Name: transport properties of ibuprofen encapsulated in cyclodextrin nanosponge hydrogels: a proton hr-mas nmr spectroscopy study
Uploaded: 2016-08-15T13:00:00.000+00:00
Duration: 10 min 10 s
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>
	<li>After about 30 min, add 6ml of Et3N to the homogeneous solution using a 10 ml graduated pipette. Keep the mixture under stirring for 15 min at RT. Plunge the flask into a water bath at RT. 
	NOTE: The reaction between &#946;-CD and EDTA is exothermic. Therefore, plunging the flask into the water bath favors the heat exchange avoiding the overheating of the reaction mixture.</li>
	<li>Add 5.124 g (20 mmol, preparation of CDNSEDTA 1:4) or 10.248 g (40 mmol, preparation of CDNSEDTA 1:8) of EDTA-dianhydride under intense stirring.</li>
	<li>After 3 hr, remove the solid material (CDNSEDTA 1:4 or CDNSEDTA 1:8) from the flask using a spatula and crush it grossly with a mortar and pestle.</li>
	<li>Wash the solid material onto filter paper with acetone at RT (100 ml &#215; 5 times), with HCl 0.1 M (200 ml &#215; 5 times), and deionized water (200 ml &#215; 3 times).</li>
	<li>Finally, dry all the solid material in air at RT for 48 hr, crush it finely into a mortar and pestle and then keep it under vacuum (&#60; 15 mbar) for 2 hr at 45 &#176;C.</li>
</ol>
<img alt="Figure 1" src="/files/ftp_upload/53769/53769fig1.jpg" /> 
Figure 1: Schematic Representation of the CDNSEDTA Polymers. Schematic synthetic route. Left: molecular structure of the monomer &#946;-cyclodextrin (&#946;-CD) and cross-linking agent EDTA-dianhydride. On the arrow the overall reaction conditions. Right: sketch of the cross-linked polymer. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53769/53769fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
2. HR-MAS NMR Measurements
<ol>
	<li>HR-MAS NMR Sample Preparation
	<ol>
		<li>Prepare a solution 0.27 M of ibuprofen sodium salt (IP) in deuterated water (99.8%).</li>
		<li>Add 20 mg of CDNSEDTA 1:4 and 2 mg of anhydrous Sodium carbonate (Na2CO3) to 150 &#181;l of the solution prepared at the point 2.1.1) into a 2 ml glass vial. Mix the content of the vial with a small spatula in order to homogenize it. Wait 2 hr before using the gel formed with this procedure. Repeat this point for the CDNSEDTA 1:8 polymer.</li>
		<li>Insert the gel in a 5mm NMR rotor suitable for HR-MAS NMR experiments using a small spatula. The total amount of gel to use depends on the internal volume of the rotor (12 &#956;l recommended).</li>
	</ol>
	</li>
	<li>HR-MAS 1H NMR Experiments
	<ol>
		<li>Set the following instrumental parameters: rotor spinning speed of 4 KHz at the MAS pneumatic control unit, sample temperature at 305 K in the variable temperature unit.</li>
		<li>Acquire the 1H HR-MAS NMR spectra of ibuprofen in CDNSEDTA (1:4) and CDNSEDTA (1:8) polymer systems using a conventional one pulse sequence on the proton resonance.
		<ol>
			<li>Create a new data set. Click on the &#34;AcquPars&#34; tab. Select the PULPROG: zg.</li>
			<li>Select the number of scans (NS = 4) and the time delay between them (D1 = 5 sec).Set the spectral width (SW = 8 ppm), the time domain (TD = 16K) and the receiver gain (RG = 32).</li>
			<li>Type &#34;zg&#34; on the console and there will be a free induction decay (FID) on the screen. To process the data click on the &#34;ProcPars&#34; tab. Set the spectral size (SI = 32K), an exponential multiplication window function (WDW = EM) and line broadening (LB = 1). Type &#34;ft&#34; to perform the Fourier transformation. Phase the spectrum using the phase tab on the screen. Obtain a high resolution well resolved spectrum.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
<img alt="Figure 2" src="/files/ftp_upload/53769/53769fig2.jpg" /> 
Figure 2: The Bipolar Pulse Pairs Longitudinal Eddy Current Delay (BPPLED) Pulse Sequence. Schematic representation of the pulse sequence used to perform the PFGSE experiments. The phase cycle for the 90&#176; pulses is: P1: (0)16, P2: (0022)4, P3: (0)4 (2)4 (1)4 (3)4, P4: (0202 2020 1313 3131), P5: (0)4 (2)4 (1)4 (3)4. The 180&#176; pulses are + x. (modified from ref.18) <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53769/53769fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="3">
	<li>HR-MAS 1H NMR PGSE Experiments 
