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Summary

Cet article décrit un processus pour le réglage de la densité de taille et de la réticulation de façon covalente réticulé nanoparticules de polyesters linéaires comportant une fonction pendentif. En adaptant les paramètres de synthèse (poids moléculaire de polymères, incorporation de fonctionnalités pendentif et RETICULATION équivalents), une densité de taille et réticulation de nanoparticules souhaitées peut être obtenue pour demandes de livraison de drogue.

Abstract

Les auteurs décrivent un protocole pour la synthèse de polyesters linéaires contenant pendentif époxyde fonctionnalité et leur incorporation dans un nanosponge avec dimensions contrôlées. Cette démarche commence par la synthèse d’une lactone fonctionnalisée qui est la clé de la fonctionnalisation de pendentif du polymère qui en résulte. Valérolactone (VL) et allyl-valérolactone (AVL) sont retrouvées ensuite à l’aide de polymérisation par ouverture. Après polymérisation modification est utilisée pour installer un groupement époxyde sur certains ou tous les groupes d’allyle. Chimie de l’époxy-amine est employé aux nanoparticules de forme dans une solution diluée de polymère et de petites molécules diamine RETICULATION basé sur la densité de réticulation et de taille nanosponge désiré. Nanosponge tailles peuvent être caractérisées par transmission d’images de microscopie électronique (met) pour déterminer la dimension et la distribution. Cette méthode fournit une voie par laquelle polyesters hautement accordables peuvent créer des nanoparticules accordables, qui peuvent être utilisés pour l’encapsulation de drogue de petite molécule. En raison de la nature de la colonne vertébrale, ces particules sont hydrolytique et enzymatiquement dégradables pour une libération contrôlée d’un large éventail de molécules petites hydrophobes.

Introduction

Tuning avec précision la taille et réticulation de la densité de nanoparticules issus d’intermoléculaire réticulation est d’une grande importance pour influencer et orienter le profil de libération de médicaments de ces nanosystèmes¹. Conception nanosponge accordabilité, c’est-à-direpréparer des particules de densité de réseau différent, est dépendante de la fonctionnalité de pendentif du polymère précurseur et les équivalents de la RETICULATION hydrophile constituée. Dans cette approche, la concentration du précurseur et RETICULATION dans le solvant est importante pour la forme de nanoparticules d’une taille discrète plutôt qu’un gel en vrac. Utilisant la spectroscopie quantitative la résonance magnétique nucléaire (RMN) comme une technique de caractérisation permet la détermination précise des fonctionnalités incorporées de pendentif et de poids moléculaire de polymères. Une fois que les nanoparticules sont forment, ils peuvent être concentrés et solubilisées dans matières organiques sans avoir le caractère d’un nanogel.

Des travaux récents NANOPARTICULE drug delivery a porté sur l’utilisation de poly (lactique-co-glycolique acide) (PLGA) self-assembled nanoparticules^2,3,4,5,6. PLGA a liens dégradables ester qui le rendent approprié pour des applications de livraison de drogue et est souvent combiné avec poly(ethylene glycol) (PEG) en raison de ses propriétés furtif⁷. Cependant, en raison de la nature auto-assemblés PLGA formation de particules, les particules ne peuvent pas être solubilisées dans organiques pour davantage de fonctionnalisation. Contrairement aux nanoparticules PLGA, la méthode proposée fournit réticulation covalente formant une NANOPARTICULE avec tailles définies et la morphologie, qui sont stables en matières organiques et se dégradent dans des solutions aqueuses¹. Avantages de cette approche sont la possibilité d’autres chimiquement fonctionnaliser la surface de la nanosponge⁸, et sa stabilité dans les solvants organiques peut être utilisée pour le chargement après des particules avec des composés pharmaceutiques^1,9. Avec cette méthode, l’encapsulation de molécules hydrophobes de petites est possible par précipitation en milieu aqueux. L’hydrophobicité du squelette polyester ainsi que de la RETICULATION hydrophile court donne à ces particules un caractère amorphe à la température corporelle. En outre, après la drogue de chargement, la particule peut former des suspensions fines en milieu aqueux pour être facilement injecté in vivo. C’est notre objectif dans ce travail d’évaluer les paramètres pour la synthèse de ces nanosponges de polyester et de déterminer celles qui sont importantes pour la conception et le contrôle de la taille et la morphologie.

