Name: the synthesis of rgd-functionalized hydrogels as a tool for therapeutic applications
Uploaded: 2016-10-07T14:00:00
Description: Watch this Scientific Journal Video about The Synthesis of RGD-functionalized Hydrogels as a Tool for Therapeutic Applications at JoVE.com

Authors would like to thank Prof. Maurizio Masi for fruitful discussion and Miss Chiara Allegretti for language editing. Authors&#39; research is supported by Bando Giovani Ricercatori 2010 (Ministero della Salute GR-2010- 2312573).

Note: The chemicals are used as received. Linear RGD is purchased, but it can be prepared by standard Fmoc solid phase peptide synthesis16,19. Solvents are of analytical grade. The dialysis requires the use of membrane with a Mw cut-off equal to 3,500 Da. The synthesized compounds are characterized by 1H NMR spectra recorded on a 400 MHz spectrometer using chloroform (CDCl3) or deuterium oxide (D2O) as solvents, and chemical shifts are reported as &#948; values in pa...

<ol>
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The PAA post-polymerization modification with alkyne moieties and the RGD functionalization with the azide group guarantee the formation of a stable bond between the polymer and the peptide. Indeed, triazole serves as a rigid linking unit among the carbon atoms, attached to the 1,4 positions of the 1,2,3-triazole ring and it cannot be cleaved hydrolytically or otherwise. In addition, triazole is extremely difficult to oxidize and reduce, unlike other cyclic structures such as benzenoids and related aromatic heterocycles<...

