This research was funded by Instituto de Salud Carlos III, Ministerio de Econom&#237;a y Competitividad, co-funded by the ESF European Social Fund and the ERDF European Regional Development Fund (MS16/00124; CP16/00124; PI17/01605), the Direcci&#243; General d&#39;Investigaci&#243;, Conselleria d&#39;Investigaci&#243;, Govern Balear (FPI/2046/2017), and PROGRAMA JUNIOR del projecte TALENT PLUS, construyendo SALUD, generando VALOR (JUNIOR01/18), financed by the sustainable tourism tax of the Balearic Islands.

Platelet Lysate (PL) is obtained as previously described in compliance with institutional guidelines3 using fresh buffy coats provided by the IdISBa Biobank as starting material. Their use for the current project was approved by its Ethics Committee (IB 1995/12 BIO).
1. EVs isolation from PL
<ol>
	<li>Larger bodies removal
	<ol>
		<li>Thaw PL at room temperature.</li>
		<li>Centrifuge PL at 1,500 x g for 15 min at 4 &#176;C. Discard the pellet as it contains cell debris.</li>
		<li>Collect the supernatant and perform two consecutive centrifugations at 10,000 x g for 30 min at 4 &#176;C. 
		NOTE: The pellet corresponds to larger EVs such as microvesicles, and in this case, it is discarded.</li>
		<li>Filter the supernatant first through 0.8 &#181;m porous membrane, and then through 0.2 &#181;m porous membrane. 
		NOTE: These steps remove all non-desired EVs.</li>
		<li>Pool the filtered PL and store at -20 &#176;C until use.</li>
	</ol>
	</li>
	<li>Size exclusion chromatography
	<ol>
		<li>Equilibrate the column coupled to chromatography equipment at the desired flow rate with filtered PBS. 
		NOTE: The flow rate used depends on the column characteristics; in this case, it is set to 0.5 mL/min.</li>
		<li>Load the processed PL (5 mL) with a syringe to the equipment.</li>
		<li>Inject the PL into the column and start collecting 5 mL fractions in 15 mL tubes.</li>
		<li>Collect the EVs enriched fractions and store them at -80 &#176;C until use. 
		&#8203;NOTE: When performing the experiment for the first time, characterize all fractions by protein quantification and immunodetection to determine the one enriched with EVs3,11. In this experiment, the 9th fraction is collected.</li>
		<li>Wash the chromatographic column with 30 mL of 0.2% NaOH solution and store it in 20% ethanol solution once it reaches equilibrium.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62781/62781fig01.jpg" /> 
Figure 1: Schematic diagram of Platelet Lysate (PL) extracellular vesicle (EVs) isolation. PL is centrifuged first at 1,500 x g, and then at 10,000 x g to remove larger bodies. The supernatant is filtered through 0.8 and 0.2 &#181;m filters. Processed PL is loaded onto the column, and EVs are separated by size exclusion chromatography. <a href="https://www.jove.com/files/ftp_upload/62781/62781fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
2. EVs characterization
NOTE: EVs characterization is necessary to perform functional studies12. Electron microscopy or western blot characterization have previously been reported13. This report will focus on the essential characterization techniques for Ti surface functionalization.
<ol>
	<li>Nanoparticle Tracking Analysis (NTA)
	<ol>
		<li>Dilute the EVs (1:1000) in 0.2 &#181;m filtered PBS. 
		NOTE: Too concentrated samples or too diluted samples will be out of range for NTA determination, and adjustment will be required.</li>
		<li>Load 1 mL of the diluted EVs with a syringe to the NTA equipment and inject them into the NTA equipment.</li>
		<li>Follow the manufacturer&#39;s protocol for particle concentration and size distribution determination.</li>
	</ol>
	</li>
	<li>Protein concentration
	<ol>
		<li>Determine the concentration using 1 &#181;L of the EVs solution. Measure the absorbance with a spectrophotometer at a wavelength of 280 nm. 
		NOTE: EVs should present low levels of proteins compared to the number of particles.</li>
		<li>Follow the manufacturer&#39;s instructions to obtain the absorbance reading using the spectrophotometer.</li>
	</ol>
	</li>
</ol>
3. Titanium surface functionalization
NOTE: In this method, machined titanium discs, c.p. grade IV, 6.2 mm diameter, and 2 mm height, are used. The discs may be manipulated with Ti tweezers, but it is important not to scratch the surface. Moreover, the machined side must face upwards during the entire process.
<ol>
	<li>Ti discs wash 
	NOTE: The volume of solutions used for Ti washing should be enough to cover Ti discs. Place Ti discs in a glass beaker and pour solutions onto them. Then, remove the solution by decanting.
	<ol>
		<li>Wash Ti implants with deionized (DI) water, and then discard the water.</li>
		<li>Wash Ti implants with ethanol 70%, and then decant to remove the solution.</li>
		<li>Place the implants in DI water and sonicate at 50 &#176;C for 5 min. Discard the water.</li>
		<li>Incubate Ti implants in a 40% NaOH solution at 50 &#176;C for 10 min with agitation. Discard the solution. 
		CAUTION: NaOH solution warms during preparation. The solution is corrosive and should be used inside a fume hood.</li>
		<li>Sonicate the implants in DI water at 50 &#176;C for 5 min, and then remove the water.</li>