	NOTE: The PGSE experiments are performed using the BPPLED pulse sequence18 reported in Figure 2. This is a pseudo two-dimensional experiment with a gradient ramp increasing linearly from 2% to 100% in the indirect dimension. The signal intensity is attenuated depending on the diffusion time &#916; and the gradient pulse &#948;. The optimization of these parameters is required before running properly a PGSE experiment. The optimization is done by running a few 1D measurements in which&#160;&#916; is kept constant, while&#160;&#948; is varied.
	<ol>
		<li>Parameters Optimization
		<ol>
			<li>Create a new data set - experiment number 1. Click on the &#34;AcquPars&#34; tab. Select the PULPROG: ledbpgp2s1d the 1D pulse sequence for diffusion optimization.</li>
			<li>Select the number of scans (NS = 16) and the time delay between them (D1 = 10 sec). Set the spectral width (SW = 8 ppm), the time domain (TD = 16K) and the receiver gain (RG = 32).</li>
			<li>Set&#160;&#916; (D20 in the sequence) equal to a constant value and&#160;&#948; (p30) to a trial value. Start value &#916; = 50 msec, &#948; = 3 msec (maximum allowed value for high resolution instruments).</li>
			<li>Read the value of spectral frequency (SFO1) from the 1H experiment and use now this value. Set the GPZ6 gradient strength to 2%. Repeat step 2.2.2.3. Use this spectrum as reference for the optimization.</li>
			<li>In the same data set create the experiment number 2. Observe all the experimental parameters. Increase the GPZ6 gradient strength to 95%. Repeat step 2.2.2.3. Compare this spectrum with the reference spectrum using the dual display icon and observe the change in the signal intensity. 
			NOTE: A well attenuated spectrum should have about 5% residual signal intensity compared with the reference spectrum. If the signal intensity is lost, reduce the value of&#160;&#948; and restart the section 2.3.1 procedure from point 2.3.1.3 until the right value for&#160;&#948; is found.</li>
			<li>Repeat the parameters optimization procedure in section 2.3.1 for all the five &#916; values. 
			NOTE: Choose five value for &#916; = 50, 80, 110, 140 and 170 msec and optimized the corresponding&#160;&#948; to 3, 2.7, 2.4, 2.1, 1.8 msec (for IP in CDNSEDTA 1:8) and to&#160;&#948; to 2.7, 2.4, 2, 1.7, 1.4 (for IP in in CDNSEDTA 1:4).</li>
		</ol>
		</li>
		<li>Acquisition of the 2D Diffusion Data Set
		<ol>
			<li>In the same data set create the experiment number 3, all the 1D experimental parameters will be loaded. Type &#34;eda&#34;. Select the PULPROG: ledbpgp2s the 2D pulse sequence and change the parmode to 2D.</li>
			<li>Set FnMODE=QF. Set the time domain TD in F2 dimension equal to 32, the number of gradient steps. All the other parameters are set correctly. Type &#34;DOSY&#34; and the gradient ramp will be generated and stored in a file. The start and final values of the ramp (2 - 95) are given as input parameters. The acquisition is now started.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Data Processing
	<ol>
		<li>Type &#34;xf2&#34; to execute the Fourier transformation in the F2 dimension. Type &#34;abs2&#34; to perform the baseline correction in the F2 dimension. Type &#34;setdiffparm&#34; to recall the experimental parameters (&#916;, &#948;, and gradient list) for the next processing step.</li>
		<li>Click &#34;T1/T2 relaxation module&#34; in the analysis tab and define the peaks to be fitted using the first spectrum of the 2D experiment. Define the peak ranges and execute the fitting. The signal intensities at each applied gradient step are obtained. 
		NOTE: The signal intensities I(q, td), for each&#160;&#916; value, depends on the experimental variables: applied pulse filed gradient (g), time variable (&#948;), magnetogyric ratio (&#947;) q= (&#947;g&#948;) according to the following equation: 
		<img alt="Equation1" src="/files/ftp_upload/53769/53769equation1.jpg" /> 
		with the molecular MSD=z2.</li>
		<li>Export the signal intensities in a spreadsheet and perform a linear fit of the data to get the value of z2 for each observed diffusion time td. 
		NOTE: The MSD value is related to the observation time td according to: <img alt="Equation2" src="/files/ftp_upload/53769/53769equation2.jpg" /></li>
		<li>Perform the log-log plot of z2 versus td for each experimental td value. The exponent &#945; value is the slope of the linear regression. A more exhaustive discussion of the physical aspects of the equations reported above can be found in ref. 21 and in the references therein. 
		NOTE: Depending on the value of the exponent &#945;, the diffusion regime is defined as: i) isotropic unrestricted diffusion for &#945; = 1, ii) anomalous subdiffusive regime for 0 &#60; &#945; &#60; 1, iii) anomalous superdiffusive regime for &#945; &#62; 1.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

