Name: transport properties of ibuprofen encapsulated in cyclodextrin nanosponge hydrogels: a proton hr-mas nmr spectroscopy study
Uploaded: 2016-08-15T13:00:00
Description: Watch this Scientific Journal Video about Transport Properties of Ibuprofen Encapsulated in Cyclodextrin Nanosponge Hydrogels: A Proton HR-MAS NMR Spectroscopy Study at JoVE.com

The authors gratefully acknowledge PRIN 2010-2011 NANOMED prot. 2010 FPTBSH and PRIN 2010-2011 PROxy prot. 2010PFLRJR_005 for funding.

1. Synthesis of CDNSEDTA Polymers
<ol>
	<li>Dry &#946;-cyclodextrin (&#946;-CD) in oven at 80 &#176;C for 4 hr before use. Dry 500 ml of dimethylsulfoxide (DMSO) and 100 ml of triethylamine (Et3N) over molecular sieves (4 &#197;) for 24 hr before using them in the protocol.</li>
	<li>Introduce 25 ml of DMSO into a 50 ml one-neck round-bottom flask. Under magnetic stirring, add 5.675 g of &#946;-CD (5 mmol). In order to reduce the formation of lumps, add the &#946;-CD powder in small portions to DMSO.</li>
	<...

<ol>
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We present an experimental methodology to determine the diffusion regime of a small drug molecule encapsulated inside two representative formulations of CDNSEDTA hydrogels. HR-MAS PGSE NMR allows the determination of the mean square displacement of small molecules in a given diffusion time (in the range of a few milliseconds up to second), then monitoring distances in the micrometer scales. In the range observed (50 - 170 msec) only one type of motion is observed for each studied system. It should be stressed, however, t...

There is a growing interest in the design and formulation of polymeric systems capable of entrapping, via non-covalent interactions, small molecules with potential biochemical activity. Such materials are expected to find applications in the transport of the active principle to selective target and release upon the action of external stimuli, such as pH variations, temperature, etc. In this context, hydrogels turned out to be versatile and powerful materials for nanomedicine in view of controlled release of drugs1. The formation of polymeric hydrogels can be achieved by interconnecting the macromolecular chains by i) physical, non-covalent interactions such as hydrogen bonds, ii) covalent cross-linking of the chains leading to a three-dimensional network able to swell in the presence of an aqueous solution or iii) a combination of the two mentioned methods2-4.
A particularly versatile class of three-dimensional, swellable polymers for the encapsulation of organic and inorganic species can be obtained starting from natural &#946;-cyclodextrin (&#946;-CD) via condensation with suitable, activated derivatives of a tetracarboxylic acid5-8 giving rise to cyclodextrin nanosponges (CDNS). The synthesis, characterization and application of CDNS is a consolidated research theme of our group. The past few years&#39; results indicate that CDNS show intriguing properties of swelling, absorption/inclusion of chemicals, and release of small drug molecules, with applications in controlled release of pharmaceutical active ingredients9-11 and environmental chemistry12-14.
Given these premises, two major issues to be addressed concern the efficient loading of the active compound in the polymeric gel and an improved understanding of solutes mobility in the gel matrices15. The literature provides both experimental studies and theories related to diffusion mechanisms of small molecules in macromolecular networks16,17. Pulsed field-gradient spin-echo (PGSE) NMR spectroscopy is a well-established structural method widely used to study the translational diffusion of small molecules in solvents18 or the self-diffusion of pure liquids. The recent developments of high resolution magic angle spinning (HR-MAS) NMR technology made it possible to collect high resolution NMR data of mobile molecules in heterogeneous suspensions19, gels and swellable polymers20,21. Indeed, the experimental setup combining HR-MAS NMR spectroscopy and the PGSE pulse sequence provides a unique opportunity to observe the solute molecules in the host&#39;s molecular environment. Important data on the transport properties of the entrapped drug molecule within a gel matrix can thus be obtained. High quality experimental data can thus be obtained allowing a more rational design of nanostructured host-guest systems.
In the present work we describe the detailed protocols for the following steps: i) synthesis and purification of two different formulation of CDNS cross-linked with EDTA polymers (Figure 1), referred to as CDNSEDTA, and characterized by different CD/cross linker molar ratio: 1:4 (CDNSEDTA 1:4) and 1:8 (CDNSEDTA 1:8); ii) the preparation of drug-loaded hydrogels for both CDNSEDTA 1:4 and CDNSEDTA 1:8. In this step we used, as model drug molecule, the popular non-steroidal anti-inflammatory ibuprofen sodium salt (IP); iii) the thorough investigation of the transport properties of IP within the CDNSEDTA hydrogels via PGSE-HRMAS NMR spectroscopy. The method we propose here is based on the measurement of the mean square displacement (MSD) of the encapsulated drug within the hydrogel followed by the analysis of the time dependence of the MSD.
We wish to stress that the methodology outlined above - which is focused on the time dependence of the drug&#39;s MDS in the matrix - provides a broader spectrum of information compared to the consolidated methodology based on the determination of the drug&#39;s diffusion coefficient only. We recently demonstrated21 that this approach allowed for the discrimination of normal and anomalous diffusion regimes experienced by IP confined in CDNS hydrogels.
We thus believe that the step-by-step description of polymer synthesis/purification, formation of the drug-loaded hydrogels, HR-MAS NMR characterization and data processing of MDS data, is a powerful toolkit for scientists interested in characterizing nanostructured systems for the confinement and release of small molecules.