Protocol

1. Synthesis and Characterization of AVL Place a magnetic stir bar inside a 2 neck 500 mL round bottom flask (Flask 1) and seal with an appropriate sized rubber septum and steel wire. Flame dry the flask to remove moisture by purging with nitrogen gas connected through an inlet needle and open outlet needle in the septum, while using a butane flame torch to gently heat the outside of the flask by moving the flame along the surface. Continue heating the entire flask by running the flame across the surface until moisture clouding the inside of the flask is not seen. Remove the flame and let the flask cool to room temperature while continuing the nitrogen flow throughout the reaction. Add 156.25 mL anhydrous tetrahydrofuran (THF) via a 30 mL syringe to Flask 1. Cool Flask 1 to -78 °C with a dry ice/acetone bath. Monitor the temperature with a thermometer throughout the reaction and add dry ice as needed to maintain -78 °C. Begin stirring the magnetic stir bar using a magnetic stir plate to create a gentle vortex. Add N,N-diisopropylethylamine (DIPA) (3.30 mL, 2.3 x 10-2 mol) via a 5 mL syringe, followed by the dropwise addition of 2.5 M n-Butyllithium (9.34 mL, 2.3 x 10-2 mol) via a 10 mL glass syringe equipped with a needle locking mechanism and metal needle to Flask 1. Continue to stir using a magnetic stir plate for 15 min at -78 °C. Flame dry (as described in step 1.1) and purge with argon gas (as described in step 1.1 with nitrogen gas) a 100 mL round bottom flask (Flask 2). Remove the argon gas inlet and outlet at the same time once the flask is cool enough to touch. Add 56 mL anhydrous THF via a 30 mL syringe and δ-valerolactone (VL) (1.97 mL, 2.1 x 10-2 mol) via a 5 mL syringe. Add the solution in Flask 2 to Flask 1 via a small cannula dropwise over 45 min while continuously stirring via a magnetic stir bar. Once Flask 2 is empty, remove the cannula and allow Flask 1 to continue stirring for 15 min at -78 °C. Flame dry (as described in step 1.1) and purge with argon gas (as described in step 1.1 with nitrogen gas) a 25 mL round bottom flask (Flask 3). Remove the argon gas inlet and outlet at the same time once the flask is cool enough to touch, to maintain positive pressure inside the flask. Add ally bromide (2.02 mL, 2.3 x 10-2 mol) and hexamethyphosphoramide (HMPA) (4.43 mL, 2.5 x 10-2 mol) each via a 5 mL syringe. Add Flask 3 to Flask 1 via a small cannula dropwise. Once Flask 3 is empty, remove the cannula. Remove as much dry ice and cold acetone as possible from the dry ice/acetone bath in which Flask 1 is submerged using a scoop and plastic cup. Replace with room temperature acetone to raise the bath to -40 °C and allow Flask 1 to continue stirring for 2 h while monitoring the temperature. If the temperature increases above -40 °C, add a few pieces of dry ice to cool the acetone. Warm to -10 °C by removing the cold acetone and dry ice and replace with room temperature acetone. Remove the septum from Flask 1 and quench the reaction with a 150 mL saturated ammonium chloride solution while stirring via a magnetic stir bar. Place Flask 1 in a freezer at -20 °C overnight for storage. Remove Flask 1 from the freezer and allow to warm to room temperature. Pour the solution from Flask 1 into a 500 mL separatory funnel. Add 50 mL dichloromethane (DCM), cap the separatory funnel, and rock gently to mix the DCM and aqueous/THF mixture to extract the product into the organic layer. Collect the organic layer into a 500 mL beaker containing an appropriately sized magnetic stir bar by opening the separatory funnel stopcock and set aside. Repeat two times. Collect all the organics into the same beaker and stir over magnesium sulfate (to remove any residual water) using a magnetic stir plate until the magnesium sulfate no longer clumps when added to solution. Discard the aqueous waste. Remove the magnesium sulfate solid from the organic product solution by using a filter paper inside a glass funnel and collect the organics in a 250 mL round bottom flask. Rinse the filter paper and solid magnesium sulfate with excess DCM to ensure that all the product is collected. Remove the solvent from the product by rotary evaporation while heating at 30 °C and using a water aspirator as the vacuum source. Purify the crude product by column chromatography10 using a silica gel as the stationary phase and eluting beginning with 3% ethyl acetate/hexanes for 1 column volume (CV), increasing the gradient to 29% ethyl acetate/hexanes over 5 CVs. Collect the fractions in 16 x 150 mm2 borosilicate glass tubes. Perform thin layer chromatography using 20% ethyl acetate/hexanes as eluent to determine the product fractions. Combine all product fractions into an appropriately sized round bottom flask and remove the solvent using rotary evaporation at 40 °C. Transfer to a pre-weighed product vial and place under a high vacuum at 0.05 torr to obtain pure AVL. Characterize the product by 1H NMR11 (CDCl3, 400 MHz): δ 5.75 (m, 1H), 5.06 (m, 2H), 4.26 (m, 2H), 2.54 (m, 3H), 2.28 (m, 1H), 2.03 (m, 1H), 1.87 (m, 1H), 1.54 (m, 1H). Yield: 2.07 g (70.56%). 