		<li>Perform several washes with DI water (at least 5) until it reaches to neutral pH. Check pH with pH indicators.</li>
		<li>Sonicate the implants in DI water at 50 &#176;C for 5 min and remove the water.</li>
		<li>Incubate Ti implants in a 50% HNO3 solution at 50 &#176;C for 10 min with agitation. Remove the solution. 
		CAUTION: HNO3 is a corrosive and oxidizer substance, and it should be used inside a fume hood.</li>
		<li>Sonicate the implants in DI water at 50 &#176;C for 5 min. Remove the water.</li>
		<li>Perform several washes with DI water (at least 5) until neutral pH is obtained. Check the pH with pH indicators.</li>
		<li>Sonicate the implants in DI water at 50 &#176;C for 5 min. Remove the water. 
		NOTE: At this point, the experiment can be stopped by storing Ti implants in a 70% ethanol solution.</li>
	</ol>
	</li>
	<li>Ti passivation 
	NOTE: Ti passivation steps are performed by completely covering Ti discs with the different solutions in the order listed below. Ti discs are placed in a glass beaker and solutions are gently poured on them. Volumes used in all wash steps must completely cover the implants and are removed via decanting.
	<ol>
		<li>Incubate the Ti implants in a 30% HNO3 solution&#160;for 30 min at room temperature under gentle agitation. Remove the solution.</li>
		<li>Perform several washes with DI water (at least 5) until it reaches to neutral pH. Check the pH with pH indicators.</li>
		<li>Incubate Ti implants overnight at room temperature in DI water.</li>
		<li>Dry off the implants under vacuum conditions at 40 &#176;C for 10 min.</li>
	</ol>
	</li>
	<li>EVs drop casting 
	NOTE: For cell functional studies, it is important to work in a cell culture cabinet.
	<ol>
		<li>Place the Ti implants in a 96-well plate, with the machined side facing up. 
		NOTE: If the implants are turned upside-down, a needle can be used to set them back.</li>
		<li>Thaw the EVs solution and mix them with agitation. Use a vortex to pulse for 3 s.</li>
		<li>Deposit the EVs on the Ti surface. In this study, drops of 40 &#181;L of EVs solution are placed onto the Ti to immobilize a maximum of 4 x 1011 EVs per implant according to the concentration determined by NTA.</li>
		<li>Place the plates containing the Ti under vacuum conditions at 37 &#176;C until drops are completely dry (~2 h). 
		&#8203;NOTE: Adjust the time depending on the number of implants and the water present in the vacuum chamber.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62781/62781fig02.jpg" /> 
Figure 2: Schematic diagram of Ti passivation and EVs functionalization by drop casting. Ti implants are passivated first by incubation for 30 min in a 30% HNO3 solution at room temperature. After several washes with DI water, pH reaches neutral. Then, Ti implants are incubated overnight at room temperature in DI water. After that, the implants are dried off under vacuum conditions at 40 &#176;C. For EVs immobilization, 40 &#181;L of EVs solution are deposited onto Ti implants. Next, implants are incubated at vacuum for 2 h until EVs are physically bound to the surface. <a href="https://www.jove.com/files/ftp_upload/62781/62781fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
4. Ti surface characterization
<ol>
	<li>Release study
	<ol>
		<li>Incubate Ti surface with 200 &#181;L of filtered PBS at 37 &#176;C. 
		NOTE: PBS is filtered to avoid interferences with the NTA measurement.</li>
		<li>Replace the PBS at different time points and store at -80 &#176;C. 
		NOTE: In this study, 2-, 6-, 10-, and 14-days&#39; time points were analyzed.</li>
		<li>Analyze stored PBS for particle studies by NTA according to the manufacturer&#39;s instructions. 
		NOTE: Particle concentration in PBS at different times is a representation of EVs release profile over time.</li>
	</ol>
	</li>
	<li>Biocompatibility studies 
	NOTE: Human umbilical cord-derived mesenchymal stem cells (hUC-MSC) are obtained from the IdISBa Biobank in compliance with institutional guidelines.
	<ol>
		<li>Maintain hUC-MSC in DMEM low glucose supplemented with 20% FBS until use. Change the medium twice per week.</li>
		<li>For cell seeding, wash the cells in flasks with 5 mL of PBS twice.</li>
		<li>Trypsinize hUC-MSC by adding 1 mL of trypsin solution. Ensure that it completely covers the monolayer of cells. Remove the trypsin solution and place the cell culture flask at 37 &#176;C for 2 min approximately. View cell detachment under the microscope. Detached cells will appear round in shape and will be in suspension.</li>
		<li>Resuspend cells in DMEM low glucose with 1% EVs depleted FBS. 
		NOTE: Prepare media supplemented with 1% FBS, and then ultracentrifuge at 120,000 x g for 18 h to remove FBS-EVs. It is important to remove EVs to avoid interferences with platelet EVs.</li>
		<li>Determine cell concentration by counting the number of cells with a Neubauer chamber14.</li>
		<li>Bring hUC-MSC to a concentration of 50,000 cells/mL.</li>
		<li>Seed 200 &#181;L of the cell solution onto the Ti implants.</li>
		<li>After 48 h, collect 50 &#181;L of media and perform the cytotoxic determination using lactate dehydrogenase (LDH) activity kit, according to the manufacturer&#39;s protocol.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

