Controlling the Size, Shape and Stability of Supramolecular Polymers in Water

The overall goal of the following experiment is to control the size, shape, and stability of super molecular dynamic nanoparticles in water. This is achieved by the design of self assembling or polymerizing dichotic amplifies whose molecular structure is coated for both attractive and repulsive interactions. This results in a frustrated growth mechanism yielding self-assembled nanoparticles in water with a controlled spherical shape.

As a second step, the salt concentration of the aqueous solution is increased, which weakens the repulsive interactions encoded in the molecular structure of the disco amphi. This induces a sphere to rod transition, temperature dependent circular di crowism or CD spectroscopic investigations or performed to reveal the underlying mechanisms of this sphere to rod transition. In addition, a combination of analytical techniques such as cryogenic transmission electron microscopy, and a nuclear magnetic resonance are used to measure and visualize the transition from one type of aggregate to the other results are obtained that show the sphere to rod transition is expressed in an enhanced cooperative in the super molecular polymerization.

This originates in a transition from an anti cooperative process in nanoparticles of restricted size to a fully cooperative nucleation elongation mechanism that leads to very large supramolecular polymers in this case, dynamic nano rods. The main advantage of combining spectroscopic techniques like CD spectroscopy, small anular, x-ray scattering and do MR with microscopic technique like cryo is that it allows to measure and visualize transitions of super molecular polymers in water. These dynamic polymers can respond to external stimuli like changes in the temperature, changes in the OnX strength or changes in pH.

The combination of these methods is widely applicable and can help answer key questions in the fields of molecular chemistry and self-assembled nanomaterials. For example, the correlation between the dimensions of the aggregates formed by the self-assembly of small molecular building blocks and the underlying formation mechanisms can be investigated. The implications of this methodology extend to a wide range of bio nano technological applications.

We've been particularly interested in the area of biomedical imaging and the development of self-assembled nano particulate contrast agents where a delicate balance between the aggregate stability, high contrast, and the ability to excrete the self-assembled contrast agents is of paramount importance for their success in clinical use. We first had the idea of combining characterization techniques when we realized that subtle changes in the molecular structure of the self assembling building blocks can lead to fundamental differences in the mechanism of the super molecular polymerization. Demonstrating the procedure will be Paul Beaumont, who's the expert and responsible for cryo 10 facilities at the technical University of Trovan.

Begin this protocol by preparing A BTA gadolinium DTPA solution in 100 millimolar citrate buffer as described in the written protocol. Accompanying this video, fill a one centimeter UV Q vet with the solution. Insert the vet into the qve holder on the circular D crowism spectrometer.

Measure a CD spectrum from 230 to 350 nanometers. Then measure a CD cooling curve at the highest intensity CD band from 363 to 283 kelvin at a rate of one kelvin per minute. Next, add the same volume of two molar sodium chloride buffered solution to the citrate buffered solution of BTA gadolinium DTPA.

This will increase the ionic strength to one molar sodium chloride and dilute the discos to half the concentration. Vortex the solution with increased ionic strength for 40 seconds following the increase in ionic strength, remeasure a CD spectrum from 230 to 350 nanometers. Then measure a CD cooling curve at the highest intensity CD band from 363 to 283 kelvin at a rate of one kelvin per minute.

Export the raw CD data into origin 8.5. Normalize the spectra by defining the CD effect at the highest measured temperature as equal to zero, and the CD effect at the lowest measured temperature as equal to one since the magnitude of the CD effect is proportional to the degree of aggregation, the normalized CD curves are proportional to the degree of aggregation. The normalized data is fitted using a non-linear curve fit option in origin Pro 8.5 with a temperature dependent self-assembly model.

In this model, a nucleation and an elongation regime are distinguished. First, fit the degree of aggregation in the elongation regime. In this equation, T represents the variable temperature.

PHI N is the net helicity, which is proportional to the degree of aggregation, and HE is the molecular enthalpy of elongation. T TE represents the elongation temperature, which is the temperature at which the self-assembly starts to become thermodynamically favorable. The normalization factor fiat is introduced to ensure that Phi N over fiat does not exceed unity, which follows from the constraint that the degree of aggregation cannot exceed.

Unity fitting allows extracting the enthalpy of elongation in jewels per mole and the elongation temperature in Kelvin that characterizes the self-assembly of the molecules for a given concentration. When fitting obey the restraint that only the degree of aggregation at temperatures below TE should be fitted since the equation is only valid in the elongation regime after fitting the first equation, the only unknown parameter in the nucleation regime equation is the activation constant ka, which describes the cooperative of the supramolecular polymerization. To find the activation constant fit, the experimentally found degree of aggregation for temperatures above TE in the nucleation regime.

Begin by preparing solutions for electron microscopy as described in the text. Briefly prepare two buffers, a 100 millimolar citrate buffer, and a 100 millimolar citrate buffer with five molar sodium chloride dissolve, BTA gadolinium DTPA in 0.1 milliliters of each of the prepared buffers to achieve a deco concentration of one millimolar. Next plasma treat a quanti foil carbon coated grid using a C Resington 2 0 8 carbon coer operating at five milliamps for 40 seconds.

The vitrification procedure is a crucial step of cryo since it ensures that a thin layer of glossy ISIS produced at is suitable for TAM analysis. The aqueous solution is applied on the grid during vitrification on an automated FEI vitro bot. This involves the application of the sample on the grid blotting of excess liquid to create a thin film of the aqueous solution on the grid and subsequent vitrification by dipping the grid very quickly in liquid Ethan.