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, that for longer observation times the transition between different diffusion regimes can be observed22. Thus, the results reported here are referred to the time-scale dictated by the hardware available for the experiments and with the instrumentation at our disposal.
For each step of this work - polymer synthesis, hydrogel loading, HR-MAS NMR data collection and processing, we list here three main problems and the corresponding solution.
Purification 
The purification of the materials according to the procedure outlined at the point 1.6 allows the elimination of DMSO, Et3N and of the unreacted EDTA eventually contained into the reaction product. In particular, washing with 0.1 M HCl aqueous solution leads to the complete removal of Et3N. Washing the materials with only acetone is not efficient for this purpose. Indeed, Et3N is in part present as Et3NH+ at the end of the reaction with the counter ions COO- provided by the cross-linked EDTA.
NMR sample preparation 
The rotor filling is the critical step for sample preparation (see 2.1.3). The presence of air bubbles must be avoided. If bubbles are present, an incorrect spinning is generally detected (e.g., maximum spinning rate not reached, spinning does not start at all, not uniform spinning rate) . In the case of non-uniform spinning or failure to start spinning the rotor, extract the sample and repeat the rotor filling more carefully.
NMR Data analysis 
In point 2.4.2 it is described how to obtain MSD values from the intensity decay curves. Very often the experimental points do not seem to follow a completely linear trend vs q2. In such case, use only the linear part of the dataset and run the linear regression.
The obtained results may be summarized as follows: 
D2O solution 
In this simple case, the MSD shows linear dependence on the diffusion time&#160;&#916; and the IP molecule undergo unrestricted diffusion in solution. This case is generally referred to as &#34;normal&#34; diffusion. The self-diffusion coefficient can be directly estimated from a single measurement at any observation time td. In a general approach, the measurement of diffusivity in isotropic, molecular solvent may be taken as reference to investigate the influence of the scaffold on the transport properties of the encapsulated guest.
CDNSEDTA 1:4 polymer 
The entrapment of IP into the polymeric hydrogel affects the drug transport properties compared to the water solution. A subdiffusive motion with &#945; = 0.64 is detected. The translational motion of the drug molecules is hampered by the presence of nanopores of different sizes originated during the cross-linking process.
CDNSEDTA (1:8) polymer 
In this case the unexpected value &#945; = 1.06 is determined, thus indicating a slightly superdiffusive motion regime. Accordingly, an acceleration effect on the particles MSD is observed (Table 1). This effect can be ascribed to the negative electric potential of the polymeric backbone generated by the carboxylic groups negatively charged in some parts of the CDNS polymer. The electrostatic interaction with the negatively charged IP molecules provides the driving force for the superdiffusive component of the motion.
The methodology described here provided information on the diffusion regime experienced by the drug in the different molecular environment corresponding to the two hydrogels formulation through the value of the &#945; exponent discussed above. This approach is of general applicability and can be confidently proposed as investigating tool of the transport properties of the encapsulated drug, with interesting fall-out for design of drug delivery-controlled release systems. However, it should be kept in mind that the results presented here suffer from the hardware limitation of the instrumentation used.
Additionally, some limiting factors for the general applicability of the method could be hypothesized: the preparation of drug-loaded gels with lipophilic and/or non-charged drugs, the possibility of strong adhesion of the drug to the polymer backbone, thus leading to unresolved NMR signals and difficulty in pH control. Finally, it should be mentioned that the diffusion NMR data span the ms time-scale, while the classic in vitro drug release experiments rely upon much longer time-windows. This is an open point object of research and debate. As a possible contribution to address this issue, we have recently examined the diffusion data of a model drug in library of carbohydrate-based hydrogels and derived a mathematical model connecting the measurements at molecular level with the kinetic data23. Larger collections of diffusion data and broader libraries of scaffolds are under investigation to refine and validate the model.

