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.
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.
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 systems3,4. 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 cargo5,6. 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 5.24×10-8 mm3 and thus number of particles microparticles that can fit the depot is ~3.2×106 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.
1. Polycaprolacyone triacrylate (PCLTA) synthesis
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.
3. Modulation of polybubble diameter
4. Centering cargo within polybubble
5. Cargo Formulation
NOTE: Polybubble formulation can house various cargo types, including small molecules, proteins, and nucleic acids.
6. Release of cargo
NOTE: Small molecule or antigen can be used as the cargo type
7. Toxicity
8. AuNR Synthesis by Kittler, S., et al.8
9. Hydrophobicization of AuNRs by Soliman, M.G., et al.9
10. NIR-activation of polybubbles
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 vial (Figure 1A,B). Partial flattening was observed when 5% CMC-based aqueous solution was used in place of DI water (Figure 1C). Subsequently, 10% CMC-based aqueous solution in the glass vial resulted in polybubble being suspended in the solution and thus successful maintenance of sphericity of the polybubble (Figure 1D).
Cargo centering
Cargo injection into the polybubble in the absence of CMC resulted in leakage causing no retention of cargo within the polybubble (Figure 3). To counter this challenge, two approaches were used: 1) viscosity of PCLTA was successfully increased using K2CO3 that was isolated after endcapping PCL triol with triacrylate (Figure 2), and 2) viscosity of the cargo was successfully increased after mixing the cargo with 5% CMC (Figure 3, Figure 4). Viscosity of the PLGADA polybubbles were sufficient to facilitate centering of the cargo and thus was not modulated using K2CO3.
Antigen functionality
HIV gp120/41 antigen was mixed with and without trehalose before injecting into the polybubble (Figure 5). Binding efficiency of antibody to the antigen (termed as functionality) with and without trehalose was observed to have no statistically significant difference.
Release studies without laser activation
Delayed burst releases were observed in PLGADA polybubbles with acriflavine in the middle on days 19 and 5 for polybubbles incubated at 37 °C (Figure 6A) and 50 °C (Figure 6B), respectively. Delayed burst releases were also observed in PCL/PCLTA polybubbles with acriflavine in the middle on days 160 and 60 for polybubbles incubated at 50 °C (Figure 7A) and 70 °C (Figure 7B), respectively. These release studies were conducted in the absence of laser-activatable AuNRs.
In vitro laser activation of polybubbles
Polybubbles with AuNRs in the shell were successfully laser activated multiple times in PLGADA polybubbles (Figure 8A) and PCL/PCLTA polybubbles (Figure 8B). Temperature changes from before and after laser activation were 10 ± 1 °C and 5 ± 1 °C in PCL/PCLTA polybubbles with higher and lower AuNR concentration in the shell, respectively. Temperature changes observed before and after laser activation were 11 ± 2 °C and 6 ± 1 °C in PLGADA polybubbles with higher and lower AuNR concentration in the shell, respectively.
Figure 1: Maintaining sphericity of polybubbles. SEM images of (A) 14 kDa PCL/300 Da PCLTA flattened polybubble due to the contact of polybubble with the bottom of the glass vial; (B) 14 kDa PCL/300 Da PCLTA polybubble from the top that was not in contact with the glass bottom; (C) the PCL/PCLTA polybubbles with lesser degree of flattening when injected into a 5% CMC solution compared to DI water solution, causing the formation of hemisphere-like shape at the point of contact with the vial; (D) polybubble that did not reach the bottom of the glass vial when injected into a 10% CMC solution, allowing for the spherical shape to be maintained. All of the scale bars indicated are 500 µm. This figure has been modified from Lee et al.7. Please click here to view a larger version of this figure.
Figure 2: Modulation of PCLTA viscosity. Concentration of K2CO3 was increased from 0 to 80 mg/mL in PCLTA and dynamic viscosity was observed to increase proportionally with the concentration of K2CO3. This figure has been modified from Arun Kumar et al10. Please click here to view a larger version of this figure.
Figure 3: Cargo injection into the polybubble with and without CMC. Top panel shows frames extracted from the video of cargo leakage during injection in the absence of CMC. Bottom panel shows frames extracted from the video of cargo retention within the polybubble in the presence of 5% CMC. This figure has been modified from Lee et al.7. Please click here to view a larger version of this figure.
Figure 4: Centered cargo. Fluorescent microscope images of (A) PCL/PCLTA polybubble with centered cargo, (B) PCL/PCLTA polybubble with cargo in the shell and centered non-fluorescent dye. This figure has been modified from Arun Kumar et al10. Please click here to view a larger version of this figure.
Figure 5: Antigen functionality with trehalose. Functionality of HIV gp120/41 with and without trehalose within the polybubble was analyzed using ELISA. The binding efficiency of an antibody to the protein is generally regarded as an indicator for the functionality of the protein. When we discuss the functionality of antigen in this study, we intend it to mean that it aids the antibodies binding the protein of interest (which is an indicator for protein functionality). No statistical significance was observed between the two groups. Confidence intervals are indicated by solid and dotted lines. This figure has been modified from Lee et al.7. Please click here to view a larger version of this figure.
Figure 6: Delayed burst release from PLGADA polybubbles. Release studies showing delayed burst releases from PLGADA polybubbles with acriflavine in the middle at (A) 37 °C, (B) 50 °C. Solid line indicates the fitted curve obtained based on the data points. This figure has been modified from Arun Kumar et al10. Please click here to view a larger version of this figure.
Figure 7: Delayed burst release from PCL/PCLTA polybubbles. Release studies showing delayed burst releases from PCL/PCLTA polybubbles with acriflavine in the middle at (A) 50 °C, (B) 70 °C. This figure has been modified from Arun Kumar et al10. Please click here to view a larger version of this figure.