Hydrogels are three-dimensional networks formed by hydrophilic cross-linked polymers, which are natural or synthetic, and characterized by a distinctive three-dimensional structure. These devices are increasingly attractive in the biomedical fields of drug delivery, tissue engineering, gene carriers and smart sensors1,2. Indeed, their high water content, as well as their rheological and mechanical properties make them suitable candidates to mimic soft tissue microenvironments and make them effective tools for water-soluble cytokine or growth factor delivery. One of the most promising use is as an injectable biomaterial carrying cells and bioactive compounds. Hydrogels may improve cell survival and control stem cell fate by holding and precisely delivering stem cell regulatory signals in a physiological relevant fashion, as observed in in vitro and in in vivo experiments3,4. The leading advantage of this is the possibility to maintain injected cells within the zone of inoculation (in situ), minimizing the amount of cells that leaves the area and extravasates into the circulatory torrent, migrating all over the body and losing the target goal5. The stability of the three-dimensional hydrogel networks is due to its cross-linking sites, formed by covalent bonds or cohesive forces among the polymer chains6.
In this framework, orthogonal selective chemistry applied to polymer chains is a versatile tool able to improve hydrogel performances7. Indeed, the modification of polymers with suitable chemical groups could help to provide appropriate chemical, physical and mechanical properties to enhance cell viability and their use in tissue formation. In the same way, among the techniques to load cells or growth factors within the gel matrix, the use of the RGD peptide allows improvements in cell adhesion and survival. RGD is a tripeptide composed of arginine, glycine and aspartic acid, which is by far the most effective and often employed tripeptide due to its ability to address more than one cell adhesion receptor and its biological impact on cell anchoring, behavior and survival8,9. In this work, the synthesis of RGD-functionalized hydrogels is studied with the aim of designing networks characterized by sufficient biochemical properties for a hospitable cell microenvironment.
The use of microwave radiation in hydrogel synthesis offers a simple procedure to minimize side reactions and obtain higher reaction rates and yields in a shorter period of time compared to the conventional thermal processes10. This method does not require purification steps and yields sterile hydrogels due to the interactions of the polymers and the absence of organic solvent in the reaction system11. Therefore, it ensures high percentages of RGD linked to the polymeric network because no modifications are required to the polymer chemical groups involved in gel formation. Carboxyl groups, from PAA and carbomer, and hydroxyl groups, from PEG and agarose, give rise to the hydrogel three-dimensional structure through a polycondensation reaction. The mentioned polymers are used for the synthesis of hydrogels in the spinal cord injury repair treatments12. These devices, as reported in previous works13,14, show high biocompatibility as well as mechanical and physicochemical properties that resemble those of many living tissues and in thixotropic nature. Moreover, they remain localized in situ, at the zone of injection.
In this work, PAA carboxyl groups are modified with an alkyne moiety (Figure 1), and a RGD-azide compound is synthesized exploiting the reactivity of the tripeptide terminal group -NH2 with a prepared chemical compound with structure (CH2)n-N3 (Figure 2). Subsequently, the modified PAA reacts with the RGD-azide derivative through CuAAC click reaction15-17 (Figure 3). The use of a copper(I) catalyst leads to major improvements in both the reaction rate and the regioselectivity. The CuAAC reaction is widely used in organic synthesis and in polymer science. It combines high efficiency and high tolerance to the functional groups, and it is unaffected by the use of organic solvents. A high selectivity, a fast reaction time and a simple purification procedure