MORPHOLOGICAL AND NANOMECHANICAL INVESTIGATIONS OF MAGNEVIST NANO-ENCAPSULATED DENDRIMERS BY ATOMIC FORCE MICROSCOPY

Citation: Libyan J Pharm & Clin Pharmacol 2012, 1: 48154 http://dx.doi.org/10.5542/LJPCP.v1i0.48154 1 (Page number is not for citation purpose). MORPHOLOGICAL AND NANOMECHANICAL INVESTIGATIONS OF MAGNEVIST NANO-ENCAPSULATED DENDRIMERS BY ATOMIC FORCE MICROSCOPY Hosam Gharib Abdelhady, College of Pharmacy, Taibah University, Al Madinah Al Munawarrah, Kingdom of Saudi Arabia, National Organization for Drug Control and research, Cairo-Egypt


INTRODUCTION:
adolinium (Gd III) is a paramagnetic metal used frequently in magnetic resonance imaging (MRI) [1-2].However, the toxicity of the free Gd III metal and its low efficiency as a contrast agent are considered as main barriers against the achievement of efficient molecular imaging.Fortunately, the rational design of the Gd III ions in water soluble chelates as diethylenetriaminepentaacetic acid (DTPA) known as (Magnevist®), Figure 1  To this end, it was found that the conjugation of Gd III chelates with polyamidoamine (PAMAM) dendrimer were not only efficient and effective in prolonging intravascular retention and circulation time of Gd III chelates due to their large sizes but also effective in modulating and relaxing water protons [7][8][9][10][11][12].Wiener E and Toth E [13][14] reported a strong increase in molecular relaxivity, which was attributed not only to the large number of Gd III-DTPA complexes attached to a single dendrimer molecule but also to a higher ionic relaxivity per Gd III.PAMAM dendrimers,Figure 2. are mono dispersed water soluble, biocompatible macromolecules with well controlled sizes, nanoscopic three dimensions and numerous surface and interior amine groups to which MRI probe scan be coupled [15][16][17].These dendrimers are constructed from various initiator cores on which each complete iterative reaction sequence results in a new dendrimer "generation".
Figure2.G4 with 2 amines on the core (green) and 64 amine groups on the surface (black).
The molecular weight (Mw) of the dendrimer is nearly doubled with the increase in generation, the number of primary amine surface groups exactly doubled and the diameter increases by ~1nm [18].Generation four, 1,4-diaminobutane core PAMAM dendrimers (G4), as an example, has 64 primary amine groups, 62 tertiary amine groups and has a Mw of 14242 Dalton (Da).However, the possibility of the aggregation of contrast agent complexes may have a profound implications on its effective molecular weight, and hence on its behavior.Thus, an accurate assessment of the morphological and nano-mechanical properties of these nanocomplexes is required.To this end, atomic force microscopy (AFM) has immerged to provide an unparalleled spatial resolution of the order of angstroms and force resolution of Pico-Newton's [19].The main advantages of AFM over other ultrahigh resolution microscopy techniques is that sample preparation is relatively simple and does not involve negative staining or shadow casting with a metal coating (as required for electron microscopy), hence AFM measurements can be made to reflect directly the natural topography and nanomechanical properties of the specimen [19][20][21][22].A well approach to measure the nanomechanical properties of different surfaces would be to record the forcedistance curves (f-d-c(s)) and measure the adhesion forces and Young's modulus directly from the force curves [21].The AFM tip is then ramped along the vertical axis and the cantilever deflection is acquired.An AFM f-d-c is the result of the tipsample interaction and the spring constant of the cantilever (Figure 3).The tip sample force is given by Hooke's law: F= -kcγc.Where F is the Force in G nano Newton (nN), γc is the cantilever deflection in nanometers (nm) and kc is the cantilever spring constant in nN/nm.Here, we suggest a simple and fast AFM method, in liquid, for the investigation of the morphological and nano-mechanical changes in G4 before and after their nanoencapsulation with Magnevist (Magnevist-G4).
Figure3.Force-distance curves (f-d-c(s)).During a force distance sequence the tip is approaching vertically from (A) to the sample surface at a constant speed until it experiences a weak attraction force at point (B).The tip continue in approaching the surface until a predetermined set point of maximum load is reached (C).The direction of motion is then reversed, and the tip is withdrawn from the sample surface.As the tip is withdrawn, it adheres to the sample (D).The extent of the adhesion force is then calculated from Hooke's law.

PREPARATION OF PAMAM DENDRIMER-COATED MICA FOR AFM IMAGING AND FORCE MEASURING.
Samples were obtained as methanolic solutions which were dried under a stream of nitrogen and subsequently placed in a vacuum (pressure ~ 0.013 mbar) for one day.The dried material was redissolved in DI water.A 0.1 µg.ml-1 aqueous stock solution was then prepared and stored at 4 ºC for a maximum of a few days.For AFM imaging, a 20 µl drop of 100 femtogram/ml ,Fg.ml1.dendrimer solution was placed on a freshly cleaved mica and left for 10 min.The substrate was then rinsed gently with DI water to remove loosely adsorbed G4 molecules.For force experiments, a 0.1µg.ml-1G4 was placed onto mica and left for 10 min.The substrate was then rinsed gently with DI water.