<table border="0" cellpadding="0" cellspacing="0" width="657">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HR-MAS probe</td>
			<td style="width:113px;">BRUKER</td>
			<td style="width:161px;">N/A</td>
			<td style="width:167px;">Probe for NMR measurements on semi-solid samples</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NMR Spectrometer</td>
			<td style="width:113px;">BRUKER</td>
			<td style="width:161px;">DRX 500</td>
			<td>FT NMR spectrometer for liquid ans semi-solis state</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">&#946;-cyclodextrin (&#946;-CD)</td>
			<td style="width:113px;">Alfa-Aesar</td>
			<td style="width:161px;">J63161</td>
			<td>Reagent</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ethylenediaminetetracetic (EDTA) dianhydride</td>
			<td style="width:113px;">Sigma-Aldrich</td>
			<td align="right" style="width:161px;">332046</td>
			<td>Reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dimethylsulfoxide (DMSO)</td>
			<td style="width:113px;">Alfa-Aesar</td>
			<td style="width:161px;">D0798</td>
			<td>Solvent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Triethylamine</td>
			<td style="width:113px;">Sigma-Aldrich</td>
			<td align="right" style="width:161px;">471283</td>
			<td>Base (reagent)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ibuprofen (IP) sadium salt</td>
			<td style="width:113px;">Sigma-Aldrich</td>
			<td style="width:161px;">I1892</td>
			<td>Antinflammatory drug</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Excel 2010</td>
			<td style="width:113px;">Microsoft</td>
			<td style="width:161px;">N/A</td>
			<td>speadsheet for data analysis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Origin 8 SR0</td>
			<td style="width:113px;">OriginLab Co.</td>
			<td style="width:161px;"></td>
			<td>speadsheet for data analysis</td>
		</tr>
	</tbody>
</table>

transport properties of ibuprofen encapsulated in cyclodextrin nanosponge hydrogels: a proton hr-mas nmr spectroscopy study

The chemical cross-linking of &#946;-cyclodextrin (&#946;-CD) with ethylenediaminetetraacetic dianhydride (EDTA) led to branched polymers referred to as cyclodextrin nanosponges (CDNSEDTA). Two different preparations are described with 1:4 and 1:8 CD-EDTA molar ratios. The corresponding cross-linked polymers were contacted with 0.27 M aqueous solution of ibuprofen sodium salt (IP) leading to homogeneous, colorless, drug loaded hydrogels.
The systems were characterized by high resolution magic angle spinning (HR-MAS) NMR spectroscopy. Pulsed field gradient spin echo (PGSE) NMR spectroscopy was used to determine the mean square displacement (MSD) of IP inside the polymeric gel at different observation times td. The data were further processed in order to study the time dependence of MSD: MSD = f(td). The proposed methodology is useful to characterize the different diffusion regimes that, in principle, the solute may experience inside the hydrogel, namely normal or anomalous diffusion. The full protocols including the polymer preparation and purification, the obtainment of drug-loaded hydrogels, the NMR sample preparation, the measurement of MSD by HR-MAS NMR spectroscopy and the final data processing to achieve the time dependence of MSD are here reported and discussed. The presented experiments represent a paradigmatic case and the data are discussed in terms of innovative approach to the characterization of the transport properties of an encapsulated guest within a polymeric host of potential application for drug delivery.