allow the obtainment of star polymers, block copolymers or chains grafting desired moieties18. This click strategy makes it possible to modify polymers after polymerization to customize the physicochemical properties according to the final biochemical application. The CuAAC experimental conditions are easily reproducible (the reaction is insensitive to water, whereas copper oxidation may occur minimally), and the nature of formed triazole ensures stability of the product. The use of copper metal can be considered a critical point, due to its potential toxic effect against cells and in the biological microenvironment, but dialysis is used as a purification method to allow the complete removal of catalytic residues. Finally, PAA modified RGD is used in hydrogel synthesis (Figure 4) and the physicochemical properties of the resulting networks are investigated, in order to check the potential functionality of these systems as cells or drugs carriers.
<img alt="Figure 1" src="/files/ftp_upload/54445/54445fig1.jpg" /> 
Figure 1: PAA modified alkyne synthesis. A scheme of PAA functionalization with alkyne group; &#34;n&#34; indicates the monomers with carboxyl group reacting with propargylamine. <a href="http://cloudflare.jove.com/files/ftp_upload/54445/54445fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54445/54445fig2.jpg" /> 
Figure 2: RGD-azide synthesis. The synthesis of RGD-azide derivative. <a href="http://cloudflare.jove.com/files/ftp_upload/54445/54445fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54445/54445fig3.jpg" /> 
Figure 3: Click reaction. Scheme of click reaction between RGD-azide derivative and alkyne-PAA. <a href="http://cloudflare.jove.com/files/ftp_upload/54445/54445fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/54445/54445fig4.jpg" /> 
Figure 4: Hydrogel synthesis. RGD functionalized hydrogel synthesis procedure. <a href="http://cloudflare.jove.com/files/ftp_upload/54445/54445fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="0" cellpadding="0" cellspacing="0" width="521">
	<tbody>
		<tr height="67">
			<td height="67" style="height:67px;width:183px;">Poly(acrylic acid) solution average Mw ~ 100,000, 35 wt % in H2O</td>
			<td style="width:108px;">Sigma Aldrich</td>
			<td style="width:87px;">523925</td>
			<td style="width:145px;">CAS 9003-01-4</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Poly(ethylene glycol) 2,000</td>
			<td>Sigma Aldrich</td>
			<td>84797</td>
			<td>CAS 25322-68-3</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Carbomeer 974P</td>
			<td>Fagron</td>
			<td>1387083</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agarose&#160;</td>
			<td>Invitrogen Corp.</td>
			<td style="width:87px;">16500-500</td>
			<td>UltraPure Agarose</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RGD peptide</td>
			<td style="width:108px;">abcam</td>
			<td>ab142698</td>
			<td style="width:145px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">4-azidobutanoic acid</td>
			<td style="width:108px;">Aurum Pharmatech</td>
			<td style="width:87px;">Z-2421</td>
			<td>&#160;CAS 54447-68-6</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Oxalyl chloride</td>
			<td style="width:108px;">Sigma Aldrich</td>
			<td>O8801</td>
			<td>CAS 79-37-8</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:183px;">Propargylamine hydrochloride 95%</td>
			<td>Sigma Aldrich</td>
			<td>P50919</td>
			<td>CAS 15430-52-1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:183px;">Copper(I) iodide</td>
			<td>Sigma Aldrich</td>
			<td>3140</td>
			<td>CAS 7681-65-4</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium ascorbate</td>
			<td>Sigma Aldrich</td>
			<td>Y0000039</td>
			<td>CAS 134-03-2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate buffered saline</td>
			<td>Sigma Aldrich</td>
			<td>P4417</td>
			<td></td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;">Dialysis Membrane</td>
			<td style="width:108px;">Spectrum Laboratories, Inc.</td>
			<td>132725</td>
			<td style="width:145px;">Spectra/Por 3 Dialysis Membrane&#160; Standard RC Tubing&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; MWCO: 3,5 kD</td>
		</tr>
	</tbody>
</table>

