Chapter Four · failure evidence
What Vaccine Delivery & Formulation got wrong, from 45 dissertations
The records detail formulation and delivery obstacles encountered during the design of vaccines and nanocarriers, including particle instability, poor encapsulation, and adverse immune interference. Many investigations also reveal that non-standard administration routes, uncoordinated delivery kinetics, and incompatible adjuvant combinations failed to trigger target protective responses. These records come from PhD theses at 16 institutions, 2021 to 2026. Each links to its thesis. They were extracted by language models reading the full text, so treat each as a lead to read, not a verdict.
Lipid and polymer carrier composition defects impair nucleic acid encapsulation and transfection
Formulation parameter choices such as low lipid to cargo ratios, suboptimal ionizable lipid chemistries, and high N to P ratios led to poor encapsulation, excessive cytotoxicity, and diminished expression. Alternative systems such as solid lipid matrices, DOPE lipoplexes, and poly beta-amino esters suffered from particle aggregation, premature cargo release, or complete failure to elicit humoral responses.
Tried and failed
nanoparticle formulation at low lipid to cargo ratio applied to lipid nanoparticles encapsulating nucleic acids. Outcome: worse than baseline. Reason: insufficient lipid mass ratio resulted in poor cargo encapsulation efficiency
Tried and failed
incorporating cationic cholesterol in lipid nanoparticles applied to intratumoral mRNA delivery. Outcome: worse than baseline. Reason: negatively enriched compared to neutral cholesterol formulations during in vivo screening
Translational Design of Lipid Nanoparticles (LNPs) to Deliver mRNA Therapies to Solid Tumors and to the Lungs · Georgia Tech
Tried and failed
permanently charged cationic lipid substitution in nanoparticles applied to RNA delivery formulations. Outcome: worse than baseline. Reason: produced significantly lower transfection expression compared to degradable ionizable lipid benchmarks
Development of Chemically-Defined Platform Materials for Localized Delivery of RNA Therapeutics · MIT
Considered and rejected
Considered and rejected: Rejected C3 LNP formulation at 20:1 N:P ratio despite higher in vitro delivery due to enhanced cytotoxicity and excess free ionizable lipid.
IONIZABLE LIPID NANOPARTICLES FOR IN UTERO GENE EDITING OF CONGENITAL DISEASE · Penn
Considered and rejected
Considered and rejected: Rejected NLC/GM and NLC/SI solid lipid formulations due to particle instability, aggregation, and unacceptable vehicle cytotoxicity.
In vitro functionality and toxicity of dacarbazine delivery nanosystem for melanoma. · Cranfield
Lost to a baseline
All PBAE formulations failed to elicit detectable N or S1 serum IgG antibody responses in vivo, whereas gene gun (gold particles) elicited high antibody titers.
New variants of poly(beta amino esters) to enhance delivery of nucleic acid medicines · University of Nottingham Repository
Considered and rejected
Considered and rejected: Rejected using mRNA Spleen SORT formulations (containing 18:1 PA) for pDNA delivery because they exhibited poor pDNA encapsulation (24%) and misdirected tropism to the lungs
Engineering Lipid Nanoparticle-Mediated Delivery of Nucleic Acids to Lymphoid Organs and Immune Cells · DSpace at UTSWMED
Considered and rejected
Considered and rejected: Rejected DOPE-dominated lipoplex formulations for in vivo gene delivery due to hexagonal phase-induced premature cargo release.
Advances in lipopolyplexes as efficient non-viral carrier for nucleic acid delivery Fortschritte bei Lipopolyplexen als effiziente nicht-virale Träger für die Übertragung von Nukleinsäuren · open_UMR Marburg DSpace 10.0
Adjuvant co-formulation incompatibility, adverse toxicity, and immune dampening
Combining adjuvants such as CpG with alum or adding TLR agonists to mRNA lipid nanoparticles unexpectedly suppressed cellular responses, accelerated degradation during storage, or reduced antibody titers. Furthermore, traditional adjuvants like alum or SAS generated severe local granulomas, systemic toxicity, or suppression of mucosal IgA when applied via non-standard routes.
