Polyanhydride-Based Microparticles for Programmable Pulsatile Release of Diphtheria Toxoid (DT) for Single-Injection Self-Boosting Vaccines

Abstract

Vaccination remains a critical tool in preventing infectious diseases, yet its effectiveness is undermined by under-immunization, particularly for vaccines requiring multiple doses that patients fail to complete. To address this challenge, the development of single-injection platforms delivering self-boosting vaccines has gained significant attention. Despite some advances, translating these platforms into clinical applications has been limited. In this study, a novel polyanhydride-based polymeric delivery platform is introduced, designed for single-injection self-boosting vaccines, replacing multiple doses. Over 20 polyanhydride polymers are synthesized and screened, ultimately down selecting to 6 for in vitro studies, and 2 for in vivo studies. Using diphtheria toxoid (DT) as a model antigen, programmed pulsatile release with a narrow window is demonstrated, ideal for self-boosting immunization. The platform effectively protects the pH-sensitive antigen before release, achieving recovery rate of 39.7% to 89.7%. The system's tunability is further enhanced by machine learning algorithms, which accurately predict release profiles, confirmed through experimental validation. In vivo studies in a mouse model reveals that the platform induces DT-specific antibody responses comparable to those generated by traditional multi-dose regimens. Collectively, these findings highlight the potential of this platform to deliver various vaccines, offering a potentially promising solution to the global challenge of under-immunization.

Keywords: antigen stability; microparticles; protein encapsulation; release kinetics; single‐administration vaccines.

Figure 1

Design and material selection for polyanhydride core‐shell MPs. a) A single‐injection vaccine platform is designed to deliver a prime bolus dose along with a group of SEAL core‐shell MPs, where the bolus dose is administered immediately, and the booster dose is released at a predetermined time. Each SEAL core‐shell MP consists of three main components: a cap, a base, and encapsulated cargo. b,c) Chemical structures of two well‐studied aromatic polyanhydride polymers, p(o‐CPPr) (b) and p(p‐CPH) (c). d) An aromatic polyanhydride library was created by diversifying hydroxybenzoic acid derivatives and dibromides via synthesis routes, with additional variation in monomer feed ratios, as shown in the generalized structure scheme.

Figure 2

Material screening, fabrication performance, and microscopic characterization of polyanhydrides for SEAL core‐shell MPs. a) The SEAL core‐shell MPs were fabricated through a multi‐step process involving polymer film pressing, base and cap pressing, cargo filling, MP sealing, and MP singulation. b) An inverted funnel approach was employed to screen 23 initial polyanhydride compositions, narrowing down to 6 compositions that demonstrated optimal fabrication performance. c,d,e) Representative high‐resolution light microscopy images showing an array of empty bases pressed from polyanhydride polymer film (c), bases filled with DT antigen, excipients, and a dye (d), and sealed MPs (e). f,g,h) Representative scanning electron microscopy (SEM) images of empty bases (f), filled bases (g), and sealed MPs (h).

Figure 3

DT antigen in vitro release profiles of polyanhydride core‐shell MPs. Cumulative release of DT antigen as determined by ELISA in an in vitro environment for a) p(o‐CPPr:p‐CPPe, 40:60), b) p(o‐CPPr:p‐CPPe, 30:70), c) p(o‐CPPr:p‐CPPe, 20:80), d) p(o‐CPPe:p‐CPPr, 60:40), e) p(o‐CPPe:p‐CPPr, 70:30), and f) p(o‐CPPe:p‐CPPr, 80:20).

Figure 4

Machine learning modeling of polyanhydride core‐shell MP profiles. a) Four features related to material selection and fabrication were used as input to construct a machine learning model, with four antigen release profile features used as output. b) Machine learning‐predicted results for the 50% and 90% release time points were compared with experimentally obtained values, with predictions falling within one standard deviation of the experimental results.

Figure 5

DT titer response of the polyanhydride core‐shell MP platform. a) Four groups were evaluated to compare in vivo delivery of DT antigen via conventional bolus injection and polyanhydride MP‐based single injection in the subcutaneous region. All groups received a prime bolus dose at the start of the study, with the booster dose designed for release at the 2‐week time point. b) ELISA results for DT‐specific titers showed that the two selected polyanhydride core‐shell SEAL MPs generated immune responses non‐inferior to the two‐bolus injection group and significantly higher than the single‐bolus injection group. c) Area under the curve (AUC) analysis of DT titer curves indicated that both polyanhydride MP groups achieved superior titer responses compared to the soluble injection control groups. d) Peak DT titer results also demonstrated that both polyanhydride MP groups outperformed the soluble groups. The data are presented as mean ± s.d. Statistical significance was evaluated using two‐tailed Student's t‐test. P ≤ 0.05 is statistically significant, with *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 and ****P ≤ 0.0001.