This protocol aims to provide clear instructions for EVs functionalization onto Ti surfaces. The method presented is based on a drop casting strategy, which is a physisorption type of functionalization. Poor bibliography exists regarding EVs functionalization on Ti surfaces, although there are few studies showing different advantages by immobilizing EVs on Ti10. Anyway, some of the strategies explored include biochemical binding8, polymeric entrapment9</su...

EVs are membrane vesicles (30-200 nm) secreted by any cell and play a key role in cell-to-cell communication by delivering their cargo. They contain a variety of active biomolecules that may include nucleic acids, growth factors, or bioactive lipids1. For these reasons, EVs have been evaluated for their potential use in therapeutics. In terms of orthopedics and bone regeneration, EVs from different sources have been tested. Among them, platelet-derived EVs have been shown to induce a differentiation effect on stem cells while maintaining a low cytotoxic profile2,3. Therefore, further research is required to explore the possibility of combining EVs with biomaterials in order to use them in daily clinical practice.
Titanium-based biomaterials are widely used as scaffolds for bone healing clinical interventions due to their mechanical properties, high biocompatibility, and long-term durability4. Nevertheless, Ti implants are a bioinert material and, therefore, present a poor capability for bonding with the surrounding bone tissue5. For this reason, titanium modifications are being studied in order to improve their performance by achieving a more functional microenvironment on its surface4,6,7. In this sense, EVs can be anchored to titanium by chemical8 or physical interactions9,10. Immobilized EVs derived from stem cells or macrophages enhance the bioactivity of Ti by promoting cellular adhesion and proliferation thereby inducing an osteogenic effect8,9,10.
This article will focus on a drop casting strategy for coating Ti surfaces with PL-derived EVs in detail. In addition, we will evaluate EVs release profile from the coated surface over time and confirm its cellular biocompatibility in vitro.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0,8 &#181;m syringe filter</td>
			<td>Sartorius</td>
			<td>16592K</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL Centrifuge tube</td>
			<td>SPL life sciences</td>
			<td>PLC60015</td>
			<td></td>
		</tr>
		<tr>
			<td>1mL syringe</td>
			<td>BD</td>
			<td>303174</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well culture plate</td>
			<td>SPL life sciences</td>
			<td>PLC30096</td>
			<td></td>
		</tr>
		<tr>
			<td>Absolut ethanol</td>
			<td>Scharlau</td>
			<td>ET0006005P</td>
			<td>Used to prepare 20 %&#160; ethanol with Milli-Q&#174; water</td>
		</tr>
		<tr>
			<td>AKTA purifier System</td>
			<td>GE Healthcare</td>
			<td>8149-30-0014</td>
			<td></td>
		</tr>
		<tr>
			<td>Allegra X-15R Centrifuge</td>
			<td>Beckman Coutler</td>
			<td>392934</td>
			<td>SX4750A swinging rotor</td>
		</tr>
		<tr>
			<td>Centrifuge 5430 R</td>
			<td>Eppendorf</td>
			<td>5428000210</td>
			<td>F-45-48-11 rotor</td>
		</tr>
		<tr>
			<td>Conical Tube, Conical Bottom, 50ml</td>
			<td>SPL life sciences</td>
			<td>PLC50050</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytotoxicity Detection Kit (LDH)</td>
			<td>Roche</td>
			<td>11644793001</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Syringes 10 ml</td>
			<td>Becton Dickinson</td>
			<td>BDH307736</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM Low Glucose Glutamax</td>
			<td>GIBCO</td>
			<td>21885025</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s PBS (1x)</td>
			<td>Capricorn Scientific</td>
			<td>PBS-1A</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS) Embrionic Certified</td>
			<td>GIBCO</td>
			<td>16000044</td>
			<td></td>
		</tr>
		<tr>
			<td>Filtropur S 0.2 &#181;m syringe filter</td>
			<td>Sarstedt</td>
			<td>83.1826.001</td>
			<td></td>
		</tr>
		<tr>
			<td>HiPrep&#160;16/60 Sephacryl&#160;S-400 HR</td>
			<td>GE Healthcare</td>
			<td>28-9356-04</td>
			<td>Precast columns</td>
		</tr>
		<tr>
			<td>human umbilical cord-derived mesenchymal stem cells (hUC-MSC)</td>
			<td>IdISBa Biobank</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nanodrop 2000 spectrophotometer</td>
			<td>ThermoFisher</td>
			<td>ND-2000</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoSight NS300 nanoparticle tracking analysis</td>
			<td>Malvern</td>
			<td>NS300</td>
			<td>Device with embedded laser at &#955;= 532 nm and camera sCMOS</td>
		</tr>
		<tr>
			<td>Needle</td>
			<td>Terumo</td>
			<td>946077135</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitric acid 69,5%</td>
			<td>Scharlau</td>
			<td>AC16071000</td>
			<td></td>
		</tr>
		<tr>
			<td>Optima L-100 XP Ultracentrifuge</td>
			<td>Beckman Coulter</td>
			<td>8043-30-1124</td>
			<td>SW-32Ti Rotor</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin Solution 100X</td>
			<td>Biowest</td>
			<td>L0022</td>
			<td></td>
		</tr>
		<tr>
			<td>pH Test strips 4.5-10.0</td>
			<td>Sigma</td>
			<td>P-4536</td>
			<td></td>
		</tr>
		<tr>
			<td>Platelet Lysate (PL)</td>
			<td>IdISBa Biobank</td>
			<td></td>
			<td>Obtained from&#160; buffy coats discarded after blood donation</td>
		</tr>
		<tr>
			<td>Polypropylene centrifuge tubs</td>
			<td>Beckman Coutler</td>
			<td>326823</td>
			<td></td>
		</tr>
		<tr>
			<td>Power wave HT</td>
			<td>BioTek</td>
			<td>10340763</td>
			<td></td>
		</tr>
		<tr>
			<td>Screw cap tube, 15 ml, (Lx&#216;): 120 x 17 mm, PP, with print</td>
			<td>Sarstedt</td>
			<td>62554502</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hidroxide</td>
			<td>Sharlau</td>
			<td>SO04251000</td>
			<td></td>
		</tr>
		<tr>
			<td>Titanium implants replicas</td>
			<td>Implantmedia, SA</td>
			<td>NA</td>
			<td>Titanium grade IV. Diameter: 6,2 mm. Height: 1,95 mm</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA 1 X</td>
			<td>Biowest</td>
			<td>L0930</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryton X100</td>
			<td>Sigma</td>
			<td>T8787</td>
			<td></td>
		</tr>
	</tbody>
</table>