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.

The PAA alkyne derivative is efficiently synthesized from polyacrylic acid and propargylamine, as showed in Figure 1 where n labels the monomers whose carboxyl groups react with the amine. The identity of the product is confirmed by 1H-NMR spectroscopy. Figure 5 shows the 1H-NMR spectrum of PAA modified with triple bond.
<img alt="Figure 5" src="/files/ftp_upload/54445/54445fig5.jpg" /> 
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Watch this Scientific Journal Video about The Synthesis of RGD-functionalized Hydrogels as a Tool for Therapeutic Applications at JoVE.com

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

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 ...

The overall goal of this methodology is to synthesize hydrogels for tissue engineering applications very easily. This method can help answer key questions in the tissue engineering field, such as the possibility to maintain high cell viability in three dimensional cell culture systems. The main advantage of this technique is that is using quick reaction, we can easily modify hydrogel systems to improve their ability to maintain cell viable.The implications of this technique extend toward the therapy of spinal cord injury because the cells loaded within the scaffold can rebuild the damaged tissue. For this method can provide significant insight into cell therapies as regenerative medicine and their implication in many methodologies. It can also be applied in several other systems like bone, heart, and cartilage.To synthesize the RGD-azide derivative, dissolve 50 milligrams of RGD in one milliliter of one molar sodium hydroxide. Then dissolve 24 milligrams of 4-azidobutanoyl chloride in two milliliters of tetrahydrofuran. Add all of the RGD solution into the 4-azidobutanoyl chloride solution at zero degrees Celsius dropwise.Return to room temperature and stir overnight. Add one milliliter of one molar HCL. Remove the solvent under reduced pressure using a rotary evaporator.Characterize the obtained product by proton nuclear magnetic resonance spectroscopy, dissolving the sample in deuterium oxide or D2O. To perform the PAA alkine modification, dissolve 200 milligrams of 35%in weight PAA solution in 15 milliliters of distilled water. Add 15.4 milligrams of propargylamine hydrochloride.Then dissolve 42.8 milligrams of HOBt in 14 milliliters of a one to one ratio of acetonytryle distilled water solution by heating to 50 degrees Celsius. Add all of the HOBt solution to the PAA solution at room temperature. Next, add 53.6 milligrams of EDC to the reaction mixture.Use one molar HCL to adjust the PH to 5.5 and stir the reaction system overnight at room temperature. Next, dissolve 11.2 grams of sodium chloride in two liters of distilled water. And then add 200 microliters of 37%weight by weight HCL.Dialyze the solution using a membrane with a molecular weight cutoff of 3.5 kilodaltons. Perform dialysis for three days. Change the dialysis solution daily with two liters of freshly prepared distilled water containing 200 microliters of 37%weight by weight HCL.Store the final solution at minus 80 degrees Celsius. Characterize the functionalized polymer by proton nuclear magnetic resonance spectroscopy, dissolving the sample in D2O. To synthesize the PAA-RGD polymer, dissolve 78 milligrams of PAA modified alkine in 10 milliliters of distilled water.Then dissolve 25 milligrams of the RGD-azide derivative in five milliliters of tetrahydrafuran. Add all of the RGD solution to the polymeric solution. Next, add 2.2 milligrams of copper iodide.And 2.2 milligrams of sodium ascorbate. Reflux the resulting mixture overnight at 60 degrees Celsius with stirring. Cool the mixture to 25 degrees Celsius.To dialyze the solution, dissolve 11.2 grams of sodium chloride in two liters of distilled water and add 200 microliters of 37 percent weight by weight HCL. Perform dialysis for three days. Change the dialysis solution daily with two liters of freshly prepared distilled water containing 200 microliters of 37%weight by weight HCL.Then place the final solution at minus 80 degrees Celsius. Characterize and store the resulting PAA-RGD polymer as done for its constituents. Blend 40 milligrams of carbomer and 10 milligrams of the functionalized PAA in nine milliters of phosphate-buffered saline at room temperature until complete dissolution.Add 400 milligrams of PEG to the solution and keep stirring for 45 minutes. Stop the stirring and allow the system to settle for 30 minutes. Then use one molar sodium hydroxide to adjust the PH to 7.4.To five milliliters of the obtained mixture, add 25 milligrams of agarose powder. Irradiate the system with microwave radiation at 500 watts for 30 seconds to one minute until boiling. And electromagnetically heat up the solution to 80 degrees Celsius.Leave the mixture exposed to room temperature until its temperature decreases to 50 degrees Celsius. Then add five milliliters of PBS to obtain a solution at a one to one volumetric ratio. Prepare a 12-multiwell plate containing steel cylinders with a diameter of 1.1 centimeters.Take 500 microliters aliquots from the solution and place them into each steel cylinder. Leave the plate at rest for 45 minutes until complete gelification of the system. Remove the cylinders using stainless steel forceps to obtain the hydrogels.See the text protocol for characterizing the physicochemical properties of the RGD-functionalized hydrogels. Carbomer 974P, PAA-RGD, and PEG, together with agarose, take part in a statistical random polycondensation after microwave irradiation. Hydroxyl groups contained in PEG and agarose react with carboxyl groups present in Carbomer 947P and PAA-RGD to form ACPEG-RGD hydrogels.Considering rheological properties, the storage modulus was found to be approximately one order of magnitude higher than the loss modulus, indicating an elastic rather than viscous material. Moreover, it is clearly visible that hydrogel behavior is dominated at low strain values by the elastic modulus. Scanning electron microscopy shows the morphology of an RGD functionalized hydrogel sample and a hydrogel without functionalization.Both hydrogels revealed that they possess a highly entangled structure of interconnected pores, with some bigger pores containing small pores and some fibular networks on the pore walls. The similar properties indicate that the presence of RGD does not alter the polymer network. After this development, this technique paved the way for research in the field of tissue engineering to explore the stability of these cell culture systems in several other diseases.After watching this video, you should have a good understanding of how to functionalize hydrogels. Improving their ability to maintain viable stem cells.

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Research

JoVE Journal

Bioengineering

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 ...

Solid Lipid Nanoparticles (SLNs) for Intracellular Targeting Applications

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter cellular signaling and gene expression. In a previous investigation, the synthesis of ultra-small solid lipid nanoparticles (SLNs) for topical drug delivery and biomarker detection applications was demonstrated. SLNs are a well-studied example of a nanoparticle delivery system that has emerged as a promising drug delivery vehicle. In this study, SLNs were loaded with a fluorescent dye and used as a model to investigate particle-cell interactions. The phase inversion temperature (PIT) method was used for the synthesis of ultra-small populations of biocompatible nanoparticles. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylphenyltetrazolium bromide (MTT) assay was utilized in order to establish appropriate dosing levels prior to the nanoparticle-cell interaction studies. Furthermore, primary human dermal fibroblasts and mouse dendritic cells were exposed to dye-loaded SLN over time and the interactions with respect to toxicity and particle uptake were characterized using fluorescence microscopy and flow cytometry. This study demonstrated that ultra-small SLNs, as a nanoparticle delivery system, are suitable for intracellular targeting of different cell types.

