we report a new molecular design and the synthesis of water-soluble organic nanocapsules from bottlebrush copolymers with triblock terpolymer side chains and their interactions with living cells. Modular control over the outer surface chemistry in these hollow cylindrical nanoparticles allowed us to probe intricate effects in their structure-dependent cellular uptake, which revealed a dramatic influence of the minor structural modifications on the nanocapsule-cell interactions. Such well-defined organic nanoparticles present a versatile platform for the preparation of robust delivery vehicles. They provide a unique combination of structural control and chemical versatility that is hardly attainable by other methods.
’ EXPERIMENTAL SECTION Materials. All chemicals were purchased from Sigma Aldrich and used as received unless otherwise noted. Amine-terminated oligoethylene glycols (OEGs) were purchased from Thermo Scientific (methylOEG4-amine) and Nanocs (methyl-OEG23-amine). HeLa cells, Wheat germ agglutinin Alexa Fluor 594 conjugate, Dulbecco’s modified Eagle medium (DMEM), Hank’s buffered salt solution (HBSS), penicillin streptomycin, and fetal bovine serum (FBS) were purchased from Invitrogen Life Technologies. Dichloromethane (DCM) and N,Ndimethylformamide (DMF) were dried using a commercial solvent purification system (Innovative, Inc.). Styrene (St) was purified by passing over basic alumina. 2,2-Azoisobutyronitrile (AIBN) and D,L-lactide (LA) were purified by recrystallization from methanol and ethyl acetate, respectively. S-1-Dodecyl-S0 -(R,R0 -dimethyl-R00 -acetic acid)trithiocarbonate (TC),40 2-cyanoprop-2-yl-4-cyanodithilbenzoate (CPD),41 and 4-(3-butenyl)styrene (BS)42 were synthesized according to literature procedures. Water was purified using a Milli-Q instrument (Millipore, Bedford, MA). Measurements. All 1H NMR spectra were recorded on a Varian Inova-500 spectrometer (500 MHz) by using CDCl3 or acetone-d6 as a solvent. Size exclusion chromatography (SEC) data were obtained using a Viscotek’s GPCmax and TDA302 Tetradetector Array system equipped with three Olexis columns (Polumer Laboratories, Varian). The detector unit contained a refractive index, UV, viscosity, low (7°), and right angle light scattering modules. Tetrahydrofuran (30 °C, 1 mL/min) was used as a mobile phase. The system was calibrated with 10 polystyrene standards from 1.2 106 to 500 g/mol. The refractive index increment (dn/dc) for poly(glycidyl methacrylate) (PGM) was measured to be 0.087 mL/g in THF (T = 30 °C, λ = 630 nm) and was used to determine the absolute molecular weight of the homopolymer. The dried nanoparticle aggregates were examined using FTIR spectroscopy (Perkin-Elmer 1760X). UVvis spectra were recorded on a Hitachi UVvis 3010 spectrophotometer. Transmission electron microscopy (TEM) images were obtained by using a JEOL 2010 TEM instrument. Samples were prepared by dip-coating a 400 mesh carboncoated copper grid from a dilute sample solution allowing the solvent to evaporate. Zeta potential and particle size distribution of the obtained nanoparticles were measured in a phosphate-buffered saline (PBS) solution by means of a Zetasizer Nano ZS (Malvern Instruments). Cell Culture. HeLa cells were grown in DMEM-supplemented 10% FBS and 1% penicillinstreptomycin at 37 °C in a humidified
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atmosphere containing 5.0% CO2. For all experiments, 5.0 104 cells/cm2 were seeded in culture dishes and grown for 24 h to 50% confluency. Confocal Microscopy. HeLa cells were seeded in 35 mm Willcodish glass-bottomed dishes for 24 h. Then, the medium was washed twice with serum-free DMEM and incubated for 13 h at 37 °C and 5.0% CO2 in serum-free DMEM containing 50 μg/mL of organic nanocapsules. Hoechst 33342 was added to obtain a concentration of 2 μg/mL during the final 20 min of incubation, and wheat germ agglutinin Alexa Fluor 594 conjugate was added to 