A Technique to Generate a Novel Delivery Platform for a Delayed Burst Release of Molecules

Overview

This video demonstrates a method for the generation of a polymer bubble or polybubble-based novel platform that enables delayed burst release. Polyester-based polymers are used to form the polybubbles with core-shell structure, and small molecules and antigens can be used as cargo.

Protocol

1. Polycaprolactone triacrylate (PCLTA) synthesis

1. Dry 3.2 mL of 400 Da polycaprolactone (PCL) triol overnight at 50 °C in an open 200 mL round bottom flask and potassium carbonate (K₂CO₃) in a glass vial at 90 °C.
2. Mix the triol with 6.4 mL of dichloromethane (DCM) and 4.246 g of K₂CO₃ under argon.
3. Mix 2.72 mL of acryloyl chloride in 27.2 mL of DCM and add dropwise to the reaction mixture in the flask over 5 min.
4. Cover the reaction mixture with aluminum foil and leave it undisturbed at room temperature for 24 h under argon.
5. After 24 h, filter the reaction mixture using a filter paper on a Buchner funnel under vacuum to discard excess reagents.
6. Precipitate filtrate from step 1.5 that contains the endcapped polymer in diethyl ether in a 1:3 (vol/vol) and rotavap at 30 °C to remove the diethyl ether.

2. Formation of the polybubble

NOTE: Injecting polymer in the deionized (DI) water would cause the polybubbles to migrate to the bottom of the vial resulting in flattened bottom. Use 10% (wt/vol) carboxymethyl cellulose (CMC) to fill the glass vial instead to avoid polybubble flattening.

1. Prepare 10% (wt/vol) CMC solution in DI water.
2. Fill a 0.92 mL glass vial with 0.8 mL of 10% CMC using a 1 mL transfer pipet.
3. Mix 1000 mg/mL of 14 kDa PCL in DCM and synthesize PCLTA in a 1:3 (vol/vol) for a total volume of 200 µL or prepare 200 µL of 1000 mg/mL of 5 kDa poly (lactic-co-glycolic acid) diacrylate (PLGADA) in chloroform.
4. Mix the 2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone (photoinitiator) with the polymer (PLGADA or PCL/PCLTA) mixture in 0.005:1 (vol/vol).
5. Load 200 µL of polymer mixture into a 1 mL glass syringe mounted on a syringe pump that is connected to a dispensing stainless-steel tube with an inner diameter of 0.016 inch.
6. Use a micromotor to control the forward and backward motion of the polymer tube to inject polymer into the 10% CMC in the glass vial to form the polybubble.
7. Cure the polybubbles under ultraviolet (UV) at 254 nm wavelength for 60 s at 2 W/cm².
8. Flash freeze the polybubbles in liquid nitrogen and lyophilize overnight at 0.010 mBar vacuum and at -85 °C.
9. Separate the polybubbles from the dried CMC using forceps and wash the polybubbles with DI water to remove any residual CMC. Note that other polymers can be used likely with modifications to alter the release kinetics.

3. Modulation of polybubble diameter

1. Fill a 0.92 mL glass vial with 10% CMC using a 1 mL transfer pipet.
2. Mix PCL/PCLTA in a 1:3 (vol/vol) with 1000 mg/mL 14kDa PCL and synthesize PCLTA. Mix the photoinitiator with a polymer mixture in a 0.005:1 (vol/vol).
3. Load the polymer mixture into a 1 mL glass syringe mounted on a syringe pump that is connected to a dispensing stainless-steel tube with an inner diameter of 0.016 inch.
4. Use a micromotor to control the forward and backward motion of the polymer tube to inject polymer into the 10% CMC in the glass vial to form the polybubble.
5. To obtain polybubbles with various diameters, vary dispensing rate from 0.0005 to 1 µL/s.
6. Take images of the vial with the polybubbles with varying diameter.
7. Use ImageJ to quantify the diameter of the polybubbles and use the size of the vial as scale.

4. Centering cargo within polybubble

1. Modulation of PCL/PCLTA viscosity using K₂CO₃:        
   NOTE: Viscosity of PLGADA does not have to be modified using K₂CO₃ because the viscosity of 5 kDa PLAGDA at 1000 mg/mL is sufficient for centering the cargo.
   1. Add K₂CO₃ (that was isolated after the PCLTA reaction) to the PCLTA at varying concentrations including 0 mg/mL, 10 mg/mL, 20 mg/mL, 40 mg/mL, and 60 mg/mL.
   2. Measure the dynamic viscosities of the solutions by changing the shear rate from 0 to 1000 1/s using rheometry.
   3. Manually inject the cargo in the middle (refer to step 4.2 to prepare the cargo mixture) of the polybubbles that were formed using the PCL/PCLTA solutions with different concentrations of K₂CO₃ (step 4.1.1). Determine the optimal concentration of K₂CO₃ by observing which solution from step 4.1.1 can result in retention of the cargo in the middle.
2. Centering of the cargo (already shown feasibility with small molecules) with CMC
   1. Mix the cargo with 5% (wt/vol) CMC in a rotator overnight to increase the viscosity of the cargo.
   2. Manually inject 2 µL of cargo mixture in the polybubble and proceed with UV curing at 254 nm wavelength for 60 s at 2 W/cm².
   3. Flash freeze the polybubbles in liquid nitrogen for 30 s and lyophilize overnight at 0.010 mBar vacuum and at -85 °C.
   4. Separate the polybubbles from the dried CMC using forceps and wash with DI water to remove any residual CMC.
   5. Cut the polybubble in half and image the halves using confocal microscopy to ensure that the cargo is centered (refer to step 6 for excitation and emission wavelengths used).