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><tbody><tr><td>HR-MAS probe</td><td>BRUKER</td><td>N/A</td><td>Probe for NMR measurements on semi-solid samples</td></tr><tr><td>NMR Spectrometer</td><td>BRUKER</td><td>DRX 500</td><td>FT NMR spectrometer for liquid ans semi-solis state</td></tr><tr><td>&amp;beta;-cyclodextrin (&amp;beta;-CD)</td><td>Alfa-Aesar</td><td>J63161</td><td>Reagent</td></tr><tr><td>Ethylenediaminetetracetic (EDTA) dianhydride</td><td>Sigma-Aldrich</td><td>332046</td><td>Reagent</td></tr><tr><td>Dimethylsulfoxide (DMSO)</td><td>Alfa-Aesar</td><td>D0798</td><td>Solvent</td></tr><tr><td>Triethylamine</td><td>Sigma-Aldrich</td><td>471283</td><td>Base (reagent)</td></tr><tr><td>Ibuprofen (IP) sadium salt</td><td>Sigma-Aldrich</td><td>I1892</td><td>Antinflammatory drug</td></tr><tr><td>Excel 2010</td><td>Microsoft</td><td>N/A</td><td>speadsheet for data analysis</td></tr><tr><td>Origin 8 SR0</td><td>OriginLab Co.</td><td></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 function of q2 (according to section 2.4). In Table 1 are reported the observed MSD values for each diffusion time &#916;. The log-log plot of z2versus td (Figure 3) gives a line with R2= 0.999 (according to section 2.4). A scaling exponent &#945; = 1 is obtained for IP dissolved in D2O solution, indicating a Gaussian motion in the liquid solution at 0.27 M concentration. Only in this case the self-diffusion coefficient can be accordingly calculated as D=&#160; 4.1*10-10 m2sec-1 and is independent of the observation time &#916;.
<img alt="Table 1" src="/files/ftp_upload/53769/53769table1.jpg" /> 
Table 1: Experimental MSD and &#916; Values. MSD (m2) measured at several&#160;&#916; (ms) for IP in D2O solution, IP in CDNS (1:4) and IP in CDNS (1:8) hydrogels. (modified from ref. 21).
<img alt="Figure 3" src="/files/ftp_upload/53769/53769fig3.jpg" /> 
Figure 3: Plot of the NMR Signal Decay and MSD Time Dependence. a) Normalized NMR signal decay I(q,td) as function of q2 for IP in D2O solution. b) Log-log plot of MSD vs diffusion time td for IP in D2O solution. (modified from ref. 21) <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53769/53769fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The experimental procedure has been used to study the diffusion motion of IP drug molecule encapsulated in CDNSEDTA 1:4 and CDNSEDTA 1:8 hydrogels. Figure 4 shows the normalized experimental signal decays I(q, td)/I(0, td) as function of q2. Use the linear part of the decay for the linear fit and then calculate the MSD values for CDNS 1:4 and CDNS 1:8 samples (Table 1) from the slope of the linear fit. The log-log plot of MSD versus td (Figure 5) gives a linear correlation with R2= 0.981 for IP in CDNSEDTA (1:4) and a scaling exponent &#945; = 0.64 indicating a sub-diffusive motion of the drug in the polymeric gel. Using a similar procedure, the motion regime for IP in CDNSEDTA (1:8) polymer was determined. The linear fit of the data gave a scaling exponent &#945; = 1.06 with R2 = 0.972. The polymer network imposes a slightly superdiffusive motion to the small drug molecule. Thus the proposed methodology gives access to the&#160;&#945; exponent of the equation reported at point 2.4.5 by a two-step processing of data. The&#160;&#945; value is a descriptor of the diffusive regime observed for the given matrix and guest molecule.
<img alt="Figure 4" src="/files/ftp_upload/53769/53769fig4.jpg" /> 
Figure 4: Plot of the NMR Signal Decay. Normalized NMR signal decay I(q,td) as function of q2 for IP in CDNSEDTA 1:4 (a) and CDNSEDTA 1:8 (b). (modified from ref. 21) <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53769/53769fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/53769/53769fig5.jpg" /> 
Figure 5: Time Dependence of MDS. Log-log plot of MSD vs diffusion time td for IP encapsulated in CDNSEDTA 1:4 (a) and CDNSEDTA 1:8 (b). (modified from ref. 21) <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53769/53769fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