platelet-derived extracellular vesicle functionalization of ti implants

Extracellular Vesicles (EVs) are biological nanovesicles that play a key role in cell communication. Their content includes active biomolecules such as proteins and nucleic acids, which present great potential in regenerative medicine. More recently, EVs derived from Platelet Lysate (PL) have shown an osteogenic capability comparable to PL. Besides, biomaterials are frequently used in orthopedics or dental restoration. Here, we provide a method to functionalize Ti surfaces with PL-derived EVs in order to improve their osteogenic properties.
EVs are isolated from PL by size exclusion chromatography, and afterward Ti surfaces are functionalized with PL-EVs by drop casting. Functionalization is proven by EVs release and its biocompatibility by the lactate dehydrogenase (LDH) release assay.

The method presented in this article allows obtaining EVs functionalized titanium discs. EVs are physically bonded to the surface, which allows a sustained release over time. The amount of EVs released can be measured by NTA on Day 2, 6, 10, and 14. The first measurements, on Day 2, show that around 109 EVs are released, followed by a sustained release on day 6 (~108 EVs); day 10 (~107 EVs), and day 14 (~107 EVs). This confirms a sustained release, despite showing a decrease in...

Watch this Scientific Journal Video about Platelet-Derived Extracellular Vesicle Functionalization of Ti Implants at JoVE.com

Platelet-Derived Extracellular Vesicle Functionalization of Ti Implants

Here, we present a method for the isolation of Extracellular Vesicles (EVs) derived from the platelet lysates (PL) and their use for coating titanium (Ti) implant surfaces. We describe the drop casting coating method, the EVs release profile from the surfaces, and in vitro biocompatibility of EVs coated Ti surfaces.

