Dendrimers have shown great promise as drug delivery vehicles in recent years because they can be synthesized with designed size and functionalities for optimal transportation, targeting, and biocompatibility. One of the most well-known termini used for biocompatibility is polyethylene glycol (PEG), whose performance is affected by its actual conformation. However, the conformation of individual PEG bound to soft materials such as dendrimers has not been directly observed. Using atomic force microscopy (AFM) and scanning tunneling microscopy (STM), this work characterizes the structure adopted by PEGylated dendrimers with the highest resolution reported to date. AFM imaging enables visualization of the individual dendrimers, as well as the differentiation and characterization of the dendrimer core and PEG shell. STM provides direct imaging of the PEG extensions with high-resolution. Collectively, this investigation provides important insight into the structure of coated dendrimers, which is crucial for the design and development of better drug delivery vehicles.
Dendrimers provide an alternative and potent means for drug delivery due to their nanometer size and the ability to incorporate various functionalities on their interior and exterior. Modern chemical synthesis capabilities allow various functionalities to be incorporated on the dendrimer exterior in order to optimize performance in terms of drug binding, transport, targeting, delivery, and biocompatibility [
The following materials were used as received: methoxy polyethylene glycol 1000 Da (PEG1000) (Sigma-Aldrich), triethylamine (TEA) (99.5%, Sigma-Aldrich), pyridine (99.8%, Sigma-Aldrich) p-nitrophenyl chloroformate (pNPCF) (98%, Sigma-Aldrich), 2,5-dihydroxybenzoic acid (2,5-DHB) (Sigma-Aldrich), anhydrous dichloromethane (DCM) (Sigma-Aldrich), dimethyl sulfoxide (DMSO) (Acros Geel), K2PtCl4 (min. 42.4% Pt, Alfa Aesar), n-octanethiol (C8) (98%, Sigma-Aldrich), deuterium oxide (D, 99.96%, Cambridge Isotope Laboratories), and phosphate buffer (10x, Lief Technologies). Fourth generation amine terminated PAMAM dendrimers were purchased as 10% by weight solutions in methanol (Sigma-Aldrich, St. Louis, MO). Silica gel 60 A 230–400 mesh ATSM (Whatman Inc) and silica gel 60 F254 plastic sheets (TLC) (Merck KGaA) were used for column and thin layer chromatography, respectively. Ultrapure water (≥18 MΩ·cm, Millipore Milli-Q) and 200 proof ethanol (Gold Shield Chemical Co.) were used for dilution and washing. Ultrapure N2 (98%, Air Gas Co.) and H2 (99.99%, Praxair, Inc.) were used for drying and flaming, respectively. Tungsten wire (
The following materials were used as received: methoxy polyethylene glycol 1000 Da (PEG1000) (Sigma-Aldrich, St. Louis, MO), triethylamine (TEA) (99.5%, Sigma-Aldrich, St. Louis, MO), pyridine (99.8%, Sigma-Aldrich, St. Louis, MO) p-nitrophenyl chloroformate (pNPCF) (98%, Sigma-Aldrich, St. Louis, MO), 2,5-dihydroxybenzoic acid (2,5-DHB) (Sigma-Aldrich, St. Louis, MO), anhydrous dichloromethane (DCM) (Sigma-Aldrich, St. Louis, MO), dimethyl sulfoxide (DMSO) (99.7%, Acros, Geel, Belgium), K2PtCl4 (min. 42.4% Pt, Alfa Aesar, Ward Hill, Massachusetts), n-octanethiol (C8) (98%, Sigma-Aldrich, St. Louis, MO), deuterium oxide (D, 99.96%, Cambridge Isotope Laboratories, Tewksbury, MA), and phosphate buffer (10x, Lief Technologies, Grand Island, NY). Fourth generation amine terminated PAMAM dendrimers were purchased as 10% by weight solutions in methanol (Sigma-Aldrich, St. Louis, MO). Silica gel 60A 230–400 mesh ATSM (Whatman Inc, Pittsburgh, PA) and silica gel 60 F254 plastic sheets (TLC) (Merck KGaA, Darmstadt, Germany) were used for column and thin layer chromatography, respectively. Ultrapure water (≥18MΩ
G4-PAMAM-NH2 dendrimers were PEGylated according to previous reports with some modifications [
The G4-PAMAM-
The molecular weight of the modified and unmodified dendrimers was determined by matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) (Ultraflex, Bruker). Spectra were acquired under positive ion reflector mode. The conjugates were dissolved in deionized water at a concentration of 1.0 mg/mL. 10 mg/mL of 2,5-DHB in methanol was used as matrix. 10
The number of PEG chains per dendrimer was determined by 1H NMR spectroscopy (MR-400, Agilent) using deuterated solvent. The deuterated solvent peak (DMSO_d6: 2.483; D2O: 4.577) in 1H NMR was set as a reference peak. The PEGylation resulted in 5.9 PEG per dendrimer, referred to as G4-PAMAM-6PEG1000, and 50.5 PEG per dendrimer, referred to as G4-PAMAM-50PEG1000.