We first applied this methodology to the IP drug molecule dissolved in water solution in order to verify the viability of this approach. A full description of the representative results can be found in ref. 21. Rather, we will focus here on the methodological aspects and the nuts-and-bolts approach to data collection and data analysis. Figure 3 shows, on a semi-logarithmic scale, the normalized experimental signal decays I(q, td)/I(0, td) as a functi...

Watch this Scientific Journal Video about Transport Properties of Ibuprofen Encapsulated in Cyclodextrin Nanosponge Hydrogels: A Proton HR-MAS NMR Spectroscopy Study at JoVE.com

Transport Properties of Ibuprofen Encapsulated in Cyclodextrin Nanosponge Hydrogels: A Proton HR-MAS NMR Spectroscopy Study

The motion regimes of ibuprofen encapsulated in &#946;-cyclodextrin nanosponges polymer network are investigated using pulsed-field-gradient spin-echo (PGSE) NMR technique. Synthesis, purification, drug loading, implementation of the NMR pulse sequence and data analysis to work out the mean square displacement of the drug at several observation times are described in detail.

The chemical cross-linking of &#946;-cyclodextrin (&#946;-CD) with ethylenediaminetetraacetic dianhydride (EDTA) led to branched polymers referred to as ...

The overall goal of this work is to show the synthesis of cyclodextrin nanosponges, convert them into drug-loaded hydrogels, and to study the drug diffusivity inside the hydrogel by high-resolution magic angle spinning, nuclear magnetic resonance spectroscopy. Cyclodextrin nanosponge are prepared by step-growth polymerization between cyclodextrins, or CDs, and the EDTA DNA dried. If we don't mention, our network of CD units is formic, capable to absorb and release organic and inorganic species.The main advantage of high-resolution magic angle spinning NMR is to monitor the transfer property of the encapsulated drug within a polymeric host with potential interest for drug delivery. The synthetic approach is very simple, and it is performed at room temperature. The step-growth polymerization reaction takes place in about 10 minutes.We have chosen ibuprofen as an example of a drug trapped in polymeric hydrogen metrics, representative of a really six car fold down delivery. Dry beta cyclodextrin in an oven at 100 degrees Celsius up to a constant weight. Also, dry 250 milliliters of DMSO, and 100 milliliters of triethylamine over four Angstrom molecular sieves for 24 hours before using them in the protocol.Introduce 25 milliliters of DMSO into a 50-milliliter, one-neck round bottom flask. Under magnetic stirring, add 5.675 grams of beta-cyclodextrin. Add the beta-cyclodextrin powder in small portions to DMSO to reduce the formation of lumps.After about 30 minutes, add six milliliters of triethylamine to the homogenous solution using a 10 milliliter graduated pipette. Plunge the flask into a water bath maintained at room temperature, and keep the mixture under stirring for 15 minutes. For preparation of cyclodextrin nanosponge one-four, add 5.124 grams of EDTA dianhydride under intense stirring.And for preparation of cyclodextrin nanosponge one-eight, add 10.248 grams of EDTA dianhydride under intense stirring. After three hours, remove the solid material, which is the cyclodextrin nanosponge, from the flask using a spatula and crush it grossly with a mortar and pestle. Wash the solid material in an acetone bath, then wash and filter the solid material on filter paper.After drying all the solid material in the air at room temperature for 48 hours, crush the dried material finely using a mortar and pestle. Then, keep it under vacuum for two hours at 45 degrees Celsius. For high-resolution magic angle spinning NMR sample preparation, first prepare a solution of 0.27 molar ibuprofen sodium salt in deuterated water.Add 20 milligrams of cyclodextrin nanosponge one-four, and two milligrams of anhydrous sodium carbonate to 150 microliters of the solution in a two milliliter glass vial. Mix the contents of the vial with a small spatula to homogenize it. Repeat this process for the cyclodextrin nanosponge one-eight polymer.Obtaining a homogenous, perfectly swollen hydrogel is the first key step for a good HR mass NMR spectrum. The hydrogel should be placed in the NMR rotor very carefully, avoiding bubbles which may cause incorrect rotor spinning. Insert the gel into a five millimeter NMR rotor suitable for HR mass experiments using a small spatula.Then, insert the rotor into the magnet of the NMR spectrometer. Once in the magnet, set the rotor