Thermostable microneedle patches for mRNA vaccine delivery

Saturday, October 14, 2023

3:15 PM – 3:30 PM PDT

Location: 336 (Center, Level 3)

Presenting Author(s)

John L. Daristotle, PhD

Postdoctoral Associate
MIT
Cambridge, Massachusetts, United States

Co-Author(s)

Av

Aurélien vander Straeten

David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, United States
MS

Morteza Sarmadi

Graduate student
MIT, United States
MK

Maria Kanelli

David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, United States
LT

Lisa Tostanoski

Center for Virology and Vaccine Research, Beth Israel Deaconess Medical Center, Harvard Medical School, United States
JC

Joe Collins

David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, United States
AP

Apurva Pardeshi

David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, United States
Jooli Han, PhD (she/her/hers)

Postdoctoral Associate
David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Massachusetts, United States
DV

Dhruv Varshney

David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, United States
BE

Behnaz Eshaghi

David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, United States
JG

Johnny Garcia

Research Associate
MIT, United States
TF

Timothy Forster

Research Associate
MIT, United States
GL

Gary Li

David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, United States
NM

Nandita Menon

Research Associate
MIT, United States
SP

Sydney Pyon

Department of Chemical Engineering, Massachusetts Institute of Technology, United States
LZ

Linzixuan Zhang

Graduate student
MIT, United States
CJ

Catherine Jacob-Dolan

Center for Virology and Vaccine Research, Beth Israel Deaconess Medical Center, Harvard Medical School, United States
OP

Olivia Powers

Center for Virology and Vaccine Research, Beth Israel Deaconess Medical Center, Harvard Medical School, United States
KH

Kevin Hall

Center for Virology and Vaccine Research, Beth Israel Deaconess Medical Center, Harvard Medical School, United States
RF

Robert Farra

Independent Consultant, United States
DB

Dan Barouch

Center for Virology and Vaccine Research, Beth Israel Deaconess Medical Center, Harvard Medical School, United States
RL

Robert Langer

Professor
MIT, United States

Last Author(s)

Ana jaklenec, PhD

Principal Research Scientist, PI
MIT
cambridge, United States

Introduction:: Dissolvable microneedle patches (MNPs) make vaccines more accessible by allowing for self-administration, but they are challenging to manufacture and difficult to load with hydrophobic cargo, such as lipid nanoparticles (LNPs). To address these issues, we developed an automated, centrifuge-free microneedle fabrication process capable of “printing” vaccine ink containing mRNA encapsulated in LNPs. As an added benefit, the formulation that we designed to preserve the activity of the mRNA-LNPs after redissolution also enhances their long-term stability. We envision deploying this manufacturing technology in a self-contained, user-friendly MNP printer that is continuous, compact, fully automated, and enclosed to maintain sterility. The combination of thermostability and ease of application provided by mRNA microneedle patches has the potential to improve vaccine equity in low- and middle-income countries.

Materials and Methods:: The prototype of the microneedle vaccine printer was assembled using commercially available parts from Thorlabs and an Epson robotic arm. Vaccine inks were formulated from mRNA (TriLink Biotechnologies) encoding for firefly luciferase (FLuc) and SARS-CoV-2 S protein receptor binding domain (RBD), sequence available in GenBank (OP839194). LNPs were synthesized using a 38.4:12.3:47.4:1.9 ratio of Lipid 5 (Organix Inc.),1-2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE, Avanti), cholesterol (MilliporeSigma), and 1-2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt) (C14-PEG2000, Avanti). To prepare the LNPs, the lipids dissolved in ethanol were mixed with mRNA solution in pH 3 citrate buffer at a volume ratio of 3:1 (aqueous:organic). To create vaccine ink, concentrated mRNA-LNPs were mixed with polymer solutions at various ratios.

Initial loading studies were performed with vaccine inks incorporating protein, DNA, or mRNA-LNPs. In vitro screens were performed with FLuc mRNA-LNPs in HeLa cells. Microneedle dissolution studies were performed using ex vivo pig skin. Compression mechanical testing was performed using an Instron 5943 with a 500 N load in compression. mRNA-LNPs were characterized using cryogenic transmission electron microscopy (cryo-TEM), capillary gel electrophoresis, Concentration and encapsulation efficiency were obtained from lysed LNPs using the Quant-iT RiboGreen assay (Thermo Fisher Scientific).

All animal procedures were approved and performed under the guidelines of the Massachusetts Institute of Technology Committee on Animal Care (1019-061-22). In vivo studies were performed in C57BL/6 or BALB/c mice. In vivo imaging was used to determine FLuc expression. SARS-CoV-2 ELISA and a Mesoscale Diagnostics (MSD) electrochemiluminescence assay were used to determine serum anti-SARS-CoV-2 RBD responses.

Results, Conclusions, and Discussions:: To support automated, compact MNP manufacturing, we desired a microneedle mold-filling process that was centrifuge-free. While vacuum is a convenient option for driving the vaccine ink into the cavities of a microneedle mold, it causes bubbles to form as air moves through the vaccine ink. We circumvented this by taking advantage of polydimethylsiloxane’s (PDMS) unique air permeability properties. A vacuum application system was designed to apply vacuum selectively to the bottom of PDMS molds, removing air trapped beneath the vaccine ink that is deposited in the microneedle mold without producing any bubbles, filling 1 mm long microneedle molds completely in approximately 15 minutes. Complementary automated dispensing and drying stages were developed to support the printing of MNPs in trays containing 100 patches, with each tray taking approximately 48 h to process from beginning to end for two-step, tip-loaded printing. A two-step tip loading process was engineered for vaccine loading as high as 25% and, depending on the configuration, up to approximately 10000 MNPs per day. To create a vaccine ink capable of supporting high mRNA-LNP activity after re-dissolution, we screened dissolvable polymers commonly used for microneedle fabrication across a wide range of mRNA to polymer ratios from 1:1 to 1:10000. Following an in vitro screen, select polymers and geometries were tested for their ability to penetrate skin, achieving up to 40% dissolution for pyramidal microneedle geometry. This yielded a vaccine ink formulation suitable for in vivo microneedle testing. Firefly luciferase and SARS-CoV-2 S protein receptor binding domain mRNA were used to demonstrate protein expression and immunogenicity of the microneedle patches in comparison to soluble controls. This study demonstrates the successful in vivo administration of mRNA-LNPs using microneedle patches. A streamlined printing process was designed to facilitate scale-up of automated microneedle manufacturing. Together, the microneedle ink formulation and printer present new opportunities for the development of mRNA based therapeutics based on solid state, intradermal delivery. We project that an mRNA microneedle printer could be developed into a standalone device that could be operated without specialized training, increasing the reach of both microneedle patches and mRNA.

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