無水物ナノ粒子のハイスループット糖鎖の合成と機能化

In this video article, we describe a novel hydro put protocol for the automated solution phase synthesis of oligosaccharides and the functionalization of poly anhy nanoparticles. With these carbohydrate based targeting agents, first carbohydrate molecules are synthesized using an automated system that utilizes solution phase synthesis instead of solid phase synthesizers. Next, the oligosaccharide intermediates are purified by florist, solid phase extraction or FSPE.

The produced carbohydrate molecules are characterized by NMR and then they're attached to the poly anhydride nanoparticles by carbo IDE conjugation. Finally, carbohydrate functionalized nanoparticles are characterized by x-ray photo electron spectroscopy and a hydro putt phenyl sulfuric acid assay. Ultimately, utilizing the hydro put methodology described here, the reaction conditions to optimize nanoparticle morphology and carbohydrate density on the particle surface have been obtained.

The main advantage of this method over the existing ones like solid face oligosaccharide synthesis, is that in this method we use substantially lower amount of building blocks In this method, typically we use two to three equivalents instead of 10 to 20 equivalents. We first have the idea for this method when we demonstrate the efficacy of surface effects, functionalization of poly hydro nanoparticles, using carbohydrates to target ceep receptors on anti denting cells. We then wanted to design a high throughput system that will enable us to screen the multiple parameters that are involved in the fabrication and application of these novel carriers.

Visual demonstration of this method is critical because the development of automated platforms for the high throughput synthesis of oligosaccharides and their attachment to polymer particles is a novel area of investigation without a lot of documentation available Prior to the automated synthesis of diano acid. A suitably protected sugar donor, typically tri chloro acetamide an accepter mainly in Alcon, fluorous alcohol are synthesized on the benchtop and prepared in chloro methane also prepare trimethyl silo, tri fluoro methane sulfonate in chloro methane, 80%methanol and 100%methanol. Ensure that the relative humidity in the room is 30%or lower in the automation chamber.

High humidity is detrimental for glycosylation reactions. The following steps are done using an automation platform. The first glycosylation is carried out with the donor and the florist alcohol.

Once done, the resulting florist tag sugar molecule is purified by FSPE. The temporary protecting group is then removed by sodium, meth oxide and methanol, and once again, the product is purified by FSPE. After that, the florist tag sugar is used as an acceptor and coupled to the same donor to get the disaccharide, the disaccharide is purified by FSPE to get started.

Place reagents, including the donor acceptor promoter, 80%methanol water, 100%methanol, sodium meth oxide in the robotic platform, and initiate the program. The robotic arm will withdraw the donor and then the acceptor from the vials and transfer them into a reaction vial. Then the mixture of donor and acceptor are stirred for 30 minutes.

After 30 minutes have passed. The robotic arm transfers catalytic trimethyl silo, tri fluoro, methane sulfonate into the mixture. The solution is then stirred for an additional 30 minutes After stirring is complete, the program pauses.

Remove an aliquot of 10 microliters to check to see if the reaction is complete by thin layer chromatography. If the reaction is complete, the acceptor molecule will not be seen. If the reaction is not complete, the acceptor on the TLC is still visible.

If this is the case, stop the program, reset the timing for glycosylation around 30 minutes, and add an excess of the promoter trimethyl silo, tri fluoro, methane sulfonate. When the reaction is complete, continue with the next step. Once the reaction is complete, the robot transfers the reaction mixture to FSPE cartridges containing C nine F 17 modified silica gel for purification.

Next, the cartridges are washed with eight milliliters of 80%methanol, followed by eight milliliters of 100%methanol. To eliminate the non floris fraction, the flow through is collected in a vial to obtain the desired florist tagged product. If additional purification is required, pause the robot and remove the appropriate reaction products.

Depending on the structure, donors can be extremely unreactive and some florist accepter molecule will be left even after adding sufficient promoter. If this is the case, FSPE will not be efficient enough for purification and additional purification via silica gel column chromatography may be performed after purification the robotic arm dispenses sodium meth oxide into the reaction vial. The reaction is then stirred for two hours in the robotic platform, if not complete as determined by TLC, extend the incubation period by approximately an hour.

After the completion of the reaction, the product is purified by FSPE as before. Next, remove the reaction product from the robot and on the benchtop subjected to dissolution in anhydrous toluene, followed by evaporation to remove residual water. Once a sample is dry, place it back in the robot the same cycle including glycosylation purification by FSPE.

Deep protection of the temporary protecting group followed by glycosylation is repeated until the desired chain length is obtained for the target molecule to de protect the protected product obtained from automation, remove the vial from the robot. The final steps of the procedure utilize explosive hydrogen gas and and must be performed outside the automation platform. On the benchtop oz, lysis of the double bond in the florist tag occurs and is followed by oxidation of the produced aldehyde into carboxylic acid.

Purify the resulting product by silica gel column chromatography on a bench top using a mixture of 30%methanol in di chloro methane. Finally, to de protect the benzo ether groups by palladium catalyzed hydrogenation, pass the product through a satellite pad to get rid of palladium, to get pure final product. Fully characterize the product using nuclear magnetic resonance or NMR spectroscopy.

High throw put polymer synthesis and nanoparticle fabrication is carried out following the protocol described by Peterson et al. As referenced in the accompanying document, the automated deposition apparatus utilized for particle functionalization consists of three ne 1000 pumps, a robotic stage integrated by two actuators, one for movement in the X direction and the other for movement in the Y direction and a second robotic stage with two adjacent racks consisting of three actuators, one for each direction the pumps and a total of five actuators are connected. In series actuators and pumps are operated by a computer using lab view software.