2. Synthesis and Characterization of VL- co- AVL Flame dry and purge with nitrogen gas a 25 mL round bottom flask equipped with a magnetic stir bar and septum. Flame dry the flask to remove moisture by purging with nitrogen gas connected through an inlet needle and open outlet needle in the septum, while using a butane flame torch to gently heat the outside of the flask by moving the flame along the surface. Continue heating the entire flask by running the flame across the surface until moisture clouding the inside of the flask is not seen. Remove the flame and let the flask cool to room temperature while continuing the nitrogen flow throughout the reaction. Quickly remove the septum from the round bottom and add tin(II) triflate (2.5 mg, 5.9  x 10-6 mol) to the very bottom of the flask using a spatula. Replace the septum. Add 3-methyl-1-butanol (72.6 µL, 6.6 x 10-3 mol) via a 100 µL microsyringe and 1.33 mL anhydrous DCM via a 2 mL syringe sequentially. Stir via a magnetic stir plate for 10 min. Add AVL (0.48 mL, 3.7 x 10-3 mol) and VL (1.37 mL, 1.4 x 10-2 mol) sequentially via a syringe. Allow to continue stirring for 18 – 20 h. NOTE: The AVL and VL percentage in the final copolymer can be altered by stoichiometry in this step. Quench the reaction by adding ~5 mL methanol (MeOH). Add ~100 mg solid-support tin scavenger and stir for 2 h. Filter by gravity filtration to remove the solid. Remove the solvent under rotary evaporation with a water aspirator as the vacuum source while heating at 30 °C until the solution is viscous. Precipitate the solution dropwise into 500 mL cold MeOH (chilled with dry ice for at least 1 h) to produce flakes of white solid. Filter the solution by vacuum filtration into a funnel containing a fritted glass disc with filter paper to collect the solid. NOTE: If precipitation produces a cloudy supernatant or fine powder which goes through the filter paper, then the product solution is too dilute. All supernatant solvent should be removed by rotary evaporation with a water aspirator as the vacuum source while heating at 30 °C until the solution is viscous and then precipitation should be performed again. Transfer the solid product to a pre-weighed vial via a spatula and dry overnight via high vacuum pressure of 0.05 torr to collect the white, flaky solid. Characterize the polymer by 1H NMR11 (CDCl3, 400 MHz): δ 5.71 (m, 1H), 5.03 (t, 2H), 4.08 (m, 4H), 3.65 (t, 2H), 2.15 – 2.50 (m, 5H), 1.47-1.78 (m, 9H), 0.91 (d, 6H). 81.42% VL, 18.58% AVL, 2,940 g/mol, 578 g/mol repeat unit of AVL. Yield: 1.43 g, (73.43%). 3. Post-polymerization Epoxidation to Produce Epoxy-valerolactone (EVL) Copolymer Units Add VL-co-AVL copolymer (500 mg, 1.7  x 10-4 mol, 2,940 g/mol) to a 6-dram vial with a magnetic stir bar. Add 6.15 mL anhydrous DCM to the vial and vortex to solubilize the polymer. Add meta-chloroperoxybenzoic acid (mCPBA) (74.53 mg, 4.3 x 10-4 mol) to a second 6-dram vial. Add 6.15 mL anhydrous DCM and vortex until the mCPBA is solubilized completely. NOTE: The conversion ratio of allyls to epoxides can be varied by altering the stoichiometry in this step. The final allyl concentration in the solvent should be 0.065 M. Transfer the mCPBA solution to the VL-AVL solution. Cap the reaction and cover with a plastic paraffin film. Allow to stir for 48 h. Transfer the reaction mixture to a 50 mL separatory funnel. Add 15 mL saturated sodium bicarbonate, cap the separatory funnel, and rock gently to mix. Collect the organic layer containing the product into a 50 mL Erlenmeyer flask with an appropriate sized magnetic stir bar (product flask). Add 5 mL DCM to the aqueous layer that is still in the separatory funnel, cap, and then rock gently. Collect the organics into the product flask. Discard the aqueous waste. Transfer the organic layer back into a separatory funnel. Repeat this