5 μg/mL during the final 10 min of incubation. For nanocapsule incubation at 4 °C, cellular incubations were carried out as described above with the solution kept at 4 °C, instead of the regular 37 °C condition. For ATP depletion experiments, cells were washed twice with serum-free DMEM and then preincubated at 37 °C and 5.0% CO2 in serum-free DMEM containing 10 mM NaN3 and 50 mM 2-deoxy-D-glucose for 30 min. Then, nanoparticles, Hoechst 33342, and wheat germ agglutinin Alexa Fluor 594 conjugate were added, and cells were incubated as described above. For endocytosis inhibition experiments, cells were washed twice with serum-free DMEM and then preincubated at 37 °C and 5.0% CO2 in serum-free DMEM containing 1 mM amiloride, 20 μg/mL chloroproamzine, or 5 mM β-cyclodextrin for 30 min. Then, nanoparticles, Hoechst 33342, and wheat germ agglutinin Alexa Fluor 594 conjugate were added, and cells were incubated as described above. After 13 h of incubation, all cells were washed five times with PBS solution. Cells were then imaged live in HBSS using a Zeiss LSM 510 meta confocal microscope. Cell Viability Assay. The cytotoxicity of water-soluble organic nanocapsules was examined by CellTilter-Blue Cell viability assay (Promega). HeLa cells were seeded in a 96-well plate at a density of 1 104 cells/well and cultured for 24 h in 100 μL of DMEM containing 10% FBS. Then, the medium was replaced with 0.1 mL of fresh medium containing different concentrations of nanoparticles. After 24 h of incubation, 20 μL/well CellTilter-Blue reagent was added. The plate was shaken for 10 s and then incubated using standard cell culture conditions for 2 h. The luminescence of each well was measured by using a SpectraMax M5Multimode plate reader (Molecular Devices). Synthesis of PGM. Glycidyl methacrylate (GM) (2 mL), CPD (36 mg), AIBN (2.4 mg), and benzene (2 mL) were mixed in a reaction vessel and degassed by three freezepumpthaw cycles. The polymerization was conducted at 60 °C for 16 h. Then, the reaction was stopped by cooling to room temperature and opening the vessel to air. The mixture was diluted with DCM and precipitated in methanol three times and dried under vacuum at 25 °C for 24 h. Yield = 1.64 g (77%). SEC (PS stds): Mn = 15 kg/mol, Mw/Mn = 1.09. PGM Hydrolysis. PGM (160 mg), THF (3.2 mL), and acetic acid (6.4 mL) were mixed in a 250 mL round-bottomed flask. The reaction mixture was stirred and placed in an oil bath at 60 °C, followed by the slow addition of 9.84 mL of water over the course of 1 h. After stirring for 24 h at 60 °C, the solvent was removed on a rotary evaporator. The isolated polymer was precipitated from THF in diethyl ether three times and dried under vacuum at 25 °C for 24 h. Yield = 0.17 g (94%). 1H NMR: Conversion = 95þ%. PLA Grafting. Hydrolyzed PGM (50 mg) and D,L-lactide (1.62 g) were added to a dried 50 mL round-bottomed flask in the glovebox. Dried DMF (10 mL) was then added under nitrogen, and the mixture was stirred until all polymer dissolved. 1,8-Diazbicyclo[5.4.0] undec-7ene (DBU, 48.6 μL) was then injected into the flask.43 After stirring at room temperature for 1.5 h, the reaction was quenched by the addition of 240 mg of benzoic acid. The resulting polymer was precipitated from THF into methanol/water (1:1) three times and dried under vacuum at 25 °C for 24 h. Yield = 1.35 g (80%). SEC (PS stds): Mn = 190 kg/mol, Mw/Mn = 1.19. 1H NMR: n(PLA) = 35. 2328
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Biomacromolecules Scheme 1
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nanocapsules was placed in a 20 mL vial. Then, 1-(30 -dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride was added to the reaction vessel. After 10 min, fluorescein derivative was added to the vessel, and the reaction was stirred overnight. The solution was then transferred to presoaked dialysis bags (MWCO 100K) and allowed to dialyze for 3 days against PBS buffer at PH 7.4.