Extracellular Vesicles (EVs) are biological nanovesicles that play a key role in cell communication. Their content includes active biomolecules such as ...

platelet-derived-extracellular-vesicle-functionalization-ti

Research

JoVE Journal

Bioengineering

2.2K Views.  University of the Balearic Islands. Here, we present a method for the isolation of Extracellular Vesicles (EVs) derived from the platelet lysates (PL) and their use for coating titanium (Ti) implant surfaces. We describe the drop casting coating method, the EVs release profile from the surfaces, and in vitro biocompatibility of EVs coated Ti surfaces.

Video: Platelet-Derived Extracellular Vesicle Functionalization of Ti Implants

Soft Lithographic Functionalization and Patterning Oxide-free Silicon and Germanium

The development of hybrid electronic devices relies in large part on the integration of (bio)organic materials and inorganic semiconductors through a stable interface that permits efficient electron transport and protects underlying substrates from oxidative degradation. Group IV semiconductors can be effectively protected with highly-ordered self-assembled monolayers (SAMs) composed of simple alkyl chains that act as impervious barriers to both organic and aqueous solutions. Simple alkyl SAMs, however, are inert and not amenable to traditional patterning techniques. The motivation for immobilizing organic molecular systems on semiconductors is to impart new functionality to the surface that can provide optical, electronic, and mechanical function, as well as chemical and biological activity. 
Microcontact printing (&mu;CP) is a soft-lithographic technique for patterning SAMs on myriad surfaces.1-9 Despite its simplicity and versatility, the approach has been largely limited to noble metal surfaces and has not been well developed for pattern transfer to technologically important substrates such as oxide-free silicon and germanium. Furthermore, because this technique relies on the ink diffusion to transfer pattern from the elastomer to substrate, the resolution of such traditional printing is essentially limited to near 1 &mu;m.10-16
In contrast to traditional printing, inkless &mu;CP patterning relies on a specific reaction between a surface-immobilized substrate and a stamp-bound catalyst. Because the technique does not rely on diffusive SAM formation, it significantly expands the diversity of patternable surfaces. In addition, the inkless technique obviates the feature size limitations imposed by molecular diffusion, facilitating replication of very small (&lt;200 nm) features.17-23 However, up till now, inkless &mu;CP has been mainly used for patterning relatively disordered molecular systems, which do not protect underlying surfaces from degradation.
Here, we report a simple, reliable high-throughput method for patterning passivated silicon and germanium with reactive organic monolayers and demonstrate selective functionalization of the patterned substrates with both small molecules and proteins. The technique utilizes a preformed NHS-reactive bilayered system on oxide-free silicon and germanium. The NHS moiety is hydrolyzed in a pattern-specific manner with a sulfonic acid-modified acrylate stamp to produce chemically distinct patterns of NHS-activated and free carboxylic acids. A significant limitation to the resolution of many &mu;CP techniques is the use of PDMS material which lacks the mechanical rigidity necessary for high fidelity transfer. To alleviate this limitation we utilized a polyurethane acrylate polymer, a relatively rigid material that can be easily functionalized with different organic moieties. Our patterning approach completely protects both silicon and germanium from chemical oxidation, provides precise control over the shape and size of the patterned features, and gives ready access to chemically discriminated patterns that can be further functionalized with both organic and biological molecules. The approach is general and applicable to other technologically-relevant surfaces.

Here we describe a simple method for patterning oxide-free silicon and germanium with reactive organic monolayers and demonstrate functionalization of the patterned substrates with small molecules and proteins. The approach completely protects surfaces from chemical oxidation, provides precise control over feature morphology, and provides ready access to chemically discriminated patterns.

The development of hybrid electronic devices relies in large part on the integration of (bio)organic materials and inorganic semiconductors through a ...

High-throughput Synthesis of Carbohydrates and Functionalization of Polyanhydride Nanoparticles

Transdisciplinary approaches involving areas such as material design, nanotechnology, chemistry, and immunology have to be utilized to rationally design efficacious vaccines carriers. Nanoparticle-based platforms can prolong the persistence of vaccine antigens, which could improve vaccine immunogenicity1. Several biodegradable polymers have been studied as vaccine delivery vehicles1; in particular, polyanhydride particles have demonstrated the ability to provide sustained release of stable protein antigens and to activate antigen presenting cells and modulate immune responses2-12.
The molecular design of these vaccine carriers needs to integrate the rational selection of polymer properties as well as the incorporation of appropriate targeting agents. High throughput automated fabrication of targeting ligands and functionalized particles is a powerful tool that will enhance the ability to study a wide range of properties and will lead to the design of reproducible vaccine delivery devices.
The addition of targeting ligands capable of being recognized by specific receptors on immune cells has been shown to modulate and tailor immune responses10,11,13 C-type lectin receptors (CLRs) are pattern recognition receptors (PRRs) that recognize carbohydrates present on the surface of pathogens. The stimulation of immune cells via CLRs allows for enhanced internalization of antigen and subsequent presentation for further T cell activation14,15. Therefore, carbohydrate molecules play an important role in the study of immune responses; however, the use of these biomolecules often suffers from the lack of availability of structurally well-defined and pure carbohydrates. An automation platform based on iterative solution-phase reactions can enable rapid and controlled synthesis of these synthetically challenging molecules using significantly lower building block quantities than traditional solid-phase methods16,17.
Herein we report a protocol for the automated solution-phase synthesis of oligosaccharides such as mannose-based targeting ligands with fluorous solid-phase extraction for intermediate purification. After development of automated methods to make the carbohydrate-based targeting agent, we describe methods for their attachment on the surface of polyanhydride nanoparticles employing an automated robotic set up operated by LabVIEW as previously described10. Surface functionalization with carbohydrates has shown efficacy in targeting CLRs10,11 and increasing the throughput of the fabrication method to unearth the complexities associated with a multi-parametric system will be of great value (Figure 1a).

