Bu içeriği görüntülemek için JoVE aboneliği gereklidir. Oturum açın veya ücretsiz deneme sürümünü başlatın.

Özet

Kovalent çapraz nano tanecikleri doğrusal polyester kolye işlevleri içeren üzerinden boyutu ve crosslinking yoğunluğunu ayarlama için bir süreç bu makalede. Sentez parametreleri (polimer molekül ağırlığı, kolye işlevselliği birleşme ve crosslinker eşdeğerleri) uyarlayarak, istenen nanopartikül boyutu ve crosslinking yoğunluğu ilaç teslim uygulamalar için elde edilebilir.

Abstract

Biz doğrusal polyester kolye Epoksit işlevsellik ve kontrollü boyutları ile bir nanosponge içine onların birleşme içeren sentezi için bir iletişim kuralı tanımlamak. Bu yaklaşım anahtarı elde edilen polimer kolye functionalization functionalized bir lactone sentezi ile başlar. Valerolactone (VL) ve allil-valerolactone (AVL) sonra yüzük-açılış polimerizasyon kullanarak copolymerized. Sonrası polimerizasyon değişiklik daha sonra bazı veya tüm kolye Alil grupların bir epoksi yan yüklemek için kullanılır. Epoksi Amin kimya polimer ve istenen nanosponge boyut ve crosslinking yoğunluk dayalı küçük molekül diamin crosslinker seyreltik bir çözümde formu nano tanecikleri için istihdam edilmektedir. Nanosponge boyutları boyut ve dağıtım belirlemek için iletim elektron mikroskobu (TEM) görüntüleme tarafından karakterize edilebilir. Bu yöntem son derece ayarlanabilir polyesterler küçük molekül uyuşturucu kapsülleme için kullanılabilir ayarlanabilir nano tanecikleri oluşturabilirsiniz bir yol sağlar. Omurga yapısı nedeniyle, bu parçacıklar hidrofobik küçük moleküller geniş bir kontrollü bir yayın için hydrolytically ve enzimatik parçalanabilir.

Introduction

Tam olarak cins crosslinking dayalı nano tanecikleri boyutu ve crosslinking yoğunluğunu ayarlama etkisi ve uyuşturucu yayın profil bu nanosystems¹rehberlik için büyük önem taşıyor. Tasarlama nanosponge ayar, Yani, farklı ağ yoğunluğunu parçacıkların hazırlanıyor, güvenen habercisi polimer kolye işlevselliğini ve dahil hidrofilik crosslinker karşılıkları. Bu yaklaşım, öncü ve solvent crosslinker konsantrasyon formu nano tanecikleri toplu jel yerine ayrı bir boyut için önemlidir. Nicel Nükleer manyetik rezonans spektroskopisi (NMR) bir karakterizasyonu teknik olarak kullanarak eklenen kolye işlevselliği ve polimer molekül ağırlığı kesin belirlenmesi için sağlar. Bir kez nano tanecikleri oluşur, konsantre ve bir nanogel karakteri kalmadan organik çözündürüldükten.

Nanopartikül ilaç dağıtım, son eser Poli kullanımı üzerinde odaklanmıştır (laktik-co-glikolik asit) (PLGA) kendi kendine monte nano tanecikleri^2,3,4,5,6. PLGA ilaç teslim uygulamalar için ideal hale parçalanabilir ester bağları vardır ve çoğunlukla poly(ethylene glycol) (PEG) nedeniyle onun stealth özellikleri⁷ile birlikte. Ancak, PLGA parçacık oluşumu kendi kendine monte yapısı nedeniyle, parçacıklar organik daha fazla functionalization için çözündürüldükten olamaz. PLGA nano tanecikleri aksine, kovalent çapraz bir nanopartikül tanımlanmış boyut ve Morfoloji, organics içinde istikrarlı ve sulu çözümler¹‘ aşağılamak ile şekillendirme önerilen yöntemdir. Bu yaklaşımın avantajları daha fazla kimyasal olarak nanosponge⁸yüzeyine functionalize yeteneği vardır ve istikrarı organik çözücüler içinde ilaç bileşikleri^1,9parçacıkların sonrası yükleme için kullanılabilir. Bu yöntemle, kapsülleme hidrofobik küçük moleküllerin yağış sulu medya içine elde edilebilir. Polyester omurga hidrofilik kısa crosslinker birlikte hydrophobicity bu parçacıklar, vücut sıcaklığında amorf bir karakter verir. Ayrıca, yükleme uyuşturucu sonra sulu ortamda kolayca enjekte içinde vivoolmak iyi süspansiyonlar parçacık oluşturabilir. Bu polyester nanosponges sentezi parametrelerini değerlendirmek ve bu tasarım ve boyut ve Morfoloji kontrolü için son derece önemli olan belirlemek için bu çalışmada hedefimizdir.

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

Nanosponge sentez parametrelerinin sonuç boyutu arasında ilişki değerlendirmek için her polimer habercisi konsantrasyon ve kolye işlevselliğini önemlidir. Şekil 1′ de, nanosponges bir successfulsynthetic düzeni reflü koşullar altında her iki öncü polimer ve diamin crosslinker DCM yılında 12 h için ekleme sonra yapılır. Epoksitler çözüm konsantrasyonu da ayrık parçacıklar şekillendirme için önemlidir. Bir kez nanosponges sentez, TE…

Discussion

Tekrarlanabilir nanosponge boyutları elde etmek ilaç teslim uygulamalarında hayati önem taşımaktadır. Birden çok parametre polimerizasyon ve nanosponge sentez ortaya çıkan Partikül boyutu ve crosslink yoğunluğunu etkiler. Üç önemli parametreler bizim analizde tespit edildi: polimer molekül ağırlığı, epoksi kolye işlevselliği ve crosslinker eşdeğerleri. Moleküler ağırlık ve epoksi işlevleri nanosponge sentezi için bir dizi üretmek için VL –co –AVL kopolimer stoichiometry değişt…

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