MAGNEVIST ENCAPSULATION FOR AFM IMAGING AND FORCE MEASURING:
Dry G4 (0.14g, 9.8 x 10-6 mol) was dissolved in 25 ml DI water to make a clear, colorless solution.Magnevist (0.47g, 5 x 10-4 mol) was then added to the dendrimer solution, and the mixture was stirred for 24 hours at room temperature.The undissolved solid was filtered.The mixture was then dialyzed against DI water for 7 hours with several water changes using a Slide-A-Lyzer Dialysis Cassettes, 7K MWCO (Thermo Scientific, Rockford, IL) and the product was lyophilized overnight.To produce Megnevist treated G4 coated mica, a 20 µl drop of 200 Picogram/ml (Pg.ml-1) dendrimer solution was placed on a freshly cleaved mica and left for 10 min.The substrate was then rinsed gently with DI water to remove loosely adsorbed molecules.For force experiments, a 0.3µg.ml-1Magnivest encapsulated G4 was placed onto mica and left for 10 min and the substrate was then rinsed gently with DI water.

AFM IMAGING EXPERIMENTS:
All tapping mode images were obtained in DI water to partially simulate the physiological environment, using a multimode AFM, with a Nanoscope IIIa controller (Veeco, Santa Barbara, CA, USA).All experiments were performed within 1 day of sample preparation.Mica, G4 and Magnivest-G4 samples were imaged by single silicon nitride (Si3N4) probes (100 µm long V-shaped cantilevers, with nominated spring constant of ~0.32 N/m, and resonant frequencies in water between 9 and 12 kHz).As imaging in liquids eliminates the capillary interaction between the AFM tip and the sample surface, imaging forces of < 0.1 nN could be achieved in order to minimize any sample deformation by the probe 21.

AFM FORCE EXPERIMENTS:
Force-distance measurements were recorded in DI water (pH ca 6.4 ± 0.1) using the same Si3N4 tip for experiments on mica, G4 and G4-Magnivest.Previous studies have highlighted the sensitivity of force measurements to differences in the nanomechanical properties at the probe-sample interface [20,22].Here, the AFM is used to differentiate between G4-Magnivest and G4.Curves were then recorded on mica to determine the background level of the interaction.The slope of the contact region of the force-distance approach curve was used to convert the measured cantilever deflection from the relative units of volt to the absolute units in nm.The loading forces were maintained at 3.5 nN to minimize any damage of the probe and/or surfaces during each experiment.The Young's Modulus for G4 and G4-Magnivest were calculated by the Scanning Probe Imaging Processor software Version 4.7, Image Metrology, Denmark.