The hydrodynamic diameter (HD) of samples was measured using dynamic light scattering (DLS) (Zetasizer Nano ZS, Melvern Instruments). The sample (1.0 mg/mL) was dissolved in phosphate buffer solution (0.1 M, pH 7.4) to maintain the pH during the measurement. HD and standard deviations were automatically calculated by built-in software. G4-PAMAM-NH2, G4-PAMAM-6PEG1000, and G4-PAMAM-50PEG1000 dendrimers were found to have a HD of 4.4 ± 1.4, 5.8 ± 2.1, and 12.2 ± 4.4 nm, respectively.
Dendrimers were immobilized on gold surfaces for AFM and STM imaging. The solutions were prepared following previously established procedures, including metal ion doping to facilitate STM imaging [
AFM images were acquired using a MFP3D-SA system (Asylum Research), which includes a closed loop capability. A silicon cantilever (AC-240, Olympus) was used for imaging and nanoshaving. The probe has a typical force constant of
STM images were taken using a walker-type scanner (UHV 300, RHK Technologies, Inc.), under ambient pressure and temperature [
The characteristic AFM tapping mode images of G4-PAMAM-50PEG1000, are shown in Figures
AFM tapping mode imaging of G4-PAMAM-50PEG1000 dendrimers. 300 × 300 nm2 topographic images of G4-PAMAM-50PEG1000 acquired at a damping set point of 42% (a) and 23% (b). 300 × 300 nm2 AFM topographic images of G4-PAMAM-NH2 at damping set points of 42% (c), and 23% (d). Cursor profile 1 is a representative G4-PAMAM-50PEG1000 and G4-PAMAM-NH2 dendrimer imaged with 42% dampening as indicated in (a) (red) and (c) (blue). Cursor profile 2 is a representative G4-PAMAM-50PEG1000 and G4-PAMAM-NH2 dendrimer imaged with 23% dampening as indicated in (b) (red) and (d) (blue).
The width of the rings, measured as full width at half of the maximum height, varies from 5.0 to 12.4 nm, with an average value of 6.9 ± 2.3 nm. The measured ring width provides an approximate view regarding the extension, or conformation, of the PEG molecules suggesting a variation in the PEG conformation. According to AFM imaging, the thickness of the PEG coating surrounding individual dendrimers is asymmetric. The difference between the thickest and thinnest regions of the PEG coating on a single dendrimer varies by 3.4% up to 47.6%. This preliminary assessment of dendrimer morphology indicates that the PEG density and extension are not uniform at the outer shell of individual dendrimers. To determine the fine structure of the PEG coating, STM imaging is performed and provides a more accurate visualization of the PEG presentation, as will be discussed in detail in Section
To verify that the features are not due to deformation upon surface immobilization, we have also imaged dendrimers at various surface coverages. At high surface coverage, as shown in Figure
300 × 300 nm2 AFM topographic images of G4-PAMAM-50PEG1000 imaged with 42% (a) and 23% (b) dampening. Cursor profile is taken from representative G4-PAMAM-50PEG1000 dendrimers in images (a) (red) and (b) (blue).
One specific difference was seen under 42% damping, at which ring contrast was observed previously; the morphology appears fragmented, analogous to flower pedals, as shown in Figure
Our previous work has demonstrated that the physical height of surface immobilized dendrimers can be measured using nanoshaving, an AFM based technique, in conjunction with topographic imaging [
Height characterization of dendrimers without and with PEGylation via nanoshaving. (a) A 300 × 300 nm2 AFM topographic image of a nanoshaved monolayer of G4-PAMAM-NH2. The (a) inset is a 1.5 × 1.5
STM provides submolecular resolution characterization of PEGylated dendrimers, revealing the morphology and structure of the PEG chains. With the overall morphology of dendrimers established by AFM, we could use STM to provide a more detailed look at the intramolecular structure especially at PEG region. Although dendrimers are not sufficiently conductive for direct STM imaging, our prior work has indicated that STM conductivity may be enhanced by coordinating metal ions into the dendrimer [
The G4-PAMAM-NH2 structure was first characterized without PEGylation. Figure
High-resolution characterization of dendrimers with varying degrees of PEGylation. (a) A 60 × 60 nm2 STM topographic image of G4-PAMAM-NH2. (b) A 15 × 15 nm2 STM topographic image of G4-PAMAM-NH2 with three representative dendrimer contact areas highlighted in red. (c) 60 × 60 nm2 image of G4-PAMAM-50PEG1000. (d) 15 × 15 nm2 image of G4-PAMAM-50PEG1000 with two representative dendrimer/PEG contact areas highlighted in red. (e) 60 × 60 nm2 image of G4-PAMAM-6PEG1000. (f) 15 × 15 nm2 image of G4-PAMAM-6PEG1000 with two representative dendrimer/PEG contact areas highlighted in red. All images were acquired at 0.7–0.9 V and 20–30 pA.
