Production of Near-Infrared Sensitive, Core-Shell Vaccine Delivery Platform

Summary

This article describes the protocols used to produce a novel vaccine delivery platform, "polybubbles," to enable delayed burst release. Polyesters including poly(lactic-co-glycolic acid) and polycaprolactone were used to form the polybubbles and small molecules and antigen were used as cargo.

Abstract

Vaccine delivery strategies that can limit the exposure of cargo to organic solvent while enabling novel release profiles are crucial for improving immunization coverage worldwide. Here, a novel injectable, ultraviolet- curable and delayed burst release- enabling vaccine delivery platform called polybubbles is introduced. Cargo was injected into polyester-based polybubbles that were formed in 10% carboxymethycellulose -based aqueous solution. This paper includes protocols to maintain spherical shape of the polybubbles and optimize cargo placement and retention to maximize the amount of cargo within the polybubbles. To ensure safety, chlorinated solvent content within the polybubbles were analyzed using neutron activation analysis. Release studies were conducted with small molecules as cargo within the polybubble to confirm delayed burst release. To further show the potential for on-demand delivery of the cargo, gold nanorods were mixed within the polymer shell to enable near-infrared laser activation.

Introduction

Limited immunization coverage results in the death of 3 million people specifically caused by vaccine-preventable diseases¹. Inadequate storage and transportation conditions lead to wastage of functional vaccines and thus contribute to reduced global immunization. In addition, incomplete vaccination due to not adhering to the required vaccine schedules also causes limited vaccine coverage, specifically in developing countries². Multiple visits to medical personnel are required within the recommended period for receiving booster shots, thus limiting the percentage of population with complete vaccination. Hence, there is a need for developing novel strategies for controlled vaccine delivery to circumvent these challenges.

Current efforts towards developing vaccine delivery technologies include emulsion-based polymeric systems^3,4. However, cargo is often exposed to greater quantity of organic solvent that can potentially cause aggregation and denaturation, specifically in the context of protein-based cargo^5,6. We have developed a novel vaccine delivery platform, "polybubbles", that can potentially house multiple cargo compartments while minimizing the volume of cargo that is exposed to solvent⁷. For example, in our polybubble core-shell platform, one cargo pocket of diameter 0.38 mm (SEM) is injected in the center of a 1 mm polybubble. In this case, surface area of cargo exposed to organic solvent would be approximately 0.453 mm². After considering the packing density of spheres (microparticles) within a sphere (cargo depot), the actual volume of microparticles (10 µm in diameter) that could fit in the depot is 0.17 mm³. The volume of one microparticle is 5.24x10^-8 mm³ and thus number of particles microparticles that can fit the depot is ~3.2x10⁶ particles. If each microparticle has 20 cargo pockets (as a result of double-emulsion) of 0.25 µm diameter, then the surface area of cargo exposed to organic solvent is 1274 mm². Cargo depot within the polybubble thus would have ~2800-fold less surface area exposed to organic solvent compared to that of organic solvent-exposed cargo in microparticles. Our polyester-based platform can thus potentially reduce the quantity of cargo exposed to organic solvent which can otherwise cause cargo aggregation and instability.

Polybubbles are formed based on phase-separation principle where the polyester in organic phase is injected into an aqueous solution resulting in a spherical bubble. Cargo in the aqueous phase can then be injected in the center of the polybubble. Another cargo compartment can potentially be achieved within the polybubble by mixing a different cargo with the polymer shell. The polybubble at this stage will be malleable and will then be cured to result in a solid polybubble structure with cargo in the middle. Spherical polybubbles were chosen over other geometrical shapes to increase the cargo capacity within the polybubble while minimizing the overall size of the polybubble. Polybubbles with cargo in the center were chosen to demonstrate delayed burst release. Polybubbles were also incorporated with near infrared (NIR)- sensitive (i.e., theranostic-enabling) agent, namely gold nanorods (AuNR), to cause increase in temperature of the polybubbles. This effect could potentially facilitate faster degradation and could be used for controlling kinetics in future applications. In this paper, we describe our approach to form and characterize polybubbles, to achieve delayed burst release from the polybubbles, and to incorporate AuNR within the polybubbles to cause NIR-activation.

Protocol

1. Polycaprolacyone triacrylate (PCLTA) synthesis

1. Dry 3.2 mL of 400 Da polycaprolacyone (PCL) triol overnight at 50 °C in an open 200 mL round bottom flask and K₂CO₃ in a glass vial at 90 °C.
2. Mix the triol with 6.4 mL of dichloromethane (DCM) and 4.246 g of potassium carbonate (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 rotovape 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) 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 1000mg/mL 14kDa PCL and synthesize PCLTA. Mix the photoinitiator with 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)⁶, polyvinylpyrrolidone (PVP), and glycopolymers⁶ 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.