Solid Lipid Nanoparticles SLNs for Intracellular Targeting Applications

In this study, a method for synthesizing ultra-small populations of biocompatible nanoparticles was described, as well as several in vitro methods by which to assess their cellular interactions.

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter ...

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 ...

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 ...

DNA Nanotubes as a Versatile Tool to Study Semiflexible Polymers

Mechanical properties of complex, polymer-based soft matter, such as cells or biopolymer networks, can be understood in neither the classical frame of flexible polymers nor of rigid rods. Underlying filaments remain outstretched due to their non-vanishing backbone stiffness, which is quantified via the persistence length (lp), but they are also subject to strong thermal fluctuations. Their finite bending stiffness leads to unique, non-trivial collective mechanics of bulk networks, enabling the formation of stable scaffolds at low volume fractions while providing large mesh sizes. This underlying principle is prevalent in nature (e.g., in cells or tissues), minimizing the high molecular content and thereby facilitating diffusive or active transport. Due to their biological implications and potential technological applications in biocompatible hydrogels, semiflexible polymers have been subject to considerable study. However, comprehensible investigations remained challenging since they relied on natural polymers, such as actin filaments, which are not freely tunable. Despite these limitations and due to the lack of synthetic, mechanically tunable, and semiflexible polymers, actin filaments were established as the common model system. A major limitation is that the central quantity lp cannot be freely tuned to study its impact on macroscopic bulk structures. This limitation was resolved by employing structurally programmable DNA nanotubes, enabling controlled alteration of the filament stiffness. They are formed through tile-based designs, where a discrete set of partially complementary strands hybridize in a ring structure with a discrete circumference. These rings feature sticky ends, enabling the effective polymerization into filaments several microns in length, and display similar polymerization kinetics as natural biopolymers. Due to their programmable mechanics, these tubes are versatile, novel tools to study the impact of lp on the single-molecule as well as the bulk scale. In contrast to actin filaments, they remain stable over weeks, without notable degeneration, and their handling is comparably straightforward.

Semiflexible polymers display unique mechanical properties that are extensively applied by living systems. However, systematic studies on biopolymers are limited since properties such as polymer rigidity are inaccessible. This manuscript describes how this limitation is circumvented by programmable DNA nanotubes, enabling experimental studies on the impact of filament rigidity.

Mechanical properties of complex, polymer-based soft matter, such as cells or biopolymer networks, can be understood in neither the classical frame of ...

Ultrathin Porated Elastic Hydrogels As a Biomimetic Basement Membrane for Dual Cell Culture

The basement membrane is a critical component of cellular bilayers that can vary in stiffness, composition, architecture, and porosity. In vitro studies of endothelial-epithelial bilayers have traditionally relied on permeable support models that enable bilayer culture, but permeable supports are limited in their ability to replicate the diversity of human basement membranes. In contrast, hydrogel models that require chemical synthesis are highly tunable and allow for modifications of both the material stiffness and the biochemical composition via incorporation of biomimetic peptides or proteins. However, traditional hydrogel models are limited in functionality because they lack pores for cell-cell contacts and functional in vitro migration studies. Additionally, due to the thickness of traditional hydrogels, incorporation of pores that span the entire thickness of hydrogels has been challenging. In the present study, we use poly-(ethylene-glycol) (PEG) hydrogels and a novel zinc oxide templating method to address the previous shortcomings of biomimetic hydrogels. As a result, we present an ultrathin, basement membrane-like hydrogel that permits the culture of confluent cellular bilayers on a customizable scaffold with variable pore architectures, mechanical properties, and biochemical composition.