aqueous wash two times. Add magnesium sulfate (to remove any residual water) to the product flask while stirring over a magnetic stir plate. Continue adding small scoops of magnesium sulfate until it no longer clumps when added. Use a glass funnel fitted with a filter paper to remove the solid magnesium sulfate while transferring the mixture to a 50 mL round bottom flask. Remove the solvent by rotary evaporation with a water aspirator as the vacuum source and heating at 25 °C. Transfer the contents of the round bottom flask to a pre-weighed product vial and remove the solvent by rotary evaporation. Place on high vacuum at 0.05 torr overnight to produce a white, waxy solid. Characterize by 1H NMR11 (CDCl3, 400 MHz): δ 5.71 (m, 1H), 5.03 (t, 2H), 4.08 (m, 4H), 3.65 (t, 2H), 2.94 (d, 1H), 2.90 (s, 1H), 2.74 (s, 1H), 2.15-2.50 (m, 5H), 1.47-1.78 (m, 9H), 0.92 (d, 6H). 84.53% VL, 9.65% AVL, 5.82% EVL, 2,855 g/mol, 1,841 g/mol repeat unit of EVL. Yield: 417.3 mg, (83.46%). 4. Nanosponge Synthesis and Characterization Dissolve the VL-co-AVL-co-EVL polymer (200 mg, 7.0 x 10-5 mol, 2,855 g/mol) in 20.01 mL anhydrous DCM for an epoxide concentration of 0.0054 M. Transfer to a 100 mL round bottom flask with a 14/20 neck. NOTE: To catalyze the reaction, 100 µL aqueous saturated sodium bicarbonate can be added in this step. Place the reaction flask in an oil bath at 50 °C. Stir the solution with a fast vortex and add 2,2'-(ethylenedioxy)bis(ethylamine) (21.45 µL, 2.9 10-4 mol) via a microsyringe dropwise. Fit the neck of the flask with a water-jacketed condenser with cool water flowing through it, fitted with a 14/20 neck adapter, and reflux the solution for 12 h. Ensure a very tight seal between the neck of the flask and the condenser to avoid solvent evaporation. Remove excess solvent from the reaction flask by rotary evaporation at 25 °C until a viscous solution is obtained. Place a large magnetic stir bar into a 2 L beaker. Cut an approximately six-inch section of 10 K molecular weight cut-off (MWCO) dialysis tubing and fold one end then close with a dialysis clip. Transfer the product to 10 K MWCO dialysis tubing. Rinse the flask with excess DCM and transfer it to the tubing.Fold the top of the tubing and close with a dialysis clip that has a wire for hanging. Hang the dialysis tubing on the side of the beaker using the wire and fill the beaker with DCM until the dialysis tubing is completely submerged. Place on a magnetic stir plate and stir gently. Cover the beaker with aluminum foil to prevent solvent evaporation. Remove the solvent by pouring into a waste container and replace with fresh DCM three times daily for 48 h to remove the unreacted polymer and crosslinker. Remove all solvent from the beaker and transfer the contents of the dialysis tubing to a 10 mL syringe fitted with a 0.45 µm polytetrafluoroethylene (PTFE) syringe filter. Push the solution through the filter directly into a pre-weighed product vial to remove any remaining impurities. Remove the solvent by rotary evaporation at 25 °C, then place on high vacuum at 0.05 torr overnight to collect the light yellow, waxy solid. Characterize by 1H NMR11 (CDCl3, 400 MHz): δ 5.71 (m, 1H), 5.03 (t, 2H), 4.08 (m, 4H), 3.60-3.67 (t, 14H), 2.94 (d, 1H), 2.90 (s, 1H), 2.74 (s, 1H), 2.15-2.50 (m, 5H), 1.47-1.78 (m, 9H), 0.92 (d, 6H). Yield: 176.9 mg (88.45%). 5. TEM Imaging of Nanosponge Morphology and Size Filter 5 mL cell culture water through a 0.2 µm PTFE syringe filter. Place 0.5 mg nanosponges into a 1.5 mL centrifuge tube. Add 1 mL filtered cell culture water. Use a probe sonicator (20 kHz, 40 a) to sonicate the solution with 2 s bursts 4 – 5 times at room temperature until the particles have developed a fine suspension. Do not use prolonged sonication or let the solution heat up as this will cause aggregation. Add 30 mg phosphotungstic acid hydrate (PTA) to 1 mL filtered cell culture water in a 1.5 mL centrifuge tube. Vortex on the highest setting for 10 s or until the PTA is completely solubilized to produce 3% PTA solution. Use a 1 mL syringe with 22 G needle to draw up 0.5 mL 3% PTA solution. Add 4 drops of the 3% PTA solution to the particles and vortex on the highest setting for 10 s. Use a pair of self-closing tweezers to pick up a TEM grid then dip it into the particles solution quickly, three times. Let the grid dry for 5 h under a cover to reduce dust collection on the grid. Perform TEM imaging12 of the sample using high contrast and 40 µm objective.