’ RESULTS AND DISCUSSION
Polystyrene Grafting. Hydroxyl end groups of the PLA brushes were then modified to install trithiocarbonate functionalities, as described in the literature.44 The modified polymer (20 mg) was then mixed with AIBN (0.11 mg), 4-(3-butenyl)styrene (BS, 0.35 mL), styrene (St, 0.25 mL), and toluene (1.2 mL) in a reaction vessel and degassed by three freezepumpthaw cycles. The polymerization was then conducted at 50 °C for 23 h. The polymer was precipitated from DCM to methanol three times and dried under vacuum. Yield = 61 mg (8%). SEC (PS stds): Mn = 592 kg/mol, Mw/Mn = 1.51. 1H NMR: n(BS) = 25, n(St) = 30. Grafting of Poly(styrene-co-maleic anhydride). Poly(GM-gLA-g-(St/BS)) (30 mg), AIBN (0.048 mg), St (0.42 mL), maleic anhydride (MA 0.36 g), and dioxane (4.67 mL) were mixed in a reaction vessel and degassed by three freezepumpthaw cycles. The reaction was then conducted at 45 °C for 1 h. The reaction was stopped by cooling to room temperature and opening the flask to air. The resulting reaction mixture was then precipitated from THF into ethyl ester three times and dried under vacuum at 25 °C for 24 h. Yield = 60 mg (4.05%). 1 H NMR: n(MA) = 65, n(St) = 65. Grafting of Poly(tert-butyl acrylate). Poly(GM-g-LA-g-(St/BS)) (10 mg), AIBN (0.015 mg), tert-butyl acrylate (t-BA, 0.32 mL), and dioxane (1.92 mL) were mixed in a reaction vessel, degassed by three freezepumpthaw cycles, and heated to 55 °C for 5 h. The polymer was precipitated from DCM into cold methanol three times and dried under vacuum. Yield = 16 mg (2.1%). 1H NMR: n(t-BA) = 146. Intramolecular Cross-Linking. Bottlebrush copolymers with triblock terpolymer side chains (20 mg) were dissolved in 18 mL mixed solvent (THF/toluene 1.3/1) under nitrogen. A solution of Grubbs’ first-generation catalyst (0.7 mg) in toluene (0.5 mL) was added to the reaction flask, and the mixture was stirred at room temperature under nitrogen for 24 h. Ethyl vinyl ether (0.5 mL) was added to the reaction mixture to quench the catalyst. The solvent was then evaporated, and shell-cross-linked polymers were precipitated in diethyl ether, redissolved in THF, and precipitated in diethyl ether. Synthesis of OEG-Modified Nanocapsules. Cross-linked bottlebrush copolymers (10 mg) were dissolved in 2 mL of mixed solvent (DMSO/pyridine 9/1). OEG-amine was added to the reaction solution, and the mixture was stirred at room temperature for 24 h. NaOH (1.0 M, 1 mL) was added to the reaction solution, and the reaction was allowed to proceed for 15 h. The mixture was then dialyzed against nanopure water for 3 days to remove residuals. General Procedure for Labeling OEG-Modified Nanocapsules with Fluorescein. A solution of OEG-amine modified