transport-properties-ibuprofen-encapsulated-cyclodextrin-nanosponge

Nanyang Technological University

Research

JoVE Journal

Bioengineering

10.2K Views.  Politecnico di Milano.  De beweging regimes ibuprofen ingekapseld in β-cyclodextrine nanosponges polymeer netwerk worden bestudeerd met behulp van pulsed-field-gradient spin-echo (PGSE) NMR-techniek. Synthese, zuivering, geneesmiddelbelading, uitvoering van de NMR pulssequentie en gegevensanalyse uitwerken de gemiddelde kwadratische verplaatsing van het geneesmiddel op verschillende observatietijdstippen worden beschreven. 

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

In Situ Monitoring of Diffusion of Guest Molecules in Porous Media Using Electron Paramagnetic Resonance Imaging

A method is demonstrated to monitor macroscopic translational diffusion using electron paramagnetic resonance (EPR) imaging. A host-guest system with nitroxide spin probe 3-(2-Iodoacetamido)-2,2,5,5-tetramethyl-1-pyrrolidinyloxy (IPSL) as a guest inside the periodic mesoporous organosilica (PMO) aerogel UKON1-GEL as a host and ethanol as a solvent is used as an example to describe the protocol. Data is shown from a previous publication, where the protocol has been applied to both IPSL and Tris(8-carboxy-2,2,6,6-perdeutero-tetramethyl-benzo[1,2-d:4,5-d&#8242;]bis(1,3)dithiole) methyl (Trityl) as guest molecules and UKON1-GEL and SILICA-GEL as host systems. 
A method is shown to prepare aerogel samples that cannot be synthesized directly in the sample tube for measurement due to a size change during synthesis. The aerogel is attached to sample tubes using heat shrink tubing and a pressure cooker to reach the necessary temperature without evaporating the solvent in the process. The method does not assume a clearly defined initial distribution of guest molecules at the start of the measurement. Instead, it requires a reservoir on top of the aerogel and experimentally determines the influx rate during data analysis.
The diffusion is monitored continually over a period of 20 hr by recording the 1d spin density profile within the sample. The spectrometer settings for the imaging experiment are described quantitatively. Data analysis software is provided to take the resonator sensitivity profile into account and to numerically solve the diffusion equation. The software determines the macroscopic translational diffusion coefficient by least square minimization of the difference between the experiment and the numerical solution of the diffusion equation.

In Situ Monitoring of Diffusion of Guest Molecules in Porous Media Using Electron Paramagnetic Resonance Imaging

A protocol for the in situ monitoring of the diffusion of guest molecules in porous media using electron paramagnetic resonance (EPR) imaging is presented.

A method is demonstrated to monitor macroscopic translational diffusion using electron paramagnetic resonance (EPR) imaging. A host-guest system with ...

Chemistry

Application and Methodology of the Non-destructive 19F Time-domain NMR Technique to Measure the Content in Fluorine-containing Drug Products

Here, we describe a protocol developed by our group that uses low-field fluorine-19 (19F) time-domain (TD) nuclear magnetic resonance (NMR) to measure the average content of fluorinated drugs in their formulated drug product forms: tablets or capsules. This method is specific to fluorinated drugs because it detects only the content of fluorine, avoiding interference from the excipients that lack fluorine. The advantages of measuring the active content of fluorinated drugs using low-field 19F TD-NMR versus high-field 19F solid-state (SS) NMR are the simplicity of the method; the low cost; and the non-destructive nature of the technique, with all samples recoverable in intact forms (e.g., powders, tablets, and capsules), making this technique affordable for any laboratory.
We have tested the method with three fluorinated drug products available on the market - cinacalcet, lansoprazole, and ciprofloxacin - with doses ranging from 15 to 500 mg. The results of the analyses, measured by low-field 19F TD-NMR, supported the reported label claims for the average drug content.
Based on the simplicity and reproducibility of the analysis, we envision this methodology being implemented in any laboratory, including manufacturing plants, as a process analytical technology (PAT) tool in the pharmaceutical industry.

Application and Methodology of the Non-destructive 19F Time-domain NMR Technique to Measure the Content in Fluorine-containing Drug Products

A simple and non-destructive technique that measures the average content of drug substances in formulated drug products containing fluorine using low-field fluorine-19 (19F) time-domain (TD) nuclear magnetic resonance (NMR) is presented here. The technique can be applied to the development and manufacturing of drugs in the pharmaceutical industry.

Here, we describe a protocol developed by our group that uses low-field fluorine-19 (19F) time-domain (TD) nuclear magnetic resonance (NMR) to measure the ...

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous number of imaging techniques currently available and the progress in instrumentation, there is still a need for highly sensitive probes that are suitable for in vivo imaging. One typical problem of available preclinical fluorescent probes is their rapid clearance in vivo, which reduces their imaging sensitivity. To circumvent rapid clearance, increase number of dye molecules at the target site, and thereby reduce background autofluorescence, encapsulation of the near-infrared fluorescent dye, DY-676-COOH in liposomes and verification of its potential for in vivo imaging of inflammation was done. DY-676 is known for its ability to self-quench at high concentrations. We first determined the concentration suitable for self-quenching, and then encapsulated this quenching concentration into the aqueous interior of PEGylated liposomes. To substantiate the quenching and activation potential of the liposomes we use a harsh freezing method which leads to damage of liposomal membranes without affecting the encapsulated dye. The liposomes characterized by a high level of fluorescence quenching were termed Lip-Q. We show by experiments with different cell lines that uptake of Lip-Q is predominantly by phagocytosis which in turn enabled the characterization of its potential as a tool for in vivo imaging of inflammation in mice models. Furthermore, we use a zymosan-induced edema model in mice to substantiate the potential of Lip-Q in optical imaging of inflammation in vivo. Considering possible uptake due to inflammation-induced enhanced permeability and retention (EPR) effect, an always-on liposome formulation with low, non-quenched concentration of DY-676-COOH (termed Lip-dQ) and the free DY-676-COOH were compared with Lip-Q in animal trials.