Representative Results

Pour évaluer la relation entre les paramètres de synthèse de la nanosponge et sa taille qui en résulte, la fonctionnalité de concentration et pendentif de chaque précurseur de polymère est importante. Dans la Figure 1, un système de successfulsynthetic de nanosponges est effectué dans des conditions de reflux après avoir incorporé les deux précurseurs polymères et diamine RETICULATION dans DCM pendant 12 h. La concentration d’époxydes dans la …

Discussion

L’obtention reproductible nanosponge tailles est vital dans les demandes de livraison de drogue. Plusieurs paramètres dans la synthèse de la polymérisation et nanosponge affectent la taille et crosslink de la densité de la particule qui en résulte. Trois paramètres importants ont été identifiés dans notre analyse : poids moléculaire de polymères, la fonctionnalité pendentif époxyde et RETICULATION équivalents. Afin de produire un éventail de poids moléculaires et fonctionnalités époxyde pour la synth…

Materials

2,2'-(Ethylenedioxy)bis(ethylamine)	Sigma-Aldrich	385506-100ML
3-methyl-1-butanol	Sigma-Aldrich	309435-100ML	anhydrous, ≥99%
Acetone	Sigma-Aldrich	179124-4L
Allyl bromide	Sigma-Aldrich	A29585-5G	≥99%
Ammonium chloride	Fisher Scientific	A661-500	saturated solution in DI water
Cell culture water	Sigma-Aldrich	W3500-500ML	Filtered through 0.45 μm syringe filter
Dichloromethane (DCM)	Sigma-Aldrich	270997-100ML	anhydrous, ≥99%, contains 40-150 ppm amylene as stabilizer
Ethyl Acetate	Fisher Scientific	E145SK-4
EZFlow 0.2 μm Syringe Filter	Foxx Life Sciences	386-2116-OEM	Hydrophillic PTFE, 13 mm
EZFlow 0.45 μm Syringe Filter	Foxx Life Sciences	386-3126-OEM	Hydrophillic PTFE, 25 mm
Fisherbrand Disposable Borosilicate Glass Test Tubes with Plain End	Fisher Scientific	14-961-31
Fisherbrand Microcentrifuge Tubes	Fisher Scientific	14-666-318	1.5 mL
Hamilton Microliter Syringe, 100 μL	Hamilton Company	80600	Model 710 N SYR, Cemented NDL, 22s ga, 2 in, point style 2
Hexamethylphosphoramide	Sigma-Aldrich	H11602-100G	≥99%, contains ≤1000 ppm propylene oxide as stabilizer
Hexanes	Fisher Scientific	H292-4
Magnesium sulfate anhydrous	Fisher Scientific	M65-500
Meta-chloroperoxybenzoic acid	Sigma-Aldrich	273031-100G	Purified to ≥99% by buffer wash
Methanol (MeOH)	Sigma-Aldrich	322415-100ML	anhydrous, ≥99%
N-butyllithium solution	Sigma-Aldrich	230707-100ML	2.5 M in hexanes
N,N-diisopropylethylamine	Sigma-Aldrich	550043-500ML	≥99%
Parafilm M	Sigma-Aldrich	P7793-1EA
PELCO Pro Reverse (Self-Closing) Tweezers	Ted Pella, Inc.	5375-NM
Phosphotungstic acid hydrate	Alfa Aesar	40116
Q55 Sonicator	Qsonica	Q55-110	55 Watts, 20 kHz
SiliaMetS Cysteine	Silicycle	R80530B-10g
SnakeSkin Dialysis Clips	Thermo Scientific	68011
SnakeSkin Dialysis Tubing, 10K MWCO	Thermo Scientific	68100
Sodium bicarbonate	Fisher Scientific	5233-500	saturated solution in DI water
TEM grid	Ted Pella, Inc.	01822-F	Ultrathin Carbon Type-A, 400 mesh, Copper, approx. grid hole size: 42µm
Tetrahydrofuran (THF)	Sigma-Aldrich	401757-1L	Anhydrous, ≥99.9%, inhibitor-free
Tin(II) trifluoromethanesulfonate	Sigma-Aldrich	388122-1G
Vortex-Genie 2	Scientific Industries	SI-0236
Whatman Filter Paper, Grade 1	Fisher Scientific	09-805H	Circles, 185 mm
δ-valerolactone	Sigma-Aldrich	389579-100ML	Purified by vacuum distillation