The copolymer systems used for particle fabrication are based on SEBA acid or SA and one six bis, paraic carboxy, oxil heane or CPH and one eight BIS para carboxy phenoxy three six dioxane or CP Teeg. Following nanoparticle fabrication place, a rack containing the 1.7 milliliter centrifugation tubes with a nanoparticle library to the linear actuator stage for the attachment of carbohydrates to the surface of poly anhy particles and a mean carboxylic acid coupling reaction consisting of two consecutive reactions is performed for the first reaction. Fill a syringe in a programmable syringe pump with an aqueous solution of EDC and ethylene diamine at a concentration of two milligrams per milligram of nanoparticles and 0.6 milligrams per milligrams of nanoparticles respectively.

Fill a second syringe in a programmable syringe pump with 2.5 milligrams per milligram of particles of NHSA total of 12 equivalents per nanoparticle sample, an aqueous solution. Next, using the lab view program, instruct the robot to deposit reagent suspensions into the nanoparticle library. The robot actuators would then move the tube holder to the correct position in the platform for dispensation of solutions, EDC and NHS.

Activate the carboxylic acid groups on the surface of poly anhydride nanoparticles and allow for a mean coupling. Next, submerge a sonication probe into each tube and sonicate each sample for 30 seconds at 40 hertz. Before moving to the next sample, clean the probe with acetone.

Once all samples are sated, the tube holder is detached from the robotic platform. Incubate the nanoparticle suspensions for nine hours with constant rotation at four degrees Celsius. Reaction times for first and second reactions can be changed to adjust the final saccharide concentration.

After the reaction is complete, centrifuge the tubes at 12, 000 times G for five minutes. In a cold environment, return to the robotic station. Reattach the tube holder to the robotic arm, and fill the second syringe in the robotic platform.

With cold water, the first syringe should remain empty. Start the robot. The supernatant in each tube is withdrawn into the empty syringe and the second pump deposits cold water into the tubes.

This step is performed to remove any unreactive reagents from the nanoparticle suspensions. Next, homogenize the nanoparticle suspension by sonication as demonstrated before. Then centrifuge the tubes at 12, 000 times G for five minutes.

Perform a second wash with cold water utilizing the robotic apparatus for the second reaction load 12 equivalents per nanoparticle sample of EDC into the first pump and 12 equivalents per nanoparticle sample of NHS into the second pump. Start the robotic platform to dispense adequate volumes into the tubes containing nanoparticles. The presence of EDC and NHS will allow the formation of an amide bond between the amine groups from the ethylene dite already attached to the nanoparticle surface and the carboxylic acid group of the deep protected sugar.

Once the deposition is complete, load 10 equivalents of a specific saccharide. In this case, lactose was used and the glycolic acid control in the two available pumps. Each saccharide is deposited into test tubes depending on the desired functionalization to achieve in each tube, which is previously programmed in the lab view program, utilized to operate the actuators and pump functions for the specific reaction employed In this study for the attachment of carbohydrates, glycolic acid is used as a linker control since deep protected saccharides already have this molecule covalently linked, which allows for further attachment to the nanoparticle surface.

Nanoparticle suspensions are homogenized by sonication as before and then incubated for nine hours with constant rotation at four degrees Celsius. After incubating wash the nanoparticle suspensions by centrifuging removing the supernatant, then Resus suspending the pellet in cold water and sonic place the tubes, which now contain the functionalized nanoparticle library in a vacuum chamber to dry for at least two hours. Functionalized nanoparticles are characterized by dynamic light scattering and other methods to assess surface composition, concentration, particle size, size distribution, and surface charge.

The fully protected diano side shown here was synthesized using the automation platform. The synthesized compound was characterized by proton NMR in a VXR 400 megahertz spectrometer. Using CDC 13 as solvent, the formation of the product can be confirmed from the presence of some characteristic peaks.

In the proton NMR diagram, the four protons from 1.79 to 2.21 and two protons at 3.38 are from the florist tag. The singlet peak at 2.16 corresponds to the acetate peak peaks at 4.94 and 5.11 are the anomeric protons to assess the effect of reaction time on the final morphology of the nanoparticles and the degree of sugar attachment achieved. The nanoparticles were functionalized with increasing reaction times as shown here, the concentration of diano on the surface of 50 50 CPT CPH nanoparticles increased with the total time of reaction and reached a maximum after 18 hours.

Nanoparticles functionalized with a 24 hour total reaction time were then used to evaluate their ability to target CLRs on mouse bone marrow derived dendritic cells using flow cytometry as shown here. Increased expression of the DC sign and the mano receptor to C type lectin receptors were observed after stimulation with non functionalized as well as lactose and dano functionalized nanoparticles. This indicates effective targeting.

However, Diano functionalized particles showed a higher level of expression indicating a specificity of this ligand for the receptors that were studied. After watching this video, you should have a good understanding of how to perform the high throughput automated synthesis of oligosaccharides as well as the functionalization of poly nanoparticles. Using these carbohydrate base at targeting agents, Once mastered the synthesis of the donor acceptor protected sugar, as well as the assembly of the apparatus can be done in 24 to 48 hours.

Of course, the time length depends on the size of the library as well as the functionalization time. While attending this method, it is important to remember that you can optimize the reaction conditions of the functionalization of biodegradable poly hydro particles depending on the nanoparticle chemistry. By following this procedure, various structures of carbohydrates can be synthesized in order to target different cell receptors to influence immunological outcome.