In this article, a high throughput method is presented for the synthesis of oligosaccharides and their attachment to the surface of polyanhydride nanoparticles for further use in targeting specific receptors on antigen presenting cells.

Transdisciplinary approaches involving areas such as material design, nanotechnology, chemistry, and immunology have to be utilized to rationally design ...

Graphene Coatings for Biomedical Implants

Atomically smooth graphene as a surface coating has potential to improve implant properties. This demonstrates a method for coating nitinol alloys with nanometer thick layers of graphene for applications as a stent material. Graphene was grown on copper substrates via chemical vapor deposition and then transferred onto nitinol substrates. In order to understand how the graphene coating could change biological response, cell viability of rat aortic endothelial cells and rat aortic smooth muscle cells was investigated. Moreover, the effect of graphene-coatings on cell adhesion and morphology was examined with fluorescent confocal microscopy. Cells were stained for actin and nuclei, and there were noticeable differences between pristine nitinol samples compared to graphene-coated samples. Total actin expression from rat aortic smooth muscle cells was found using western blot. Protein adsorption characteristics, an indicator for potential thrombogenicity, were determined for serum albumin and fibrinogen with gel electrophoresis. Moreover, the transfer of charge from fibrinogen to substrate was deduced using Raman spectroscopy. It was found that graphene coating on nitinol substrates met the functional requirements for a stent material and improved the biological response compared to uncoated nitinol. Thus, graphene-coated nitinol is a viable candidate for a stent material.

Graphene offers potential as a coating material for biomedical implants. In this study we demonstrate a method for coating nitinol alloys with nanometer thick layers of graphene and determine how graphene may influence implant response.

Atomically smooth graphene as a surface coating has potential to improve implant properties. This demonstrates a method for coating nitinol alloys with ...

Multi-Scale Modification of Metallic Implants With Pore Gradients, Polyelectrolytes and Their Indirect Monitoring In vivo

Metallic implants, especially titanium implants, are widely used in clinical applications. Tissue in-growth and integration to these implants in the tissues are important parameters for successful clinical outcomes. In order to improve tissue integration, porous metallic implants have being developed. Open porosity of metallic foams is very advantageous, since the pore areas can be functionalized without compromising the mechanical properties of the whole structure. Here we describe such modifications using porous titanium implants based on titanium microbeads. By using inherent physical properties such as hydrophobicity of titanium, it is possible to obtain hydrophobic pore gradients within microbead based metallic implants and at the same time to have a basement membrane mimic based on hydrophilic, natural polymers. 3D pore gradients are formed by synthetic polymers such as Poly-L-lactic acid (PLLA) by freeze-extraction method. 2D nanofibrillar surfaces are formed by using collagen/alginate followed by a crosslinking step with a natural crosslinker (genipin). This nanofibrillar film was built up by layer by layer (LbL) deposition method of the two oppositely charged molecules, collagen and alginate. Finally, an implant where different areas can accommodate different cell types, as this is necessary for many multicellular tissues, can be obtained. By, this way cellular movement in different directions by different cell types can be controlled. Such a system is described for the specific case of trachea regeneration, but it can be modified for other target organs. Analysis of cell migration and the possible methods for creating different pore gradients are elaborated. The next step in the analysis of such implants is their characterization after implantation. However, histological analysis of metallic implants is a long and cumbersome process, thus for monitoring host reaction to metallic implants in vivo an alternative method based on monitoring CGA and different blood proteins is also described. These methods can be used for developing in vitro custom-made migration and colonization tests and also be used for analysis of functionalized metallic implants in vivo without histology.

Multi-Scale Modification of Metallic Implants With Pore Gradients, Polyelectrolytes and Their Indirect Monitoring In vivo

In this video, we will demonstrate modification techniques for porous metallic implants to improve their functionality and to control cell migration. Techniques include development of pore gradients to control cell movement in 3D and production of basement membrane mimics to control cell movement in 2-D. Also, a HPLC-based method for monitoring implant integration in-vivo via analysis of blood proteins is described.