Nanocapsule Synthesis. Coreshell bottlebrush copolymers can be converted to organic nanocapsules by cross-linking the shell and selective removal of the core (Scheme 1). To control the surface chemistry of the nanoobjects, we introduced a third layer, composed of a functional copolymer, which remains on the outer side of the nanocapsules after the above-mentioned transformations. The bottlebrush copolymer precursor with triblock terpolymer side-chains was synthesized by a combination of controlled radical and ring-opening polymerizations. Coreshell bottlebrushes with a polylactide (PLA) core and a poly(styrene-co-(4butenylstyrene)) (PSB) shell were synthesized as previously described from a GM backbone with an average degree of polymerization of 225 and a polydispersity index of 1.09 (Scheme 2 and Figure S1, Supporting Information).25 Every branch was composed of a PLA block with an average of 35 repeat units and a PSB block with an average of 55 units. The third layer, composed of a poly(styrene-co-maleic anhydride) (PSMA),37,45,46 was grafted by the reversible additionfragmentation chain transfer (RAFT) polymerization47 from the PLAPSB core shell bottlebrush precursor P0 (Scheme 2). The formation of the PSMA layer was confirmed by FTIR spectroscopy (ν = 1855 and 1778 cm1) and by 1H NMR spectroscopy (δ 3.5) analyses (Figure 1 and Figure S2, Supporting Information). Every PSMA branch had an average length of 65 repeat units, as measured by 1H NMR. The final bottlebrushes P1 contained a degradable PLA core, a cross-linkable PSB middle layer, and a functional PSMA outer layer. The bottlebrushes with triblock terpolymer side chains were converted to cylindrical nanoparticles by high dilution intrabottlebrush cross-linking of the PSB layer in the presence of the Grubbs’ catalyst. The formation of organic nanoparticles was confirmed by TEM (Figure S3, Supporting Information). Such cylindrical nanoparticles contain an outer PSMA shell, whose anhydride groups can serve as reactive anchor points for further modifications. Thus, this method allows for the modular control of the outer surface chemistry by converting anhydride groups to a variety of different functionalities. A collection of different nanoparticles was prepared by reacting the anhydride groups with either water or amino-terminated OEG with varying lengths (Scheme 3). In addition, nanoparticles N3 with a poly(acrylic acid) coating were prepared by using a poly(tert-butyl acrylate) third layer instead of PSMA.25 The subsequent hydrolysis of the PLA core yielded hollow nanocapsules that were easily dispersible in water. The removal of the PLA core was evidenced by the disappearance of the PLA carbonyl group vibration at 1759 cm1 in the FTIR spectrum of the sample after degradation (Figure 1C). In addition, FTIR analysis of the organic nanotubes confirmed complete disappearance of cyclic anhydride groups (1855 cm1) as well as the presence of carboxylic acid groups (1700 and 1727 cm1) for sample N2 and amide groups (ca. 1650 cm1) for samples N4N7 (Figure S4, Supporting Information). Hollow 2329
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Scheme 2
Figure 1. FTIR spectra of bottlebrush copolymers P0 (A) and P1 (B) and nanocapsules N5 (C).
