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/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/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, 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>
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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 ...

transport-properties-ibuprofen-encapsulated-cyclodextrin-nanosponge

Research

JoVE Journal

Bioengineering

10.2K Views.  Politecnico di Milano. The motion regimes of ibuprofen encapsulated in β-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.

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

Tracking Hypoxic Signaling within Encapsulated Cell Aggregates

In Diabetes mellitus type 1, autoimmune destruction of the pancreatic &beta;-cells results in loss of insulin production and potentially lethal hyperglycemia. As an alternative treatment option to exogenous insulin injection, transplantation of functional pancreatic tissue has been explored1,2. This approach offers the promise of a more natural, long-term restoration of normoglycemia. Protection of the donor tissue from the host's immune system is required to prevent rejection and encapsulation is a method used to help achieve this aim.
Biologically-derived materials, such as alginate3 and agarose4, have been the traditional choice for capsule construction but may induce inflammation or fibrotic overgrowth5 which can impede nutrient and oxygen transport. Alternatively, synthetic poly(ethylene glycol) (PEG)-based hydrogels are non-degrading, easily functionalized, available at high purity, have controllable pore size, and are extremely biocompatible,6,7,8. As an additional benefit, PEG hydrogels may be formed rapidly in a simple photo-crosslinking reaction that does not require application of non-physiological temperatures6,7. Such a procedure is described here. In the crosslinking reaction, UV degradation of the photoinitiator, 1-[4-(2-Hydroxyethoxy)-phenyl]-2-hydroxy-2-methyl-1-propane-1-one (Irgacure 2959), produces free radicals which attack the vinyl carbon-carbon double bonds of dimethacrylated PEG (PEGDM) inducing crosslinking at the chain ends. Crosslinking can be achieved within 10 minutes. PEG hydrogels constructed in such a manner have been shown to favorably support cells7,9, and the low photoinitiator concentration and brief exposure to UV irradiation is not detrimental to viability and function of the encapsulated tissue10. While we methacrylate our PEG with the method described below, PEGDM can also be directly purchased from vendors such as Sigma.
An inherent consequence of encapsulation is isolation of the cells from a vascular network. Supply of nutrients, notably oxygen, is therefore reduced and limited by diffusion. This reduced oxygen availability may especially impact &beta;-cells whose insulin secretory function is highly dependent on oxygen11-13. Capsule composition and geometry will also impact diffusion rates and lengths for oxygen. Therefore, we also describe a technique for identifying hypoxic cells within our PEG capsules. Infection of the cells with a recombinant adenovirus allows for a fluorescent signal to be produced when intracellular hypoxia-inducible factor (HIF) pathways are activated14. As HIFs are the primary regulators of the transcriptional response to hypoxia, they represent an ideal target marker for detection of hypoxic signaling15. This approach allows for easy and rapid detection of hypoxic cells. Briefly, the adenovirus has the sequence for a red fluorescent protein (Ds Red DR from Clontech) under the control of a hypoxia-responsive element (HRE) trimer. Stabilization of HIF-1 by low oxygen conditions will drive transcription of the fluorescent protein (Figure 1). Additional details on the construction of this virus have been published previously15. The virus is stored in 10% glycerol at -80&deg; C as many 150 &mu;L aliquots in 1.5 mL centrifuge tubes at a concentration of 3.4 x 1010 pfu/mL. 
Previous studies in our lab have shown that MIN6 cells encapsulated as aggregates maintain their viability throughout 4 weeks of culture in 20% oxygen. MIN6 aggregates cultured at 2 or 1% oxygen showed both signs of necrotic cells (still about 85-90% viable) by staining with ethidium bromide as well as morphological changes relative to cells in 20% oxygen. The smooth spherical shape of the aggregates displayed at 20% was lost and aggregates appeared more like disorganized groups of cells. While the low oxygen stress does not cause a pronounced drop in viability, it is clearly impacting MIN6 aggregation and function as measured by glucose-stimulated insulin secretion15. Western blot analysis of encapsulated cells in 20% and 1% oxygen also showed a significant increase in HIF-1&alpha; for cells cultured in the low oxygen conditions which correlates with the expression of the DsRed DR protein.