Metallic implants, especially titanium implants, are widely used in  clinical applications. Tissue in-growth and integration to these implants in  the ...

Lipid Bilayer Vesicle Generation Using Microfluidic Jetting

Bottom-up synthetic biology presents a novel approach for investigating and reconstituting biochemical systems and, potentially, minimal organisms. This emerging field engages engineers, chemists, biologists, and physicists to design and assemble basic biological components into complex, functioning systems from the bottom up. Such bottom-up systems could lead to the development of artificial cells for fundamental biological inquiries and innovative therapies1,2. Giant unilamellar vesicles (GUVs) can serve as a model platform for synthetic biology due to their cell-like membrane structure and size. Microfluidic jetting, or microjetting, is a technique that allows for the generation of GUVs with controlled size, membrane composition, transmembrane protein incorporation, and encapsulation3. The basic principle of this method is the use of multiple, high-frequency fluid pulses generated by a piezo-actuated inkjet device to deform a suspended lipid bilayer into a GUV. The process is akin to blowing soap bubbles from a soap film. By varying the composition of the jetted solution, the composition of the encompassing solution, and/or the components included in the bilayer, researchers can apply this technique to create customized vesicles. This paper describes the procedure to generate simple vesicles from a droplet interface bilayer by microjetting.

Microfluidic jetting against a droplet interface lipid bilayer provides a reliable way to generate vesicles with control over membrane asymmetry, incorporation of transmembrane proteins, and encapsulation of material. This technique can be applied to study a variety of biological systems where compartmentalized biomolecules are desired.

Bottom-up synthetic biology presents a novel approach for investigating and reconstituting biochemical systems and, potentially, minimal organisms. This ...

Surface Functionalization of Hepatitis E Virus Nanoparticles Using Chemical Conjugation Methods

Virus-like particles (VLPs) have been used as nanocarriers to display foreign epitopes and/or deliver small molecules in the detection and treatment of various diseases. This application relies on genetic modification, self-assembly, and cysteine conjugation to fulfill the tumor-targeting application of recombinant VLPs. Compared with genetic modification alone, chemical conjugation of foreign peptides to VLPs offers a significant advantage because it allows a variety of entities, such as synthetic peptides or oligosaccharides, to be conjugated to the surface of VLPs in a modulated and flexible manner without alteration of the VLP assembly.
Here, we demonstrate how to use the hepatitis E virus nanoparticle (HEVNP), a modularized theranostic capsule, as a multifunctional delivery carrier. Functions of HEVNPs include tissue-targeting, imaging, and therapeutic delivery. Based on the well-established structural research of HEVNP, the structurally independent and surface-exposed residues were selected for cysteine replacement as conjugation sites for maleimide-linked chemical groups via thiol-selective linkages. One particular cysteine-modified HEVNP (a Cys replacement of the asparagine at 573 aa (HEVNP-573C)) was conjugated to a breast cancer cell-specific ligand, LXY30 and labeled with near-infrared (NIR) fluorescence dye (Cy5.5), rendering the tumor-targeted HEVNPs as effective diagnostic capsules (LXY30-HEVNP-Cy5.5). Similar engineering strategies can be employed with other macromolecular complexes with well-known atomic structures to explore potential applications in theranostic delivery.

We have engineered the capsid protein of hepatitis E virus as a theranostic nanoparticle (HEVNP). HEVNP self-assembles into a stable icosahedral cage in mucosal delivery. Here, we describe the modification of HEVNPs for tumor targeting by mutating surface-exposed residues to cysteines, which conjugate synthetic ligands that specifically bind tumor cells.

Virus-like particles (VLPs) have been used as nanocarriers to display foreign epitopes and/or deliver small molecules in the detection and treatment of ...

Environmental Dynamic Mechanical Analysis to Predict the Softening Behavior of Neural Implants

When using dynamically softening substrates for neural implants, it is important to have a reliable in vitro method to characterize the softening behavior of these materials. In the past, it has not been possible to satisfactorily measure the softening of thin films under conditions mimicking body environment without substantial effort. This publication presents a new and simple method that allows dynamic mechanical analysis (DMA) of polymers in solutions, such as phosphate buffered saline (PBS), at relevant temperatures. The use of environmental DMA allows measurement of the softening effects of polymers due to plasticization in various media and temperatures, which therefore allows a prediction of the materials behavior under in vivo conditions.

To allow reliable predictions of the softening of polymeric substrates for neural implants in an in vivo environment, it is important to have a reliable in vitro method. Here, the use of dynamic mechanical analysis in phosphate buffered saline at body temperature is presented.

When using dynamically softening substrates for neural implants, it is important to have a reliable in vitro method to characterize the softening behavior ...