cylindrical nanoparticles prepared by this method were also visualized by TEM (Figure 2 and Figure S3 in Supporting Information). The average length of the nanoparticles was measured to be 35 ( 5 nm, whereas the pore diameter was ∼5 nm. Dynamic light scattering (DLS) analysis of the nanocapsules in a PBS solution revealed hydrodynamic diameters of the nanoparticles to be 107135 nm (Table 1). Slight variations in nanoparticle sizes for samples N2N7 may be attributed to the differential swelling of the outer layer affected by grafted OEG chains of different lengths. The discrepancy between the sizes obtained from TEM and DLS arises from the fact that TEM measurements are conducted on dried nanoparticle samples and the outer layer is invisible because of a low electron contrast. Surface charge densities of the water-soluble nanocapsules in a PBS solution were obtained by zeta potential measurements. As expected, all of the synthesized water-soluble nanocapsules exhibited negatively charged surfaces (Table 1), which is due to the ionization of carboxylic acid groups. Such moderately charged surfaces also ensured a good stability of the nanoparticle dispersions in aqueous environment. Zeta potential
values for OEG-modified nanocapsules (N4N7) were slightly smaller (less negative) than the values obtained for the poly(acrylic acid)-functionalized nanocapsules (N3), which can be ascribed to the decrease in the number of ionized carboxylic acid groups. The prepared organic nanocapsules were labeled with a fluorescent dye for confocal fluorescence microscopy measurements. Fluoresceinamine was incorporated into the nanocapsules by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimidemediated coupling reaction with the surface carboxylic acid groups.10,48 The UVvis spectrum of the prepared nanocapsules showed an absorption band at 488 nm, corresponding to fluorescein. No obvious size, zeta potential or morphological variations were observed for the fluorescein-functionalized nanoparticles, as evidenced by DLS and TEM. Cellular Uptake. The transport through a cellular membrane is one of the key steps in the delivery of therapeutics and molecular probes. Thus, we investigated the cellular uptake of negatively charged organic nanocapsules to establish their potential as delivery vehicles. HeLa cells were incubated at 37 °C in the presence of organic nanocapsules for 2 h, followed by washing to remove unincorporated nanoparticles. Because flow cytometry cannot differentiate between cell attachment and internalization, we used confocal laser scanning microscopy (CLSM) to observe the localization of the fluorescein-labeled nanocapsules (green). Cells were treated with two additional fluorescent markers to visualize the nucleus (Hoechst 33342, blue) and the cell membrane (wheat germ agglutinin Alexa Fluor 594 conjugate, red) prior to CLSM analysis. As seen from Figure 3, nanocapsules with a hydrolyzed PSMA outer surface (N2), as well as those functionalized with short OEG chains (N4 and N5), were efficiently internalized by the cells. The green spots, corresponding to nanocapsules, appeared to accumulate in the intracellular environment around the nuclei in most cases. No intracellular accumulation was observed for nanocapsules functionalized with longer OEG chains (N6 and N7) and those functionalized with a poly(acrylic acid) coating (N3). The striking difference between the cellular uptake behavior of nanocapsules N2 and N3, which differ only by the presence of phenyl groups on the surface coating of N2, suggests a delicate 2330
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Scheme 3
Table 1. Structural Characteristics of the Organic Nanocapsules in a PBS Solution nanotube
Dh (nm)a
PDa
ζ potential (mv)
cellular uptakeb
N2 N3
126 ( 7 135 ( 8
0.15 0.16
34 ( 2 45 ( 4
yes no
N4
111 ( 6
0.19
28 ( 2
yes
N5
107 ( 4
0.15
27 ( 3
yes
N6
129 ( 5
0.16
22 ( 2
no
N7
135 ( 6
0.19
30 ( 3
no
a
Hydrodynamic diameter (Dh) and polydispersity (PD) measured by DLS. b Uptake by HeLa cells at 37 °C.
Figure 2. Transmission electron micrograph and the size distribution of nanocapsules N5 deposited from an aqueous solution without staining.
balance between hydrophilic (carboxylic acid) and hydrophobic (phenyl) groups on the surface. Another evidence of the importance of such a balance can be obtained by following the cellular uptake of a series of nanocapsules with progressively longer OEG side chains: N4, N5, N6, and N7. A sharp transition seems to occur at the OEG side chain length of three ethylene glycol repeat units, where nanocapsules N5 functionalized with OEG2 were efficiently internalized by HeLa cells, whereas nanocapsules N6 functionalized with OEG4 did not undergo any cellular uptake. Synthetic organic nanocarriers with negatively charged surfaces are relatively unexplored for intracellular transport applications. Becker et al. reported that negatively charged shell-crosslinked nanoparticles (with mostly poly(acrylic acid) coating) cannot be internalized by living cells unless conjugated to a protein transduction domain.49 Mail€ander et al. reported efficient cellular uptake (HeLa) of polystyrene nanospheres with carboxylic-acid-functionalized surfaces.20,50 The authors noted that the optimum uptake was obtained for particles with the carboxylic group surface density of ∼0.5 nm2, but the uptake decreased when the concentration of carboxylic acid on the surface was further increased. The authors did not elaborate on the reason for such behavior. Dai et al. reported cellular uptake of functionalized and negatively charged carbon nanotube transporters.13 The authors hypothesized that unmodified regions of carbon nanotubes nonspecifically associate with hydrophobic regions of the cell surface, triggering endocytosis.