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

The use of fluorophores for in vivo imaging can be greatly limited by opsonization, rapid clearance, low detection sensitivity and cytotoxic effects on the host. Encapsulation of fluorophores in liposomes by film hydration and extrusion leads to fluorescence quenching and protection which enables in vivo imaging with high detection sensitivity.

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous ...

A Rapid and Quantitative Fluorimetric Method for Protein-Targeting Small Molecule Drug Screening

We demonstrate a new drug screening method for determining the binding affinity of small drug molecules to a target protein by forming fluorescent gold nanoclusters (Au NCs) within the drug-loaded protein, based on the differential fluorescence signal emitted by the Au NCs. Albumin proteins such as human serum albumin (HSA) and bovine serum albumin (BSA) are selected as the model proteins. Four small molecular drugs (e.g., ibuprofen, warfarin, phenytoin, and sulfanilamide) of different binding affinities to the albumin proteins are tested. It was found that the formation rate of fluorescent Au NCs inside the drug loaded albumin protein under denaturing conditions (i.e., 60 &#176;C or in the presence of urea) is slower than that formed in the pristine protein (without drugs). Moreover, the fluorescent intensity of the as-formed NCs is found to be inversely correlated to the binding affinities of these drugs to the albumin proteins. Particularly, the higher the drug-protein binding affinity, the slower the rate of Au NCs formation, and thus a lower fluorescence intensity of the resultant Au NCs is observed. The fluorescence intensity of the resultant Au NCs therefore provides a simple measure of the relative binding strength of different drugs tested. This method is also extendable to measure the specific drug-protein binding constant (KD) by simply varying the drug content preloaded in the protein at a fixed protein concentration. The measured results match well with the values obtained using other prestige but more complicated methods.

A protocol for small molecular drug screening based on in-situ synthesis of ultrasmall fluorescent gold nanoclusters (Au NCs) using drug-loaded protein as template is presented. This method is simple to determine the binding affinity of drugs to a target protein by a visible fluorescent signal emitted from the protein-templated Au NCs.

We demonstrate a new drug screening method for determining the binding affinity of small drug molecules to a target protein by forming fluorescent gold ...

Magnetic and Thermal-sensitive Poly(N-isopropylacrylamide)-based Microgels for Magnetically Triggered Controlled Release

Magnetically and thermally sensitive poly(N-isopropylacrylamide) (PNIPAAm)/Fe3O4-NH2 microgels with the encapsulated anti-cancer drug curcumin (Cur) were designed and fabricated for magnetically triggered release. PNIPAAm-based magnetic microgels with a spherical structure were produced via a temperature-induced emulsion followed with physical-crosslinking by mixing PNIPAAm, polyethylenimine (PEI), and Fe3O4-NH2 magnetic nanoparticles. Because of their dispersity, the Fe3O4-NH2 nanoparticles were embedded inside the polymer matrix. The amine groups exposed on the Fe3O4-NH2 and PEI surface supported the spherical structure by physically crosslinking with the amide groups of the PNIPAAm. The hydrophobic anti-cancer drug curcumin can be dispersed in water after encapsulation into the microgels. The microgels were characterized by transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FT-IR), and UV-Vis spectral analysis. Furthermore, magnetically triggered release was studied under an external high frequency magnetic field (HFMF). A significant &#34;burst release&#34; of curcumin was observed after applying the HFMF to the microgels due to the magnetic inductive heating (hyperthermia) effect. This manuscript describes the magnetically triggered controlled release of Cur-PNIPAAm/Fe3O4-NH2 encapsulated curcumin, which can be potentially applied for tumor therapy.

Magnetic and Thermal-sensitive PolyN-isopropylacrylamide-based Microgels for Magnetically Triggered Controlled Release

This manuscript describes the preparation of magnetic and thermal-sensitive microgels via a temperature-induced emulsion without chemical reaction. These sensitive microgels were synthesized by mixing poly(N-isopropylacrylamide) (PNIPAAm), polyethylenimine (PEI) and Fe3O4-NH2 nanoparticles for the potential use in magnetically and thermally triggered drug-release.

Magnetically and thermally sensitive poly(N-isopropylacrylamide) (PNIPAAm)/Fe3O4-NH2 microgels with the encapsulated anti-cancer drug curcumin (Cur) were ...