7. Toxicity

1. Quantifying chlorine content in polybubbles using neutron activation analysis (NAA)
   1. Use polybubbles that were lyophilized for 2, 4, 6, 20, and 24 h for this study at 0.010 mBar vacuum and at -85 °C.
   2. Measure 5-9 mg of polybubbles and place them on LDPE irradiation vials.
   3. Prepare 1000 g/mL of chlorine calibration solution from national institute of standards and technology (NIST)-traceable calibration solution.
   4. Use 1- megawatts Triga reactor to carry out neutron irradiations on each sample at neutron fluence rate of 9.1 × 10¹² /cm²·s for 600 s.
   5. Transfer the polybubbles to unirradiated vials.
   6. Use HPGe detector to obtain gamma-ray spectra for 500 s after 360 s decay intervals.
   7. Use NAA software by canberra Industries to analyze the data.
2. Quantifying chlorine content released from polybubbles using NAA
   1. Incubate polybubbles that were lyophilized overnight (at 0.010 mBar vacuum and at -85 °C) in 400 µL of PBS at 37 °C.
   2. Collect the supernatants at weeks 1, 2, and 3 after incubation.
   3. Analyze the supernatants for chlorine content using NAA using the same method as described above in step 7.1.

8. AuNR Synthesis by Kittler, S., et al.⁸

1. Prepare AuNR seeding solution by mixing 250 µL of 10 mM chloroauric acid (HAuCl₄), 7.5 mL of 100 mM cetrimonium bromide (CTAB), and 600 µL of 10 mM ice cold sodium borohydride (NaBH₄).
2. Prepare Growth solution by mixing 40 mL of 100 mM CTAB, 1.7 mL of 10 mM HAuCl₄, 250 µL of silver nitrate (AgNO₃), and 270 µL of 17.6 mg/mL ascorbic acid to a tube.
3. Vigorously mix 420 µL of seed solution with the growth solution at 1200 rpm for 1 min. Then leave the mixture undisturbed to react for 16 h.
4. Remove the excess reagents from the mixture by centrifuging at 8000 × g for 10 min and discard the supernatant.

9. Hydrophobicization of AuNRs by Soliman, M.G., et al.⁹

1. Adjust pH of 1.5 mL of synthesized CTAB-stabilized AuNRs to 10 using 1 mM sodium hydroxide (NaOH).
2. Stir the solution with 0.1 mL of 0.3 mM methylated PEG (mPEG) thiol at 400 rpm overnight.
3. Mix PEGylated AuNRs with 0.4 M dodecylamine (DDA) in chloroform at 500 rpm for 4 days.
4. Pipet out the top organic layer containing hydrophobicized AuNRs and store at 4 °C until future use.

10. NIR-activation of polybubbles

1. Mix the polymer (PLGADA or PCL/PCLTA) solution with hydrophobicized AuNRs in a 1:9 (vol/vol).
2. Add photoinitiator to the polymer-AuNR mixture in a 0.005:1 (vol/vol).
3. Form polybubbles by injecting the polymer-AuNR mixture into a 0.92 mL glass vial with 10% CMC (wt/vol) (refer to step 2).
4. Cure the polybubbles at 254 nm wavelength for 60 s at 2 W/cm².
5. Flash freeze in liquid nitrogen for 30 s and lyophilize overnight at 0.010 mBar vacuum and at -85 °C.
6. Separate the dried polybubbles using forceps and wash with DI water to remove any residual CMC.
7. Incubate the polybubbles in 400 µL of PBS at 37 °C.
8. Activate the polybubbles using 801 nm NIR laser at 8A for 5 min every Monday, Wednesday, and Friday.
9. Take forward-looking infrared (FLIR) images of the polybubble before and after laser activation to obtain temperature values.
10. Calculate temperature differences between before and after laser activation based on the temperature values from the FLIR images.

Results

Polybubbles were extensively characterized using SEM and NAA. Cargo was successfully centered to result in a delayed burst release. Polybubbles were also successfully laser-activated because of the presence of AuNRs within the polybubbles.

Polybubble characterization
Polybubbles injected in an aqueous solution without CMC resulted in a flattened polybubble due to their contact with the bottom of the glass ...

Discussion

Current technologies and challenges
Emulsion-based micro- and nanoparticles have been commonly used as drug delivery carriers. Although release kinetics of the cargo from these devices have been extensively studied, controlling burst release kinetics has been a major challenge¹¹. Cargo versatility and functionality is also limited in emulsion-based systems owing to the exposure of cargo to excess aqueous and organic solvents. Protein-based cargo are often not compatible with...

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
Bromosalicylic acid	Acros Organics	AC162142500
Carboxymethylcellulose (CMC)	Millipore Sigma	80502-040
Centrimonium bromide (CTAB)	MP Biomedicals	ICN19400480
Chloroform	Fisher Chemical	C2984
Coating buffer	Abcam	ab210899
Dichloromethane (DCM)	Sigma Aldrich	270997-1L
Diethyl ether	Fisher Chemical	E1384
Dodeacyl Amine	Acros Organics	AC117665000
Doxorubicin hydrochloride	Fisher BioReagents	BP251610
L-ascorbic acid	Acros Organics	A61 100
Legato 100 Syringe Pump	KD Scientific	14 831 212
mPEG thiol	Laysan Bio	NC0702454
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
Potassium carbonate	Acros Organics	AC424081000
Recombinant HIV1 gp120 + gp41 protein	Abcam	ab49054
Silver nitrate	Acros Organics	S181 25
Sodium borohydride	Fisher Chemical	S678 10
Tetrachloroauric acid	Fisher Chemical	G54 1
Trehalose	Acros Organics	NC9022655
Triethyl amine	Acros Organics	AC157910010