A method for photo-encapsulation of cells in a crosslinked PEG hydrogel is described. Hypoxic signaling within encapsulated murine insulinoma (MIN6) aggregates is tracked using a fluorescent marker system. This system allows serial examination of cells within a hydrogel scaffold and correlation of hypoxic signaling with changes in cell phenotype.

In Diabetes mellitus type 1, autoimmune destruction of the pancreatic &beta;-cells results in loss of insulin production and potentially lethal ...

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 ...

In Situ Mapping of the Mechanical Properties of Biofilms by Particle-tracking Microrheology

Bacterial cells are able to form surface-attached biofilm communities known as biofilms by encasing themselves in extracellular polymeric substances (EPS). The EPS serves as a physical and protective scaffold that houses the bacterial cells and consists of a variety of materials that includes proteins, exopolysaccharides and DNA. The composition of the EPS may change, which remodels the mechanic properties of the biofilm to further develop or support alternative biofilm structures, such as streamers, as a response to environmental cues. Despite this, there are little quantitative descriptions on how EPS components contribute to the mechanical properties and function of biofilms. Rheology, the study of the flow of matter, is of particular relevance to biofilms as many biofilms grow in flow conditions and are constantly exposed to shear stress. It also provides measurement and insight on the spreading of the biofilm on a surface. Here, particle-tracking microrheology is used to examine the viscoelasticity and effective crosslinking roles of different matrix components in various parts of the biofilm during development. This approach allows researchers to measure mechanic properties of biofilms at the micro-scale, which might provide useful information for controlling and engineering biofilms.

In Situ Mapping of the Mechanical Properties of Biofilms by Particle-tracking Microrheology

Particle-tracking microrheology investigates the viscoelasticity of materials. Here, the technique is used to determine the viscoelasticity, creep compliance and effective crosslinking roles of different matrix components of a bacterial biofilm. The matrix consists of polymeric substances secreted by the bacteria and its components determine biofilm structure and mechanical properties.

Bacterial cells are able to form surface-attached biofilm communities known as biofilms by encasing themselves in extracellular polymeric substances ...

The Synthesis of RGD-functionalized Hydrogels as a Tool for Therapeutic Applications

The use of polymers as biomaterials has provided significant advantages in therapeutic applications. In particular, the possibility to modify and functionalize polymer chains with compounds that are able to improve biocompatibility, mechanical properties, or cell viability allows the design of novel materials to meet new challenges in the biomedical field. With the polymer functionalization strategies, click chemistry is a powerful tool to improve cell-compatibility and drug delivery properties of polymeric devices. Similarly, the fundamental need of biomedicine to use sterile tools to avoid potential adverse-side effects, such as toxicity or contamination of the biological environment, gives rise to increasing interest in the microwave-assisted strategy.
The combination of click chemistry and the microwave-assisted method is suitable to produce biocompatible hydrogels with desired functionalities and improved performances in biomedical applications. This work aims to synthesize RGD-functionalized hydrogels. RGD (arginylglycylaspartic acid) is a tripeptide that can mimic cell adhesion proteins and bind to cell-surface receptors, creating a hospitable microenvironment for cells within the 3D polymeric network of the hydrogels. RGD functionalization occurs through Huisgen 1,3-dipolar cycloaddition. Some PAA carboxyl groups are modified with an alkyne moiety, whereas RGD is functionalized with azido acid as the terminal residue of the peptide sequence. Finally, both products are used in a copper catalyzed click reaction to permanently link the peptide to PAA. This modified polymer is used with carbomer, agarose and polyethylene glycol (PEG) to synthesize a hydrogel matrix. The 3D structure is formed due to an esterification reaction involving carboxyl groups from PAA and carbomer and hydroxyl groups from agarose and PEG through microwave-assisted polycondensation. The efficiency of the gelation mechanism ensures a high degree of RGD functionalization. In addition, the procedure to load therapeutic compounds or biological tools within this functionalized network is very simple and reproducible.