Our results are consistent with the hypothesis that the presence of hydrophobic groups on the outer surface is important for the nonspecific cellular uptake of nanocarriers. It appears that a certain balance must be achieved between the hydrophilic groups, necessary to keep these nano-objects in aqueous solutions, and the hydrophobic groups, necessary for the nonspecific association with the cellular membrane. From the CLSM images shown in Figure 3, one can also observe a significant accumulation of nanocapsules at the cellular membrane, which would support the idea of nonspecific association of these nanocarriers as the first step toward their internalization. Poly(ethylene glycol) (PEG) coatings are often used to reduce the immunogenicity of the synthetic nanocarriers and to improve their blood circulation times.51,52 In a recent study, Dai et al. demonstrated that single-walled carbon nanotubes functionalized with long PEG chains (5400 Da) exhibited a reduced cellular uptake than those functionalized with shorter PEG chains (2000 Da).17 The authors reasoned that longer PEG molecules are covering the hydrophobic surface of the nanotubes, thus, preventing their association with hydrophobic domains in the cell membrane. Kataoka et al. also observed that the PEG palisade surrounding polyplex micelles hampered their transfection efficiencies.53 Our findings on the cellular uptake of OEG-functionalized nanocapsules N4N7 accentuate the effect of OEG chain length in a more dramatic fashion due to the modular surface chemistry control achievable by the utilized synthetic method. It appears that a surface coating of welldistributed shorter OEG chains is sufficient to completely shut down nonspecific cellular internalization of organic nanocarriers. A detailed investigation of the cellular uptake mechanism and cytotoxicity was carried out using N5 nanocapsules. To differentiate between the passive and active cellular uptake, we 2331
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Figure 5. Schematic representation of different pathways for endocytosis (A): clathrin-mediated (1), caveolae-mediated (2), and macropinocytosis (3), and live cell images of HeLa cells incubated with organic nanocapsules N5 (50 μg/mL) in the presence of endocytosis inhibitors: β-cyclodextrin (B), chloroproamzine (C), and amiloride (D).
Figure 3. Live cell images of HeLa cells incubated with organic nanocapsules (50 μg/mL) N2 (A), N3 (B), N4 (C), N5 (D), N6 (E), and N7 (F) for 2 h at 37 °C. Nanocapsules were tagged with fluorescein and appear green. Cell membranes were stained with wheat germ agglutinin Alexa Fluor 594 conjugate (red) and nuclei were stained with Hoechst 33342 (blue).
Figure 4. Live cell images of HeLa cells incubated with organic nanocapsules N5 (50 μg/mL) at 4 °C (A) and in the presence of NaN3/2-deocy-D-glucose (B).