Current bilayer culture models do not allow for functional in vitro studies that mimic in vivo microenvironments. Using polyethylene glycol and a zinc oxide templating method, this protocol describes the development of an ultrathin biomimetic basement membrane with tunable stiffness, porosity, and biochemical composition that closely mimics in vivo extracellular matrices.

The basement membrane is a critical component of cellular bilayers that can vary in stiffness, composition, architecture, and porosity. In vitro studies ...

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 ...

Synthesis of Near-Infrared Emitting Gold Nanoclusters for Biological Applications

Over the past decade, fluorescent gold nanoclusters (AuNCs) have witnessed growing popularity in biological applications and enormous efforts have been devoted to their development. In this protocol, a recently developed, facile method for preparation of water soluble, biocompatible, and colloidally stable near-infrared emitting AuNCs have been described in detail. This room-temperature, bottom-up chemical synthesis provides easily functionalizable AuNCs capped with thioctic acid and thiol-modified polyethylene glycol in aqueous solution. The synthetic approach requires neither organic solvents or additional ligand exchange nor extensive knowledge of synthetic chemistry to reproduce. The resulting AuNCs offer free surface carboxylic acids, which can be functionalized with various biological molecules bearing a free amine group without adversely affecting the photoluminescent properties of the AuNCs. A quick, reliable procedure for flow cytometric quantification and confocal microscopic imaging of AuNC uptake by HeLa cells also been described. Due to the large Stokes shift, proper setting of filters in flow cytometry and confocal microscopy is necessary for efficient detection of near-infrared photoluminescence of AuNCs.

A reliable and easily reproducible method for preparation of functionalizable, near-infrared emitting photoluminescent gold nanoclusters and their direct detection inside HeLa cells by flow cytometry and confocal laser scanning microscopy is described.

Over the past decade, fluorescent gold nanoclusters (AuNCs) have witnessed growing popularity in biological applications and enormous efforts have been ...

Simultaneous Quantification of Selected Kynurenines Analyzed by Liquid Chromatography-Mass Spectrometry in Medium Collected from Cancer Cell Cultures

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and cancer biology. An in vitro cell culture assay is often used to learn about the contribution of different tryptophan catabolites in a disease mechanism and for testing therapeutic strategies. Cell culture medium that is rich in secreted metabolites and signaling molecules reflects the status of tryptophan metabolism and other cellular events. New protocols for the reliable quantification of multiple kynurenines in the complex cell culture medium are desired to allow for a reliable and quick analysis of multiple samples. This can be accomplished with liquid chromatography coupled with mass spectrometry. This powerful technique is employed in many clinical and research laboratories for the quantification of metabolites and can be used for measuring kynurenines.
Presented here is the use of liquid chromatography coupled with single quadrupole mass spectrometry (LC-SQ) for the simultaneous determination of four kynurenines, i.e., kynurenine, 3-hydroxykynurenine, 3-hydroxyanthranilic, and xanthurenic acid in the medium collected from in vitro cultured cancer cells. SQ detector is simple to use and less expensive compared to other mass spectrometers. In the SQ-MS analysis, multiple ions from the sample are generated and separated according to their specific mass-to-charge ratio (m/z), followed by the detection using a Single Ion Monitoring (SIM) mode.
This paper draws the attention on the advantages of the reported method and indicates some weak points. It is focused on critical elements of LC-SQ analysis including sample preparation along with chromatography and mass spectrometry analysis. The quality control, method calibration conditions and matrix effect issues are also discussed. We described a simple application of 3-nitrotyrosine as one analog standard for all target analytes. As confirmed by experiments with human ovary and breast cancer cells, the proposed LC-SQ method&#160;generates reliable results and can be further applied to other in vitro cellular models.

Described here is a protocol for the determination of four different tryptophan metabolites generated in the kynurenine pathway (kynurenine, 3-hydroxykynurenine, xanthurenic acid, 3-hydroxyanthranilic acid) in the medium collected from cancer cell cultures analyzed by liquid chromatography coupled with a single quadrupole mass spectrometry.

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and ...