5. Cargo Formulation

NOTE: Polybubble formulation can house various cargo types, including small molecules, proteins, and nucleic acids.

1. Based on previous studies, in the case of protein cargo, use excipients including polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), and glycopolymers to improve the stability of protein during polybubble formulation.
2. Form polybubbles based on the protocol in step 2.
3. Prepare the antigen solution by adding 17.11 g of trehalose to 625 µL of HIV gp120/41 antigen.
4. Manually inject 1 µL of antigen solution in the middle of the polybubble.
5. Open polybubbles on days 0, 7, 14, and 21, and record the fluorescence of antigen with excitation and emission wavelengths 497 nm and 520 nm, respectively.
6. Determine the functionality of the antigen using enzyme-linked immunosorbent assay (ELISA) and use 5% nonfat milk as a blocking buffer.

6. Release of cargo

NOTE: Small molecule or antigen can be used as the cargo type

1. Small molecule
   1. Incubate polybubbles with centered acriflavine in 400 µL of phosphate buffer saline (PBS) at 37 °C, 50 °C for PLGADA polybubbles and at 37 °C, 50 °C, 70 °C for PCL/PCLTA polybubbles.        
      NOTE: The reason why we recommend testing above body temperatures is to a) simulate the temperature (50 °C) at which the polybubble reaches while lasering the gold nanorods (AuNRs) within PCL and PLGA; and b) accelerate the degradation process of PCL (50 °C, 70 °C).
   2. At each time point, collect the supernatants and replace with 400 µL of fresh PBS.
   3. Use a plate reader to quantify the fluorescence intensities in the collected supernatants.
      NOTE: Use ex/em of 416 nm/514 nm for acriflavine.
2. Antigen
   1. Incubate polybubbles with centered bovine albumin serum (BSA)-488 in 400 µL of PBS at 37 °C, 50 °C for PLGADA polybubbles and at 37 °C, 50 °C for PCL/PCLTA polybubbles.
   2. At each time point, collect the supernatants and replace with 400 µL fresh PBS.
   3. Use a plate reader to quantify the fluorescence intensities in the collected supernatants. Use ex/em of 497 nm/520 nm for BSA-488.
      NOTE: Release study at 70 °C for PCL/PCLTA polybubbles should not be conducted to avoid exposing the antigen to extreme temperature.

Materials

Name	Company	Catalog Number	Comments
1-Step Ultra Tetramethylbezidine (TMB)-Enzyme-Linked Immunosorbent Assay (ELISA) Substrate Solution	Thermo scientific	34028
2-Hydroxy-2-methylpropiophenone	TCI AMERICA	H0991
450 nm Stop Solution for TMB Substrate	Abcam	ab17152
Acryloyl chloride	Sigma Aldrich	A24109-100G
Acriflavine	Chem-Impex International	22916
Anhydrous ethyl ether	Fisher Chemical	E138-500
Anti-HIV1 gp120 antibody conjugated to horseradish peroxidase (HRP)
Bovine serum albumin (BSA)	Fisher BioReagents	BP9700100
BSA-CF488 dye conjugates	Invitrogen	A13100
Carboxymethylcellulose (CMC)	Millipore Sigma	80502-040
Chloroform	Fisher Chemical	C2984
Coating buffer	Abcam	ab210899
Dichloromethane (DCM)	Sigma Aldrich	270997-1L
Diethyl ether	Fisher Chemical	E1384
Legato 100 Syringe Pump	KD Scientific	14 831 212
Nonfat dry milk	Andwin Scientific	NC9022655
Potassium carbonate	Acros Organics	AC424081000
Phosphate saline buffer	Fisher BioReagents	BP3991
(Poly(caprolactone)	Sigma Aldrich	440744-250G
(Poly(caprolactone) triol	Acros Organics	AC190730250
Poly (lactic-co-glycolic acid) diacrylate	CMTec	280050
Recombinant HIV1 gp120 + gp41 protein	Abcam	ab49054
Trehalose	Acros Organics	NC9022655