After vitrification transfer the treated grid into liquid nitrogen to preserve it, then manually transfer the sample grid to an autoloader cassette, which is also cooled with liquid nitrogen. The next step is to insert the cassette into the TUE cryo Titan, TEM auto Loader. The TUE Cryo Titan is equipped with a field emission gun operating at 300 kilovolts.

Recording the TAM images requires experience and fast handling. This is because the high energy electron beam damages the specimen during imaging Record images using a CCD camera equipped with a post column gatin energy filter. Since gadolinium is highly paramagnetic and proton signals would thereby be broadened significantly, a different disco was used where gadolinium was substituted with diamagnetic yttrium and a solution of BT atrium.

DTPA is prepared. Calculate how many milligrams of BT atrium DTPA at a molecular weight of 2, 979 grams per mole is necessary to achieve a target concentration of one millimolar. Proceed to dissolve the determined amount of BT atrium DTPA in 50 millimolar derated succinate buffer in D two O after pipetting 0.6 milliliters of the resulting solution into a wilm, a LABAs NMR tube.

Insert the sample into a variant unity in Nova 500 spectrometer equipped with a five millimeter I-D-P-F-G probe from Varian. Perform the DOI experiments as discussed in the text after recording a standard proton NMR adapting the 90 degree pulse and optimizing the mixing times accordingly. The DOI one-shot pulse sequence from is used after determining the self diffusion of HDO in ovarian reference probe, and in the sample the diffusion coefficient of the aggregates is determined from which the hydrodynamic radius can be calculated.

Finally, calculate the hydrodynamic radii RH of the aggregates using the Stokes Einstein relation for the diffusion of a spherical particle. The ionic character of the peripheral gadolinium DTPA complexes introduces frustration in the one dimensional growth of the disco monomers whose core is designed to polymerize into elongated rod like aggregates. The balance between attractive and repulsive interactions controls the size and the shape of the aggregates.

A powerful technique to determine the size and the shape of particles and solution is synchrotron source. Small angle x-ray scattering or sucks. BTA gadolinium DTPA was dissolved in citrate buffer solution and the sucks profiles were recorded at two different concentrations.

A slope approaching zero in the low Q region indicates a lack of shape and isotropy in the aggregate suggesting the presence of spherical objects. The data measured at different concentrations were fitted using a homogenous mono disperse spherical form factor leading to a calculated radius are of 3.2 nanometers. The calculated geometric radius of monomeric disco, BTA gadolinium DTPA is 3.0 nanometers, which the presence ofer aggregates with an aspect ratio of close to one in order to provide further evidence for the spherical shape and nanometer size of the self-assembled objects.

Proton diffusion ordered NMR spectroscopy was performed. DOI NMR allows determination of the diffusion coefficients of the super molecular aggregates from which their hydrodynamic radius can be calculated. The diffusion coefficient of the aggregated diamagnetic dichotic amplify in a deuterated succinate buffer was determined to be 0.69 times 10 to the power minus 10 meter squared per second via the stokes Einstein relation.

A hydrodynamic radius of 2.9 nanometers was calculated for the discreet objects of spherical size. This size is an excellent agreement with the value obtained from SOX data for BTA gadolinium DTPA. Further evidence for successful control over one dimensional stack length was obtained from cryo TEM micrographs.

The BTA gadolinium DTPA produces the expected spherical objects with diameters close to six nanometers at a one millimolar concentration, which confirms the results from so and DOI measurements. The formation of high aspect ratio rod, like supramolecular polymers is clearly observed in cryo te micrographs at high ionic strength. Electrostatic screening is the most likely explanation for this finding.

The shape changes from a spherical aggregate of around six nanometers in diameter to elongated rods with a diameter of six nanometers and a length of up to several hundred nanometers. The room temperature CD spectra of BTA Gadolinium DTPA with increasing salt concentration are shown here. The BTA gadolinium DTPA concentration is eight times 10 to the power minus three millimolar at low ionic strength and four times 10 to the power minus three millimolar at high ionic strength.

Although a significantly lower concentration is applied for the CD measurements, the clear cotton effect indicates the presence of intact aggregates even at micromolar concentrations. The shape of the CD spectrum changes upon increasing the salt concentration, which is a good indication for reduced repulsive interactions at the periphery of the stacks and enhanced packing of the decos. In addition, the CD cooling curves of the same solutions show distinct differences in shape.

The temperature at which aggregation starts shifts to higher temperatures at higher salt concentration. An increasingly cooperative mechanism also becomes apparent as characterized by a more abrupt increase in the CD effect, whereas the cooling curve at zero molar sodium chloride is best described by an ISO smic self-assembly model indicative of an anti cooperative process. The cooling curve at 1.0 molar sodium chloride is typical for a cooperative self-assembly process and can be described by a nucleation elongation model quantifying the thermodynamic parameters of the self-assembly of BTA gadolinium DTPA at zero and one molar sodium chloride.

Using a cooperative model clearly reveals the decrease in ka, which is the dimensionless activation.Constant. Lower values for KA indicate a higher degree of cooperative in the self-assembly process, which is expressed in the formation of highly elongated supramolecular polymers. As observed in cryo TEM While attempting the demonstrated experimental techniques, it is important to remember that only a combination of experimental methods will lead to a meaningful overall description of the dynamic nanomaterials under investigation.

After Watching this video, you should have a good understanding of how widely applicable our combined experimental approach is and how it can help answer key questions in the field of self-assembled nanomaterials and super poems.