repeated the internalization experiments at low temperatures and under ATP depletion conditions. The cellular uptake of N5 nanocapsules was completely inhibited when the incubation was carried out at 4 °C or at 37 °C in the presence of NaN3/2-deocyD-glucose (Figure 4). There still appeared to be nanoparticles
accumulating at the cellular membrane, but no internalization was observed. The inhibition of cellular uptake under both of these conditions indicate that the internalization process is energy-dependent and occurs by endocytosis.54 The process of endocytosis can be classified into two main categories: phagocytosis (uptake of large particles) and pinocytosis (uptake of fluid and solutes).55 Phagocytosis is typically restricted to specialized mammalian cells (e.g., macrophages), whereas pinocytosis occurs in all cells. There are at least four types of pinocytosis mechanisms: clathrin-mediated endocytosis, caveolae-mediated endocytosis, macropinocytosis, and clathrin/ caveolae-independent endocytosis (Figure 5a). Because the mechanism of clathrin/caveolae-independent endocytosis remains poorly understood, we focus our study on the other three major pathways. In this work, three types of inhibitors (amiloride - inhibitor for the macropinocytosis, chloroproamzine - inhibitor for clathrin-mediated endocytosis, and β-cyclodextrin - inhibitor for caveolae-mediated endocytosis) were used to investigate whether cellular uptake of organic nanocapsules occurred through a specific endocytic pathway.56 As shown in Figure 5, the uptake of N5 nanocapsules was not inhibited in the presence of 5 mM β-cyclodextrin or 20 μg/mL chloropromazine (Figure 5b,c). Cellular internalization of N5 nanocapsules was completely shut down in the presence of 1 mM amiloride (Figure 5d). The results suggest that macropinocytosis is the predominant pathway by which N5 nanocapsules are uptaken into HeLa cells, which is consistent with their relatively large size and the absence of specific receptors. To assess the cytotoxicity of the negatively charged organic nanotubes, we carried out CellTiter-Blue cell viability assays (Promega). HeLa cells were incubated with nanocapsules N5 at different concentrations for 24 h. As shown in Figure 6, no loss of cell viability was observed in the studied concentration range 2332
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’ AUTHOR INFORMATION Corresponding Author
*E-mail: jrzayev@buffalo.edu.
’ ACKNOWLEDGMENT This work was supported by the start-up funds from the University at Buffalo and by the National Science Foundation (DMR-0846584).
Figure 6. Viability of HeLa cells after 24 h of incubation with N5 nanocapsules at different concentrations.
(up to 300 μg/mL), indicating that the nanocapsules were not causing cell death within the tested period.
’ CONCLUSIONS We have developed a new method for the preparations of organic nanocapsules with controlled surface chemistry. Bottlebrush copolymers with triblock terpolymer side chains, which consisted of the PLA inner block, PSB middle block, and PSMA outer block, have been converted to standalone tubular objects with well-defined lengths by selective intramolecular cross-linking of the PBS shell, followed by the degradation of the PLA core. The anhydride groups of the PSMA outer coating were reacted with amine-terminated OEGs of varying lengths, allowing for the precise control of the outer surface characteristics. The synthesized negatively charged tubular nano-objects were efficiently internalized by HeLa cells. The cellular uptake of the nanocarriers was surface-chemistry-dependent and revealed a number of interesting trends that were consistent with the hypotheses proposed for carbon nanotube transporters. The presence and accessibility of the hydrophobic groups on the nanocapsule surface was crucial for their association with cellular membrane and subsequent endocytosis, whereas longer OEG chains on the nanocapsule surface completely inhibited the uptake. The use of various selective inhibitors suggested that the organic nanoparticles were internalized by HeLa cells by the mechanism of macropinocytosis. These negatively charged nanocarriers did not exhibit any cytotoxicity, as confirmed by the CellTiter-Blue cell viability assay. The precise structural and functional control attainable by the reported methodology will not only help in elucidating nanocarriercell interactions but also enable broader utilization of these nanocarriers in therapeutic and diagnostic applications. In addition, the accessible internal cavity will allow for the encapsulation of therapeutic and diagnostic agents. Surfacedependent cellular uptake is the first step toward developing effective drug delivery systems. Also of paramount importance is in vivo behavior of the nanocapsules, in particular, their circulation times and immunogenicity, which will be the focus of future studies. ’ ASSOCIATED CONTENT
bS
Supporting Information. GPC and NMR analyses of polymers and TEM and FTIR characterization of the nanocapsules. This material is available free of charge via the Internet at http://pubs.acs.org.
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