Synthesis of Stimuli-responsive Nanogels using Aqueous One-step Crosslinking and Co-nanopolymerization

Nanogels consisting of crosslinked-polymeric nanoparticles have been developed for the delivery of numerous chemical and biological therapeutics, owing to their versatile bottom-up synthesis and biocompatibility. While various methods have been employed for nanogel synthesis to date, very few have achieved it without the use of harsh organic solvents or high temperatures that can damage the integrity of the biological payload. In contrast, the methodology presented here accomplishes the synthesis of sub-100 nm sized, protein-loaded nanogels using mild reaction conditions. Here, we present a method for the non-covalent encapsulation of protein-based payloads within nano-gels that were synthesized using an aqueous-based, single-step, crosslinking copolymerization technique. In this technique, we initially electrostatically bind a protein-based payload to a cationic quaternary ammonium monomer and simultaneously cross-link and co-polymerize it using ammonium persulfate and N,N,N&#39;,N&#39;-tetramethylethylenediamine to form nanogels that entrap the protein payload. The size and polydispersity index of the nanogels is determined using dynamic light scattering (DLS), while the surface morphology is assessed by transmission electron microscopy (TEM). The mass of protein entrapped within nanogels is determined by calculating the encapsulation efficiency. Furthermore, the controlled-release ability of the nanogels via the gradual degradation of redox-responsive structural elements is also assessed in bioreduction assays. We provide examples of nanoparticle optimization data to demonstrate all caveats of nanogel synthesis and characterization using this technique. In general, uniformly sized nanogels were obtained with an average size of 57 nm and a polydispersity index value of 0.093. A high encapsulation efficiency of 76% was achieved. Furthermore, the nanogels exhibited controlled release of up to 86% of the encapsulated protein by gradual degradation of novel redox-responsive components in the presence of glutathione over 48 h.

Nanogels are an excellent and versatile nanoparticle platform for the delivery of biologics. Stimuli-responsive poly(ethylene) glycol-based polymeric nano-gels, capable of encapsulating protein-based payloads, were synthesized using a one-step cross-linking co-nanopolymerization strategy in aqueous conditions. The optimal fabrication and characterization of these novel nanoparticles are presented here.

Nanogels consisting of crosslinked-polymeric nanoparticles have been developed for the delivery of numerous chemical and biological therapeutics, owing to ...

Nanosensors to Detect Protease Activity In Vivo for Noninvasive Diagnostics

Proteases are multi-functional enzymes that specialize in the hydrolysis of peptide-bonds and control broad biological processes including homeostasis and allostasis. Moreover, dysregulated protease activity drives pathogenesis and is a functional biomarker of diseases such as cancer; therefore, the ability to detect protease activity in vivo may provide clinically relevant information for biomedical diagnostics. The goal of this protocol is to create nanosensors that probe for protease activity in vivo by producing a quantifiable signal in urine. These protease nanosensors consist of two components: a nanoparticle and substrate. The nanoparticle functions to increase circulation half-life and substrate delivery to target disease sites. The substrate is a short peptide sequence (6-8 AA), which is designed to be specific to a target protease or group of proteases. The substrate is conjugated to the surface of the nanoparticle and is terminated by a reporter, such as a fluorescent marker, for detection. As dysregulated proteases cleave the peptide substrate, the reporter is filtered into urine for quantification as a biomarker of protease activity. Herein we describe construction of a nanosensor for matrix metalloproteinase 9 (MMP9), which is associated with tumor progression and metastasis, for detection of colorectal cancer in a mouse model.

Nanosensors to Detect Protease Activity In Vivo for Noninvasive Diagnostics

Proteases are tightly regulated enzymes involved in fundamental biological processes, and dysregulated protease activity drives progression of complex diseases such as cancer. This method&#39;s goal is to create nanosensors that measure protease activity in vivo by producing a cleavage signal that is detectable from host urine and discriminates disease.

Proteases are multi-functional enzymes that specialize in the hydrolysis of peptide-bonds and control broad biological processes including homeostasis and ...