Tried and failed
co-delivering TLR7 agonist adjuvant with mRNA vaccine applied to lipid nanoparticle mRNA vaccine formulation. Outcome: worse than baseline. Reason: failed to increase antibody titers and actively suppressed CD8+ T cell immune responses
Next-Generation Lipid Nanoparticle Formulations for Non-Liver Delivery of Nucleic Acid-Based Therapies and Vaccines · Georgia Tech
Tried and failed
alum adjuvant supplementation applied to mucosal and systemic antibody induction. Outcome: worse than baseline. Reason: alum dampened mucosal IgA response, impairing protection
The development of humoral breadth and protection against influenza · Harvard
Tried and failed
combining TLR agonist with alum adjuvant applied to protein subunit nanoparticle vaccine immunization. Outcome: worse than baseline. Reason: CpG addition unexpectedly reduced target-specific binding and neutralizing antibody titers compared to alum alone
RBD-HBsAg conjugated virus-like particle vaccine protects rhesus macaques from SARS-CoV-2 challenge · Harvard
Lost to a baseline
RBD-VLP formulated with alum + CpG 1018 elicited lower binding antibody titers and ~30% less human ACE2 binding than the formulation with alum alone after storage.
Considered and rejected
Considered and rejected: Alum salts rejected as adjuvants due to causing severe inflammation/granulomas on mucosal surfaces.
Development of a Nanoparticle Vaccine Delivery System with Polymeric Oral Adjuvants for Poultry · Virginia Tech
Considered and rejected
Considered and rejected: Rejected aluminum hydroxide adjuvant in transdermal/pulmonary formulations due to skin irritation, nodule formation, and incompatibility with spray-freeze drying
Considered and rejected
Considered and rejected: Replacing Sigma Adjuvant System (SAS) with aluminum hydroxide (alum) adjuvant in the longitudinal stepwise immunization study due to severe SAS-induced lethality and glomerulonephritis in VH1-2/LCL mice.
Vaccine responses in the context of impaired self-tolerance · Harvard
Administration route selection failures hinder localized or systemic immune responses
Selecting intramuscular injection failed to establish mucosal IgA or skin-resident T cell responses compared to targeted local administration. Conversely, inhalational and mucosal routes suffered from rapid clearance of uncoated particles, practical delivery barriers, and uneven lung distribution.
Tried and failed
uncoated protein nanoparticle delivery applied to intranasal mucosal vaccination. Outcome: worse than baseline. Reason: lack of mucoadhesive or mucodiffusive coatings led to rapid clearance and poor long-term systemic humoral responses
Surface Engineering of Protein Nanoparticles for Intranasal Delivery · Georgia Tech
Tried and failed
intramuscular vaccination to induce mucosal antibody responses applied to respiratory viral mucosal immunity. Outcome: no signal. Reason: intramuscular delivery failed to boost or maintain long-term virus-specific mucosal IgA levels
Immediate and delayed immune and inflammatory responses to COVID-19 · Imperial
Lost to a baseline
Intranasal immunization with Flu-Q11 formulations produced less dramatic viral load reduction than subcutaneous immunization.
Designing Multi-Epitope Supramolecular Nanofiber Vaccines for Autoimmunity and Infectious Disease · DukeSpace
Lost to a baseline
Intramuscular immunization generated lower skin resident CD4+ T cell responses and reduced parasite burden control compared to intradermal vaccination upon L. major challenge.
Synthetic Dna Technology As A Tool To Generate Vaccine Immunity In The Skin · Penn
Considered and rejected
Considered and rejected: Intranasal route for QIV influenza vaccination was discarded because it did not improve T cell response compared to intraperitoneal application in the established mouse model.
Considered and rejected
Considered and rejected: Rejected intra-tracheal vaccination route for human translation due to procedural impracticality and uneven lung distribution compared to aerosol/intranasal delivery
The systemic-prime, mucosal-boost vaccine regimen for protection against respiratory viruses · Oxford
Considered and rejected
Considered and rejected: Aerosol route of BCG administration for practical field vaccination in calves (rejected in favor of s.c. administration due to aerosol inoculation being time-consuming and labor-intensive)
Functional and epigenetic aspects of BCG-induced trained immunity in bovine innate immune cell populations · Iowa State
Antigen presentation, targeting ligand, and multi-antigen co-delivery interference
Uncoated protein nanoparticles and unstable metal-chelating liposomes failed to trigger robust cellular immunity or retain antigenic peptides in biological media. Similarly, co-delivering multiple antigens or immunosuppressive drugs caused antigenic competition and blunted antibody titers relative to simpler benchmarks.