RESULTS AND DISCUSSION:
The loading of Magnevist in G4 was determined based on the weight gain in dendrimers after their interaction with Magnevist and subsequent washing, dialysis and drying.The weight gain was found to be (0.282 mg).This indicates the attachment of ~ 30.5 Magnevist molecules per one G4 molecule.Figure 4 (a-c) show topographic AFM images of the adsorption of G4 at the watermica interface following the exposure of mica to a 40µl of 100 fg.ml-1 aqueous solution of G4.In contrast to the essentially featureless images of the atomically flat mica surface, Figure 4 (a), globular features were observed.The average of their measured diameters and heights were 15±3.5 nm and 0.9±0.2nm respectively, Figure 4 (b).The decrease in height of these dendritic structures when compared to their theoretical diameter (4.5 nm) is implying the flattening of individual G4 on mica due to its open flexible behaviour.This behaviour was previously reported [22][23][24] where dendrimers exist as an open plate like molecules in which the dendrimer branches of neighboring molecules can inter-penetrate each other to make aggregates.These aggregates were confirmed by the analysis of their volume.On the basis of the dome shape determined from our images, a molecular volume can be calculated from the following equation: V= 1/6h(h2 + 3/4d2) where h is the height and d is the diameter of the cap [25].The number of molecules per feature was then calculated by dividing the calculated volume by the theoretical value of the individual dendrimer molecule.The average number of G4 molecules per aggregate was found to be 3.56 ± 0.53 molecules.Figure 4(c) shows images of Magnevist-G4 complexes adsorbed onto mica from 20 Pg.ml-1 aqueous solution.Well defined spherical structure were obtained.The average of their measured diameters and heights were 40±13 and 4.38±0.54nm respectively.Both G4 and Magnevist-G4 complexes on mica are depicted in three dimensions (3D) as shown in Figure 5   It is obvious that most of the G4 appears as small aggregates above the mica substarct while few G4 makes larger aggregates.This indicates that G4 exists in a relatively open planar structure that interpenetrates each other to make a film on mica [22].Any extra G4 molecules then may lie upon that film and make aggregates [22][23][24].In contrast, most of the magnives-G4 complexes appears as similar discreat spherical features that most likely reflect the rigidity of the complex due to the loading of Magnevist within G4.This loading enabled the molecules to retain their shape on mica surface and appear as discrete spherical features.Since Magnevist has carboxylic acid groups (Figure 1), it forms amine salts with both the surface primary amine and the interior tertiary amine groups of G4 [26].Theoretically, the formation of Magnevist complexes with G4 will increase the diameter of the G4 molecule by ~ 2 nm [27]and increases the volume of the complex to be ~ 143.72 nm3 (considering a sphere).Experimentally, the volume analysis of the Magnevist-G4 complexes was 1221.48 nm3.This indicates the presence of ~ 8.49 ± 0.32 molecules forming an individual MRI particle.Since most of the Magnevist-G4 complexes appear as nearly similar 3D features, thus, the packing for 9 spheres of equal sizes may be suggested as in Figure 6.This packing increases the size and hence the contrast of the MRI complex.New et al [6] reported that G6 dendrimers has the highest increase in relaxivity based on its individual molecular size (7.2 nm diameter) and surface amine number (256) if compared to smaller dendrimers.However, as the individual dendrimers size increases, its toxicity increases and its biocompatibility decreases [11].In contrast, the attachment of 9 G4 molecules in one particle may provide more biocompatibility by having the particle dissociated to 9 small G4 molecules and excreted via the kidney [11].Under conditions employed in our experiments (i.e.DI water, pH ca.6.6±0.1) it is therefore likely that both the G4 and Magnevist-G4 surfaces would have protonated surface primary amine groups (PKa ~ 10.7) [28].Thus, the pull off forces recorded between the tip and both G4 and Magnevist-G4 surfaces could be attributed to the electrostatic attractive interactions between the available protonated primary amine groups (R-NH3)+ and the silicon tip (behaved as silicon oxide).The decrease in the pull off forces on Magnevist-G4 (0.58±0.16) surface if compared with G4 surface (0.78±0.10 nN) is most probably attributed to the interaction of Magnevist molecules with some of the surface (R-NH3) + on G4.This interaction then, decreases the number of the (R-NH3)+ available for the interaction with the silicon tip during the force measurements.As a result, smaller pull off forces were recorded on the Magnevist-G4 surface than that recorded on the G4 surface.Another possible explanation may attributes the smaller pull off forces recorded on Magnevist-G4 to the decrease of the Tip-Magnevist-G4 interacting area, as a result of an increase in the rigidity of this system compared to the G4 system, Figure 8 shows f-d-c(s) done on G4 and Magnevist-G4 surfaces (only the approach curves are shown).The difference in rigidity was seen as differences in the slops of the force curves after the tip-sample contact, where the harder surface (Magnevist-G4) repels the tip stronger than the softer (G4) surface.The yield will be a very steep slope on Magnevist-G4 complexes with a Young's modulus of 2.04 x108 Pa versus less steep slope for the G4 with a Young's modulus of 9.94 x107Pa G4.

CONCLUSION:
The AFM successfully monitored the loading of Magnevist (≥ 30 molecules) within individual G4 molecules.The loading has increased the rigidity of the G4 and enhanced the physical stability of the loaded G4 spheres on mica.This observation indicates the ability of AFM imaging and force measuring to detect the loading of Magnevist within G4 molecules and to investigate its effect on the G4 morphology and nanomechanical properties in liquid.

ACKNOWLEDGMENT AND FUNDING SOURCES:
This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

Figure1.
Figure1.Magnevist molecule.However, clinically used Gd III chelates still suffer from nonspecificity, low relaxivity, rapid extravasation and rapid whole body clearance [6].To this end, it was found that the conjugation of Gd III chelates with polyamidoamine (PAMAM) dendrimer were not only efficient and effective in prolonging intravascular retention and circulation time of Gd III chelates due to their large sizes but also effective in modulating and relaxing water protons[7][8][9][10][11][12].Wiener E and Toth E[13][14] reported a strong increase in molecular relaxivity, which was attributed not only to the large number of Gd III-DTPA complexes attached to a single dendrimer molecule but also to a higher ionic relaxivity per Gd III.PAMAM dendrimers,Figure2.are mono dispersed water soluble, biocompatible macromolecules with well controlled sizes, nanoscopic three dimensions and numerous surface and interior amine groups to which MRI probe scan be coupled[15][16][17].These dendrimers are constructed from various initiator cores on which each complete iterative reaction sequence results in a new dendrimer "generation".

Figure6.
Figure6.Packing of 9 Magnevist-G4 molecules.Where one G 4 molecule is shown at the core, 4 G 4 molecules are shown below the core and 4 G 4 molecules are shown Above the core.Magnivest molecules pack these G 4 molecules together.