Evaluation of the Storage Stability of Extracellular Vesicles

Extracellular vesicles (EVs) are promising targets in current research, to be used as drugs, drug-carriers, and biomarkers. For their clinical development, not only their pharmaceutical activity is important but also their production needs to be evaluated. In this context, research focuses on the isolation of EVs, their characterization, and their storage. The present manuscript aims at providing a facile procedure for the assessment of the effect of different storage conditions on EVs, without genetic manipulation or specific functional assays. This makes it possible to quickly get a first impression of the stability of EVs under a given storage condition, and EVs derived from different cell sources can be compared easily. The stability measurement is based on the physicochemical parameters of the EVs (size, particle concentration, and morphology) and the preservation of the activity of their cargo. The latter is assessed by the saponin-mediated encapsulation of the enzyme beta-glucuronidase into the EVs. Glucuronidase acts as a surrogate and allows for an easy quantification via the cleavage of a fluorescent reporter molecule. The present protocol could be a tool for researchers in the search for storage conditions that optimally retain EV properties to advance EV research toward clinical application.

Here we present a readily applicable protocol to assess the storage stability of extracellular vesicles, a group of naturally occurring nanoparticles produced by cells. The vesicles are loaded with glucuronidase as a model enzyme and stored under different conditions. After storage, their physicochemical parameters and the activity of the encapsulated enzyme are evaluated.

Extracellular vesicles (EVs) are promising targets in current research, to be used as drugs, drug-carriers, and biomarkers. For their clinical ...

In Vesiculo Synthesis of Peptide Membrane Precursors for Autonomous Vesicle Growth

Compartmentalization of biochemical reactions is a central aspect of synthetic cells. For this purpose, peptide-based reaction compartments serve as an attractive alternative to liposomes or fatty acid-based vesicles. Externally or within the vesicles, peptides can be easily expressed and simplify the synthesis of membrane precursors. Provided here is a protocol for the creation of vesicles with diameters of ~200 nm based on the amphiphilic elastin-like polypeptides (ELP) utilizing dehydration-rehydration from glass beads. Also presented are protocols for bacterial ELP expression and purification via inverse temperature cycling, as well as their covalent functionalization with fluorescent dyes. Furthermore, this report describes a protocol to enable the transcription of RNA aptamer dBroccoli inside ELP vesicles as a less complex example for a biochemical reaction. Finally, a protocol is provided, which allows in vesiculo expression of fluorescent proteins and the membrane peptide, whereas synthesis of the latter results in vesicle growth.

Presented here are protocols for the creation of peptide-based small unilamellar vesicles capable of growth. To facilitate in vesiculo production of the membrane peptide, these vesicles are equipped with a transcription-translation system and the peptide-encoding plasmid.

Compartmentalization of biochemical reactions is a central aspect of synthetic cells. For this purpose, peptide-based reaction compartments serve as an ...

Mitigation of Blood Borne Cell Attachment to Metal Implants through CD47-Derived Peptide Immobilization

The key complications associated with bare metal stents and drug eluting stents are in-stent restenosis and late stent thrombosis, respectively. Thus, improving the biocompatibility of metal stents remains a significant challenge. The goal of this protocol is to describe a robust technique of metal surface modification by biologically active peptides to increase biocompatibility of blood contacting medical implants, including endovascular stents. CD47 is an immunological species-specific marker of self and has anti-inflammatory properties. Studies have shown that a 22 amino acid peptide corresponding to the Ig domain of CD47 in the extracellular region (pepCD47), has anti-inflammatory properties like the full-length protein. In vivo studies in rats, and ex vivo studies in rabbit and human blood experimental systems from our lab have demonstrated that pepCD47 immobilization on metals improves their biocompatibility by preventing inflammatory cell attachment and activation. This paper describes the step-by step protocol for the functionalization of metal surfaces and peptide attachment. The metal surfaces are modified using polyallylamine bisphosphate with latent thiol groups (PABT) followed by deprotection of thiols and amplification of thiol-reactive sites via reaction with polyethyleneimine installed with pyridyldithio groups (PEI-PDT). Finally, pepCD47, incorporating terminal cysteine residues connected to the core peptide sequence through a dual 8-amino-3,6-dioxa-octanoyl spacer, are attached to the metal surface via disulfide bonds. This methodology of peptide attachment to metal surface is efficient and relatively inexpensive and thus can be applied to improve biocompatibility of several metallic biomaterials.

Presented here is a protocol for appending peptide CD47 (pepCD47) to metal stents using polybisphosphonate chemistry. Functionalization of metal stents using pepCD47 prevents the attachment and activation of inflammatory cells thus improving their biocompatibility.

The key complications associated with bare metal stents and drug eluting stents are in-stent restenosis and late stent thrombosis, respectively. Thus, ...