We present a protocol for the synthesis of RGD-functionalized hydrogels as devices for cell and drug delivery. The procedure involves copper catalyzed alkyne-azide cycloaddition (CuAAC) between alkyne-modified polyacrylic acid (PAA) and a RGD-azide derivative. The hydrogels are formed using microwave-assisted polycondensation and their physicochemical properties are investigated.

The use of polymers as biomaterials has provided significant advantages in therapeutic applications. In particular, the possibility to modify and ...

Light-mediated Formation and Patterning of Hydrogels for Cell Culture Applications

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In particular, light-mediated click reactions, such as photoinitiated thiol&#8722;ene and thiol&#8722;yne reactions, afford spatiotemporal control over material properties and allow the design of systems with a high degree of user-directed property control. Fabrication and modification of hydrogel-based biomaterials using the precision afforded by light and the versatility offered by these thiol&#8722;X photoclick chemistries are of growing interest, particularly for the culture of cells within well-defined, biomimetic microenvironments. Here, we describe methods for the photoencapsulation of cells and subsequent photopatterning of biochemical cues within hydrogel matrices using versatile and modular building blocks polymerized by a thiol&#8722;ene photoclick reaction. Specifically, an approach is presented for constructing hydrogels from allyloxycarbonyl (Alloc)-functionalized peptide crosslinks and pendant peptide moieties and thiol-functionalized poly(ethylene glycol) (PEG) that rapidly polymerize in the presence of lithium acylphosphinate photoinitiator and cytocompatible doses of long wavelength ultraviolet (UV) light. Facile techniques to visualize photopatterning and quantify the concentration of peptides added are described. Additionally, methods are established for encapsulating cells, specifically human mesenchymal stem cells, and determining their viability and activity. While the formation and initial patterning of thiol-alloc hydrogels are shown here, these techniques broadly may be applied to a number of other light and radical-initiated material systems (e.g., thiol-norbornene, thiol-acrylate) to generate patterned substrates.

We describe a sequential process for light-mediated formation and subsequent biochemical patterning of synthetic hydrogel matrices for three-dimensional cell culture applications. The construction and modification of hydrogels with cytocompatible photoclick chemistry is demonstrated. Additionally, facile techniques to quantify and observe patterns and determine cell viability within these hydrogels are presented.

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In ...

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung tissue from porcine, rat, or mouse, the tissue is perfused and submerged in subsequent chemical detergents to remove the cellular debris. Histological comparison of the tissue before and after processing confirms removal of over 95% of double stranded DNA and alpha galactosidase staining suggests the majority of cellular debris is removed. After decellularization, the tissue is lyophilized and then cryomilled into a powder. The matrix powder is digested for 48 hr in an acidic pepsin digestion solution and then neutralized to form the pregel solution. Gelation of the pregel solution can be induced by incubation at 37 &#176;C and can be used immediately following neutralization or stored at 4 &#176;C for up to two weeks. Coatings can be formed using the pregel solution on a non-treated plate for cell attachment. Cells can be suspended in the pregel prior to self-assembly to achieve a 3D culture, plated on the surface of a formed gel from which the cells can migrate through the scaffold, or plated on the coatings. Alterations to the strategy presented can impact gelation temperature, strength, or protein fragment sizes. Beyond hydrogel formation, the hydrogel stiffness may be increased using genipin.

This is a method to create a 3-dimensional cell culture scaffold from pulmonary extracellular matrix. Intact lung is processed into hydrogels that can support the growth of cells in three-dimensions.

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung ...

Targeted Plasma Membrane Delivery of a Hydrophobic Cargo Encapsulated in a Liquid Crystal Nanoparticle Carrier