Flash NanoPrecipitation for the Encapsulation of Hydrophobic and Hydrophilic Compounds in Polymeric Nanoparticles

The formulation of a therapeutic compound into nanoparticles (NPs) can impart unique properties. For poorly water-soluble drugs, NP formulations can improve bioavailability and modify drug distribution within the body. For hydrophilic drugs like peptides or proteins, encapsulation within NPs can also provide protection from natural clearance mechanisms. There are few techniques for the production of polymeric NPs that are scalable. Flash NanoPrecipitation (FNP) is a process that uses engineered mixing geometries to produce NPs with narrow size distributions and tunable sizes between 30 and 400 nm. This protocol provides instructions on the laboratory-scale production of core-shell polymeric nanoparticles of a target size using FNP. The protocol can be implemented to encapsulate either hydrophilic or hydrophobic compounds with only minor modifications. The technique can be readily employed in the laboratory at milligram scale to screen formulations. Lead hits can directly be scaled up to gram- and kilogram-scale. As a continuous process, scale-up involves longer mixing process run time rather than translation to new process vessels. NPs produced by FNP are highly loaded with therapeutic, feature a dense stabilizing polymer brush, and have a size reproducibility of &#177; 6%.

Flash NanoPrecipitation (FNP) is a scalable approach to produce polymeric core-shell nanoparticles. Lab-scale formulations for the encapsulation of hydrophobic or hydrophilic therapeutics are described.

The formulation of a therapeutic compound into nanoparticles (NPs) can impart unique properties. For poorly water-soluble drugs, NP formulations can ...

A Freeze-Thawing Method to Prepare Chitosan-Poly(vinyl alcohol) Hydrogels Without Crosslinking Agents and Diflunisal Release Studies

Chitosan-poly(vinyl alcohol) hydrogels can be produced by the freeze-thawing method without using toxic crosslinking agents. The applications of these systems are limited by their characteristics (e.g., porosity, flexibility, swelling capacity, drug loading and drug release capacity), which depend on the freezing conditions and the kind and ratio of polymers. This protocol describes how to prepare hydrogels from chitosan and poly(vinyl alcohol) at 50/50 w/w % of polymer composition and varying the freezing temperature (-4 &#176;C, -20 &#176;C, -80 &#176;C) and freeze-thawing cycles (4, 5, 6 freezing cycles). FT-IR spectra, SEM micrograph and porosimetry data of hydrogels were obtained. Also, the swelling capacity and drug loading and release of diflunisal were assessed. Results from SEM micrographs and porosimetry show that the pore size decreases, while the porosity increases at lower temperatures. The swelling percentage was higher at the minor freezing temperature. The release of diflunisal from the hydrogels has been studied. All the networks maintain the drug release for 30 h and it has been observed that a simple diffusion mechanism regulates the diflunisal release according to Korsmeyer-Peppas and Higuchi models.

A Freeze-Thawing Method to Prepare Chitosan-Polyvinyl alcohol Hydrogels Without Crosslinking Agents and Diflunisal Release Studies

The freezing-thawing method is used to produce chitosan-poly(vinyl alcohol) hydrogels without crosslinking agents. For this method, it is important to consider the freezing conditions (temperature, number of cycles) and polymer ratio, which can affect the properties and applications of the obtained hydrogels.

Chitosan-poly(vinyl alcohol) hydrogels can be produced by the freeze-thawing method without using toxic crosslinking agents. The applications of these ...

Preparation and Characterization of Nanoliposomes for the Entrapment of Bioactive Hydrophilic Globular Proteins

Liposome nanocapsules have been applied for many purposes in the pharmaceutical, cosmetic, and food industries. Attributes of liposomes include their biocompatibility, biodegradability, non-immunogenicity, non-toxicity, and ability to entrap both hydrophilic and hydrophobic compounds. The classical hydration of thin lipid films in an organic solvent is applied herein as a technique to encapsulate tarin, a plant lectin, in nanoliposomes. Nanoliposome size, stability, entrapment efficiency, and morphological characterization are described in detail. The nanoliposomes are prepared using 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (ammonium salt; DSPE-MPEG 2000), and cholesterylhemisuccinate (CHEMS) as the main constituents. Lipids are first dissolved in chloroform to obtain a thin lipid film that is subsequently rehydrated in ammonium sulfate solution containing the protein to be entrapped and incubated overnight. Then, sonication and extrusion techniques are applied to generate nanosized unilamellar vesicles. The size and polydispersity index of the nanovesicles are determined by dynamic light scattering, while nanovesicle morphology is assessed by scanning electron microscopy. Entrapment efficiency is determined by the ratio of the amount of unencapsulated protein to original amount of initially loaded protein. Homogeneous liposomes are obtained with an average size of 155 nm and polydispersity index value of 0.168. A high entrapment efficiency of 83% is achieved.

This study describes classical hydration using the thin lipid film method for nanoliposome preparation followed by nanoparticle characterization. A 47 kDa-hydrophilic and globular protein, tarin, is successfully encapsulated as a strategy to improve stability, avoid fast clearance, and promote controlled release. The method can be adapted to hydrophobic molecules encapsulation.

Liposome nanocapsules have been applied for many purposes in the pharmaceutical, cosmetic, and food industries. Attributes of liposomes include their ...

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

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