Tried and failed
immunization with soluble and uncoated protein nanoparticles applied to inducing cellular immune response. Outcome: no signal. Reason: antigen presentation without surface coating or adjuvant failed to stimulate cellular immunity over baseline
Development and Immunological Evaluation of Protein-Based Nanoparticle Vaccines · Georgia Tech
Lost to a baseline
scFv-conjugated LNP formulations failed to outperform monoclonal antibody (104D2 mAb) conjugated LNPs in delivery
Lost to a baseline
Nanoparticle vaccine without drugs (OVA-E no DRUG) yielded higher Ova-specific total IgG and IgG1 titers than the formulation with encapsulated AICAR and rapamycin (p = 0.003)
Considered and rejected
Considered and rejected: Rejected Ni-NTA and Co-NTA liposome formulations due to lack of stable binding of his-tagged peptides and antigens in biological media.
Developing a Novel Liposomal Adjuvant for Malaria Transmission-Blocking Vaccines · DSpace at SUNY Buffalo
Tried and failed
co-delivery of multiple antigens in nanoparticle formulations applied to dual-pathogen subunit vaccination. Outcome: worse than baseline. Reason: antigen competition or immunological interference reduced antigen-specific serum IgG titers relative to single-antigen formulations
Utilizing Combinatory Adjuvant-Loaded Chitosan-Derived Nanoparticles for a Joint SARS-CoV-2/Influenza Vaccine · Georgia Tech
Lost to a baseline
Conjugate vaccine OXY-CRM197 + Alum elicited higher IgG antibody titers than the lipid-PLGA hNP-based OXY-CRM197-hNP(Alum) formulation
Precision Adjuvant Design Enabling Tailored Nanoparticle Immunization Platforms for Oxycodone and Other Substance Abuse Vaccines · Virginia Tech
Physical destabilization, aggregation, and manufacturing processing failures
Chemical additives in copolymer nanogels and silica particles triggered colloidal aggregation across testing pH conditions and rapid clearance. Processing methods such as ambient humidity drying and rapid precursor gelation caused immediate nozzle clogging or irreversible destruction of viral infectivity.
Tried and failed
incorporating methyl methacrylate in emulsion copolymerization applied to pH-responsive cationic nanogels. Outcome: unstable. Reason: formulations aggregated across the testing pH range during colloidal stability measurements
Considered and rejected
Considered and rejected: Rejected direct syringe-loading of commercial Kineret into MAMC double emulsions due to emulsion destabilization caused by formulation excipients.
Cellular And Mechano-Active Material Approaches To Improve Disc Repair After Herniation · Penn
Considered and rejected
Considered and rejected: Aqueous phase CAT-SiNP formulation was replaced with reverse emulsion due to poor colloidal stability and rapid aggregation leading to RES clearance.
Hybrid Enzyme-Loaded Silica Nanoparticles for Potential Therapeutic Applications · DSpace at UTSWMED
Tried and failed
controlled ambient humidity drying of polymer matrices applied to solid-state viral vector formulations. Outcome: unstable. Reason: sorption-desorption cycling and water movement caused complete loss of viral infectivity despite returning to baseline moisture
Formulation, bioprocessing, and manufacturing of viral vectors in unique dosage forms · UT Austin
Tried and failed
dual-syringe delivery of fast-gelling hydrogel precursor applied to in situ hydrogel formulation. Reason: gelation occurred too rapidly inside the mixing nozzle, causing immediate clogging and preventing injection
Release kinetic mismatches and poor translation between in vitro and in vivo models
Asynchronous dosing of bolus adjuvants alongside escalating antigen schedules failed to coordinate germinal center responses, while slow localized depot release failed to trigger systemic immune activation. In addition, polyphenol complexes failed to retain oligonucleotide adjuvants, and high-throughput in vitro delivery screens showed no correlation with in vivo performance.