Upon PEGylation, the STM images reveal significant structural changes from that of core particles. Figure
In order to determine the effect of the packing density of the PEG at the dendrimer surfaces, various PEG : core ratios were prepared. Figure
The observed coverage dependence of PEG conformation may be understood by taking into account PEG-PEG, PEG-solution, and PEG-surface interactions. On solid flat surfaces PEG1000 chains adopt various conformations depending on the interplay between these interactions. Three characteristic conformations have been described previously, known as pancake, mushroom, and brush, with the extension length of 0.5, 1.0, and 2.5 nm, respectively [
Results from STM investigations indicate that the incorporation of Pt2+ ions leads to sufficient conductivity for high-resolution STM imaging, despite PEGylation, which in principle should hinder metal ion doping. In comparison to PAMAM dendrimers, the incorporation of metal ions follows slower kinetics. In the case of G4-PAMAM-OH, for example, each dendrimer was saturated by Pt2+ within 48 hrs, under 1 : 70 dendrimer : Pt2+ molar ratio, at a 1
High-resolution characterization of dendrimers doped at 1 : 70 and 1 : 700 dendrimer to Pt2+ molar ratios. (a–c) 15 × 15 nm2 STM topographic images of G4-PAMAM-50PEG1000, G4-PAMAM-6PEG1000, and G4-PAMAM-NH2 doped in a solution containing a 1 : 70 molar ratio of dendrimer to Pt2+. (d–f) 15 × 15 nm2 STM topographic images of G4-PAMAM-50PEG1000, G4-PAMAM-6PEG1000, and G4-PAMAM-NH2 doped in a solution containing a 1 : 700 molar ratio of dendrimer to Pt2. Cursors 1–3 provide the lateral and apparent height dimensions of individual representative G4-PAMAM-50PEG1000, G4-PAMAM-6PEG1000, and G4-PAMAM-NH2 dendrimers doped at a 1 : 70 Pt2+ ratio. Cursors 4–6 provide the lateral and apparent height dimensions of individual representative G4-PAMAM-50PEG1000, G4-PAMAM-6PEG1000, and G4-PAMAM-NH2 dendrimers doped at a 1 : 700 Pt2+ ratio. All images were acquired at 0.7–0.9 V and 20–30 pA.
Using AFM and STM, we have characterized the morphology and structure of PEGylated dendrimers. AFM investigation allows for the visualization of individual dendrimers on surfaces and provides accurate height measurements. In addition, AFM studies reveal that the PAMAM core and PEG shell can be visualized under tapping mode imaging to ascertain the uniformity and distribution of PEGylation on individual dendrimers. Further, the results indicate that PEG chains among adjacent dendrimers could interdigitate, in contrast to the dendrimer cores. STM imaging enables direct visualization of the PEG extensions with high-resolution. The PEG chains at the exterior PAMAM cores adopt various conformations including pancake, mushroom, and brush, similar to that at the solid and flat surfaces. Unique to high coverage PEGylated dendrimers, a greater variation in PEG structure and degree of extension is observed with the PEG podia, up to 4.5 nm from the core. To the best of our knowledge, this work is among the first to reveal high-resolution information on the local structure of PEGylated dendrimers. Collectively, this investigation provides important insight into the structure of coated dendrimers, which shall be important to guide the design and development of better drug delivery vehicles. Work is in progress to correlate structural information with the efficacy of drug delivery.
All the authors of this paper declare that there is no conflict of interests with any financial organization regarding the material discussed in this paper.
This work is supported by NSF (CHE-0809977, DMR-1104260, and CBET-0933144), W. M. Keck Foundation, and Nano@WSU Incubator. The authors thank Dr. Chris Fleming of BST NanoCarbon, Ms. Susan Stagner, and Drs. Ming Zhang and Jianli Zhao of UC Davis for helpful discussion and assistance in paper preparation.