Figure 8: NIR laser activation of polybubbles. Temperature change observed before and after NIR laser activation in (A) PLGADA polybubbles, (B) PCL/PCLTA polybubbles with higher and lower concentration of AuNRs in the polymer shell. This increase in temperature could be leveraged to potentially expedite the polymer degradation leading to earlier release of the cargo. This figure has been modified from Arun Kumar et al10. Please click here to view a larger version of this figure.
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 micro-and nanoparticles due to the possibility of cargo denaturation and aggregation12. In addition to cargo stability, cargo kinetics is especially important in the context of vaccines because of the need for booster shots leading to seroconversion. Previous efforts to address these challenges in vaccine delivery have not been sufficiently successful, as the notion of single injection vaccine systems have been around for a couple of decades and has not yet been clinically translated.
Our polybubble vaccine delivery platform can potentially overcome the challenges with increased exposure of cargo to organic solvent by minimizing the exposed cargo volume. This technology can potentially accommodate at least two cargo compartments: cargo in the shell and cargo in the center. Polybubbles with centered cargo can be used to control the burst release of the cargo while being compatible to different cargo types, including small molecules and antigen. In this study, we used polyesters with varying degradation times, PLGADA (shorter degradation time) and PCL/PCLTA (longer degradation time), as the polymer carriers and acriflavine (small molecule) as the cargo type to demonstrate delayed burst release. In the following sections we describe the crucial steps in forming polybubbles that are capable of enabling both delayed burst release and NIR activation, especially for future on-demand delivery applications.
Cargo centering within the polybubble
Cargo centering was one of the significant challenges that was encountered during the formulation of the polybubbles. Immediately after injection, cargo would migrate to the surface and the cargo pocket would be stabilized without bursting into the aqueous 10% CMC solution. Polybubbles with such off-centered cargo can result in earlier release due to the non-uniform thickness of the polymer surrounding the cargo. Modulating the viscosity of the polymer and the cargo was thus crucial in resolving issues related to cargo centering. Viscosity of the cargo was increased by mixing the cargo solution with 5% CMC. To increase the viscosity of the polymer, molecular weight of the polymer could have been modified. However, increasing molecular weight often results in slower polymer degradation thus causing further delay in cargo release. Viscosity of the polymer was thus modified by increasing the concentration of the polymer. Higher concentration (1000 mg/mL) was sufficient to increase the viscosity of PLGADA. However, viscosity of PCL/PCLTA was not adequate to retain the cargo in the middle. Thus, K2CO3 that was isolated after the endcapping reaction of PCLTA was used to increase the viscosity of PCLTA.
Novel delayed release
Delayed burst release was observed from the release studies conducted using the polybubbles with centered cargo. Small molecule (acriflavine) was used as centered cargo in the polybubbles to study the release profile. Unique release profiles were observed based on the polyester used due to the difference in the degradation time of the polymers. Burst release was observed earlier in PLGADA polybubbles compared to that of PCL/PCLTA polybubbles. Early cargo release was observed in PLGADA polybubbles because PLGA degrades faster compared to PCL13. Upon successful modulation of release kinetics with two types of polyesters, we further wanted to engineer the polybubble to potentially enable on-demand release of the cargo.
NIR-activation of polybubbles
On-demand release of the cargo with respect to timing of the patients' needs has been challenging to achieve using current delivery strategies14. We hypothesized that expediting the cargo release on-demand could be possible by accelerating the polymer degradation through the use of NIR- sensitive (i.e., theranostic-enabling) agents. AuNRs have been extensively studied for their ability to be activated using NIR laser that can travel few centimeters through the skin15. CTAB-stabilized AuNRs were thus prepared based on the protocol by Kittler, S, et al. and were hydrophobicized based on the methods published by Solimon, M.G., et al. Polybubbles with hydrophobicized AuNRs in the shell were then irradiated with NIR laser at desired time points for 5 min to observe temperature change. Temperatures before and after laser were measured based on the FLIR images. Cured polymer shell helped preserve the shape of AuNRs during the laser activation thus enabling multiple NIR activations of polybubbles. This is an interesting observation because in previous literature, AuNRs have often been known to lose their rod-like shape (crucial for NIR activation) due to laser activation16. The successful laser-activation of the polybubbles with AuNRs could pave the way to control on-demand release of the cargo in the next generation of polybubbles.
Significance and future applications
The results obtained from this study thus shows that polybubbles have the potential to be used as a novel vaccine delivery platform. Preparation of polybubbles described in this paper will further enable other researchers to use polybubbles as a delivery platform for other therapeutic applications. For example, in addition to vaccine delivery, polybubbles can also be potentially used to delivery synergistic therapeutic agents with varying release kinetics. Furthermore, polybubbles are made of polyesters that are biodegradable and have been used in many FDA-approved medical devices. We further validated the safety of polybubbles by showing that the chlorine released from polybubbles are well within the safety levels recommended by the EPA17. Thus, our novel, injectable, UV-curable polybubble platform has the potential to be used as a safe and effective drug delivery platform for a variety of cargo types.
Limitations of this technology
The polybubble platform technology can be used as a vaccine delivery platform potentially enabling controlled release. Our studies highlight the versatility of this platform capable of delivering different cargo types, including antigens and small molecules. However, one of the current limitations of this technology is that the cargo is currently being injected manually. For scaling purposes, we are currently engineering an automated platform that will enable injection (i.e., as an array) of cargo within the polybubble and will potentially help alleviate the concerns regarding translatability of this technology.
The authors have nothing to disclose.
We would like to thank Dr. Bryan E. Tomlin affiliated with the elemental analysis lab within the department of chemistry at TAMU who assisted with the neutron activation analysis (NAA).
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 |