The controlled delivery of drug/imaging agents to cells is critical for the development of therapeutics and for the study of cellular signaling processes. Recently, nanoparticles (NPs) have shown significant promise in the development of such delivery systems. Here, a liquid crystal NP (LCNP)-based delivery system has been employed for the controlled delivery of a water-insoluble dye, 3,3&#8242;-dioctadecyloxacarbocyanine perchlorate (DiO), from within the NP core to the hydrophobic region of a plasma membrane bilayer. During the synthesis of the NPs, the dye was efficiently incorporated into the hydrophobic LCNP core, as confirmed by multiple spectroscopic analyses. Conjugation of a PEGylated cholesterol derivative to the NP surface (DiO-LCNP-PEG-Chol) enabled the binding of the dye-loaded NPs to the plasma membrane in HEK 293T/17 cells. Time-resolved laser scanning confocal microscopy and F&#246;rster resonance energy transfer (FRET) imaging confirmed the passive efflux of DiO from the LCNP core and its insertion into the plasma membrane bilayer. Finally, the delivery of DiO as a LCNP-PEG-Chol attenuated the cytotoxicity of DiO; the NP form of DiO exhibited ~30-40% less toxicity compared to DiOfree delivered from bulk solution. This approach demonstrates the utility of the LCNP platform as an efficient modality for the membrane-specific delivery and modulation of hydrophobic molecular cargos.

A liquid crystal nanoparticle (LCNP) nanocarrier is exploited as a vehicle for the controlled delivery of a hydrophobic cargo to the plasma membrane of living cells.

The controlled delivery of drug/imaging agents to cells is critical for the development of therapeutics and for the study of cellular signaling processes. ...

Synthesis of Hydrogels with Antifouling Properties As Membranes for Water Purification

Hydrogels have been widely utilized to enhance the surface hydrophilicity of membranes for water purification, increasing the antifouling properties and thus achieving stable water permeability through membranes over time. Here, we report a facile method to prepare hydrogels based on zwitterions for membrane applications. Freestanding films can be prepared from sulfobetaine methacrylate (SBMA) with a crosslinker of poly(ethylene glycol) diacrylate (PEGDA) via photopolymerization. The hydrogels can also be prepared by impregnation into hydrophobic porous supports to enhance the mechanical strength. These films can be characterized by attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) to determine the degree of conversion of the (meth)acrylate groups, using goniometers for hydrophilicity and differential scanning calorimetry (DSC) for polymer chain dynamics. We also report protocols to determine the water permeability in dead-end filtration systems and the effect of foulants (bovine serum albumin, BSA) on membrane performance.

This paper reports practical methods to prepare hydrogels in freestanding films and impregnated membranes and to characterize their physical properties, including water transport properties.

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 culture systems lack a three-dimensional (3D) matrix, resulting in a significant disconnect between results collected in vitro and in vivo. To address this limitation, researchers have engineered 3D hydrogel tissue culture platforms that can mimic the biochemical and biophysical properties of the in vivo cell microenvironment. This research has motivated the need to develop material platforms that support 3D cell encapsulation and downstream biochemical assays. Recombinant protein engineering offers a unique toolset for 3D hydrogel material design and development by allowing for the specific control of protein sequence and therefore, by extension, the potential mechanical and biochemical properties of the resultant matrix. Here, we present a protocol for the expression of recombinantly-derived elastin-like protein (ELP), which can be used to form hydrogels with independently tunable mechanical properties and cell-adhesive ligand concentration. We further present a methodology for cell encapsulation within ELP hydrogels and subsequent immunofluorescent staining of embedded cells for downstream analysis and quantification.

Recombinant protein-engineered hydrogels are advantageous for 3D cell culture as they allow for complete tunability of the polymer backbone and therefore, the cell microenvironment. Here, we describe the process of recombinant elastin-like protein purification and its application in 3D hydrogel cell encapsulation.

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 all liposomes of the ensemble to be identical. However, new experimental platforms able to observe liposomes at the single-particle level have made it possible to perform highly sophisticated and quantitative studies on protein-membrane interactions or drug carrier properties on individual liposomes, thus avoiding errors from ensemble averaging. Here we present a protocol for preparing, detecting, and analyzing single liposomes using a fluorescence-based microscopy assay, facilitating such single-particle measurements. The setup allows for imaging individual liposomes in a massive parallel manner and is employed to reveal intra-sample size and compositional inhomogeneities. Additionally, the protocol describes the advantages of studying liposomes at the single liposome level, the limitations of the assay, and the important features to be considered when modifying it to study other research questions.

This protocol describes the fabrication of liposomes and how these can be immobilized on a surface and imaged individually in a massive parallel manner using fluorescence microscopy. This allows for the quantification of the size and compositional inhomogeneity between single liposomes of the population.

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