in rotation by a pneumatic unit up to 4, 000 hertz. To perform HR mass proton NMR experiments, set up the instrumental parameters with a rotor spinning speed of four kilohertz at the mass pneumatic control unit, and a sample temperature at 305 kelvin in the variable temperature unit.Run a one-dimensional spectrum, quire the diffusion HR mass spectra of ibuprofen in cyclodextrin nanosponge one-Four and cyclodextrin nanosponge one-eight hydrogels by using the pulsed gradient spin-echo pulse sequence on the proton resonance. The diffusion experiments are performed by using the BP led pulse sequence. This is a pseudo two-dimensional experiment with a gradient ramp increasing linearly from 2%to 100%gradient strength in the indirect dimension.The signal intensity is attenuated depending on the diffusion time and gradient pulse P30. The optimization of these parameters is required before properly running an experiment. The optimization is done by running two one-dimensional measurements, in which the diffusion time is kept constant while P30 is varied.Variable diffusion time measurements are carried out by incrementing the diffusion time in the range of 50 to 200 milliseconds. For each experiment, an array of 32 spectra is collected. Then, process the data as described in the text protocol.The basic pulsed field gradient spin-echo experiment allows for measurement of the molecular mean-squared displacement of ibuprofen in the hydrogels. The NMR signal intensity decays as the gradient strength increases. The decay fitting provides the molecular mean-squared displacement, indicated as Z-squared.In turn, the mean-squared displacement is related to the type of diffusion regime. The alpha exponent is obtained experimentally by a log-log linear regression of the mean-squared displacement as a function of the diffusion time. The value of the alpha exponent is a descriptor of the diffusive regime observed for ibuprofen in the hydrogel matrix.Isotropic unrestricted diffusion exists when alpha equals one. A subdiffusive regime is indicated for an alpha value of less than one. And a superdiffusive regime is represented by an alpha value of greater than one.Ibuprofen entrapped in the hydrogel matrix shows two different diffusion regimes according to the polymer preparation. Hydrogels of nanosponges one-four are represented by a subdiffusive regime. Conversely, hydrogels of nanosponges one-eight are characterized by a slightly superdiffusive regime.Once mastered, this reaction can be done in high-yield and purity in one hour if it is performed properly. The materials show interesting capability of water uptake by forming viscous hydrogels, in which, biologically active molecules can be easily incorporated. The molecular state in the transfer properties of a biologically active molecule encapsulated in such polymeric matrixes can be studied experimentally by using HMS NMR techniques.The NMR results on the diffusion of ibuprofen in the hydrogels prepared from cyclodextrine nanosponges indicate that the diffusion can be modulated by a suitable preparation of the polymers. Ibuprofen shows both subdiffusive and superdiffusive regime in cyclodextrin nanosponges hydrogels. This finding can be exploited for original design of the release properties of cyclodextrin nanosponges hydrogen scaffold.

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Hydrogels have been widely utilized to enhance the surface hydrophilicity of membranes for water purification, increasing the antifouling properties and ...

Production of Elastin-like Protein Hydrogels for Encapsulation and Immunostaining of Cells in 3D

Two-dimensional (2D) tissue culture techniques have been essential for our understanding of fundamental cell biology. However, traditional 2D tissue ...

A Quantitative Fluorescence Microscopy-based Single Liposome Assay for Detecting the Compositional Inhomogeneity Between Individual Liposomes

Most research employing liposomes as membrane model systems or drug delivery carriers relies on bulk read-out techniques and thus intrinsically assumes ...

Characterization of Intra-Cartilage Transport Properties of Cationic Peptide Carriers

Several negatively charged tissues in the body, like cartilage, present a barrier to the targeted drug delivery due to their high density of negatively ...

Fragmenting Bulk Hydrogels and Processing into Granular Hydrogels for Biomedical Applications

Granular hydrogels are jammed assemblies of hydrogel microparticles (i.e., &#34;microgels&#34;). In the field of biomaterials, granular hydrogels have ...