Tried and failed
in vitro screening to predict in vivo delivery applied to lipid nanoparticle nucleic acid formulations. Outcome: did not generalise. Reason: In vitro delivery efficacy showed no correlation with in vivo delivery across hundreds of formulation variants
UTILIZING HIGH-THROUGHPUT IN VIVO NANOPARTICLE SCREENS TO IMPROVE DRUG DELIVERY · Georgia Tech
Considered and rejected
Considered and rejected: Rejected (aPDL1-FN3)4-ELPdepot depot formulation in favor of (aPDL1-FN3)4-ELPsoluble because localized slow release failed to elicit required systemic immune activation
Developing Modular Protein Therapeutics as Alternatives to Monoclonal Antibodies for Cancer Immunotherapy · DukeSpace
Tried and failed
bolus adjuvant with escalating dose antigen schedule applied to vaccine immune response induction. Outcome: worse than baseline. Reason: asynchronous delivery failed to coordinate immune stimulation needed for germinal center and Tfh cell responses
Tried and failed
polyphenol-mediated depot formation for local retention applied to oligonucleotide adjuvant delivery. Reason: polyphenol complexation failed to retain oligonucleotide cargo at injection site unlike protein antigens
Engineering translational vaccine delivery systems with the polyphenol tannic acid · MIT
Left open by the authors
Problems the authors named and did not get to.
Left open
Screen alternative ionizable lipids to replace DOTAP in lipid nanoparticles for protein delivery without causing in vivo toxicity or clotting. Blocker: Requires wet lab microfluidic formulation, in vitro encapsulation assays, and in vivo toxicity/clotting testing.
Left open
Evaluate how RNA design and LNP formulations impact antigen and inflammatory kinetics in mRNA vaccines. Blocker: Requires a wet lab to formulate lipid nanoparticles (LNPs), design RNA sequences, and measure in vivo or in vitro kinetics.
Left open
Evaluate mRNA delivery by barcoded lipid nanoparticles to intratumoral immune cell subtypes in immunocompetent mice with spontaneous tumors. Blocker: Requires wet lab facilities, barcoded LNP formulations, and in vivo animal tumor models
Translational Design of Lipid Nanoparticles (LNPs) to Deliver mRNA Therapies to Solid Tumors and to the Lungs · Georgia Tech
Left open
Evaluate scFv and VHH ligand formats lacking Fc regions for targeting CD117 lipid nanoparticles. Blocker: Requires wet lab facilities, microfluidic formulation equipment, protein engineering, and in vivo/in vitro RNA delivery assays.
Left open
Test therapeutically relevant nucleic acid payloads formulated in lipid nanoparticles in an in vivo atherosclerosis mouse model. Blocker: Requires wet lab facilities, microfluidic LNP formulation, and in vivo animal disease models
Design and Formulation Optimization of Lipid Nanoparticles Carrying MRNA to the Heart and the Pancreas · Georgia Tech
Left open
Develop lipid nanoparticle-based mRNA delivery strategies for Cas9-retron genome editing systems. Blocker: Requires a wet lab for lipid nanoparticle formulation, mRNA synthesis, and in vitro/in vivo delivery assays
Discovery of novel systems for genome editing · UT Austin
Left open
Evaluate in vivo biodistribution and systemic cytokine cascade leakage of LbL nanoparticles across heterogeneous tumor phenotypes. Blocker: Requires wet-lab facilities, animal models, and physical nanoparticle formulations.
Left open
Load cGAMP-STINGΔTM vaccine component onto the LbL-coated microneedle and evaluate its therapeutic efficacy in mouse melanoma models. Blocker: Requires wet lab drug formulation, microneedle fabrication, and in vivo mouse melanoma experiments
Layer-by-layer coated microneedles for cancer immunotherapy · MIT
Left open
Develop microneedle patches with time-changing degrading patterns to indicate elapsed time post-administration. Blocker: Requires wet-lab polymer formulation, microneedle fabrication, and physical degradation testing
Microscale polymeric-based technologies for controlled vaccine delivery · MIT
Left open
Design de novo protein scaffolds or multi-component nanoparticle displays across sequential immunizations to minimize off-target anti-scaffold antibody responses. Blocker: Requires wet lab protein engineering, nanoparticle formulation, and in vivo immunization assays
Rationally designed immunogens enable immune focusing following SARS-CoV-2 spike imprinting · Harvard
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