Every entry summarises what the study set out to test, what it found and why it changes how we design delivery systems. Browse research and reviews or search the collection.
A review of blood–brain barrier transport mechanisms and emerging lipid nanoparticle strategies for carrying nucleic acid and other complex therapies into the central nervous system.
This review surveys lipid nanoparticle composition, ionizable lipid synthesis and strategies to target delivery of fragile RNA therapeutics while addressing tissue selectivity and immune activation.
Ca Brimacombe, Ja Kulkarni, Mhy Cheng, K An, D Witzigmann, Pr Cullis
A review of the physical principles and development history behind siRNA lipid nanoparticles for liver gene silencing and mRNA lipid nanoparticles for protein expression.
Yue Wang, Shi-Kun Zhou, Yan Wang, Zi-Dong Lu, Yue Zhang, Cong-Fei Xu, Jun Wang
PD-1/PD-L1 blockade therapy is successful but often yields poor benefits due to insufficient T-cell infiltration and low intratumoral concentrations of PD-1/PD-L1 inhibitors. While strategies exist to increase either T-cell recruitment or inhibitor delivery, none actively recruit T cells while achieving tumor-specific delivery of PD-L1 inhibitors to specifically eliminate inhibition of tumor-infiltrating T cells. A strategy that addresses both. ### Nanoparticle Characterization | Parameter | Value | |---------------|-----------| | Hydrodynamic diameter | 107.2 nm | | PDI | ~0.2 | | Zeta potential | +15.3 mV | | Stability in 10% FBS | ≥5 days (size/PDI stable).
Bispecific T-cell engagers (BiTEs) are promising but require large amounts of purified protein, have high manufacturing costs, poor in vivo stability, and short serum half-lives. mRNA delivery could enable continuous endogenous production of BiTEs, but efficient and safe delivery systems are needed. The study developed a novel ionizable lipid nanoparticle (LNP) for mRNA encoding B7H3×CD3 BiTE to achieve prolonged half-life and potent antitumor. LNP@GFP-mRNA showed transfection efficiency comparable to Lipofectamine 8000 in 293T, AML12, and LO2 cells; serum presence did not affect transfection. - After IV LNP@Luc-mRNA, strongest luciferase signal was in liver.
Xuexiang Han, Ningqiang Gong, Lulu Xue, Margaret M. Billingsley, Rakan El-Mayta, Sarah J. Shepherd, Mohamad-Gabriel Alameh, Drew Weissman, Michael J. Mitchell
Liver fibrosis has no approved antifibrotic therapies. Activated hepatic stellate cells (HSCs) drive fibrosis by producing collagen, and HSP47 is a key collagen chaperone in these cells. Targeted delivery of siRNA to activated HSCs remains challenging, so a ligand-tethered LNP platform was developed to deliver RNA selectively to activated fibroblasts/HSCs. Lead AA-T3A-C12 LNP achieved >80% GFP knockdown at 50 nM in activated 3T3-GFP fibroblasts; knockdown significantly reduced by haloperidol (p = 0.009), confirming sigma receptor dependence. - In fibroblast/hepatocyte.
Sydney R. Shannon, Elena Ben-Akiva, Jordan J. Green
Gene therapies are transforming treatment for many diseases, but clinical efficacy and safety depend on both the delivery material and the cargo. Non-viral delivery to immune cells remains especially challenging, and no clinical translation breakthrough has yet been achieved for non-viral gene editing. This mini-review addresses that gap by surveying biomaterial-based delivery to immune cells, CRISPR/Cas9 cargo options, and how the two fields can be integrated for cancer immunotherapy. --- - PBAE nanoparticles with anti-CD3ε Fab2, MTAS, and NLS delivered DNA encoding leukemia-specific CARs to T cells at 34% efficiency *in vivo*, programming functional antigen recognition and anti-tumor effects. - PBAE mRNA CAR nanoparticles (anti-CD8, PGA-coated) transiently transfected T cells at 10% efficiency *in vivo*. - CART polymers: ~80% mRNA transfection in Jurkat T cells *in vitro*, but only ~1.5% *in vivo*; mixed hydrophobic blocks outperforme
CAR-T cell therapy has shown impressive success in hematological malignancies, but systemic toxicity (CRS, ICANS, on-target/off-tumor effects) and the complex, costly, individualized manufacturing process of autologous CAR-T cells hinder broader application. Universal allogeneic CAR-T cells have encountered safety concerns, with FDA halting some clinical trials. There is an urgent need for new strategies to overcome these barriers. --- - In vivo CAR-T induction with PBAE nanoparticles: Matthias Stephan's team achieved stable and transient expression of CD19-specific CAR in T cells via CAR-DNA and CAR-mRNA nanoparticles, respectively. Antitumor efficacy comparable to conventional lab-manufactured CAR-T cells without systemic toxicity. - Lentiviral in vivo CAR-T: Buchholz and colleagues induced in situ CAR-T cells in NSG mice with antitumor activity, but observed CRS and unexpected CAR-positive NK and NKT cells due to non-specific lentivi
Ann E. Metzloff, Margaret M. Billingsley, Michael J. Mitchell
mRNA vaccines have entered global use, but delivering mRNA to T cells in vivo remains challenging because T cells are non-phagocytic, reside largely in lymphoid tissues, and intravenously administered lipid nanoparticles (LNPs) tend to accumulate in the liver. There is a need for precise, scalable strategies to target mRNA to disease-specific T cells rather than all T cells, to avoid off-target inflammation and maintain self-tolerance. --- - UV-exchanged pMHCI APNs performed comparably to conventionally refolded pMHCI APNs for targeting and delivering mRNA to antigen-specific cytotoxic T cells in three mouse models. - Simultaneous targeting of three antigen-specific cytotoxic T cell populations was achieved by injecting a mixture of three UV-exchanged APNs, each carrying a different influenza A antigenic peptide. - mRNA delivery to the three antigen-specific cytotoxic T cell populations occurred at significantly higher rates compared wi
Joel G. Rurik, István Tombácz, Amir Yadegari, Pedro O. Méndez Fernández, Swapnil V. Shewale, Li Li, Toru Kimura, Ousamah Younoss Soliman, Tyler E. Papp, Ying K. Tam, Barbara L. Mui, Steven M. Albelda, Ellen Puré, Carl H. June, Haig Aghajanian, Drew Weissman, Hamideh Parhiz, Jonathan A. Epstein
Fibrosis affects millions of people with cardiac disease and contributes to heart failure. While adoptive transfer of CAR T cells targeting fibroblast activation protein (FAP) has shown promise in reducing cardiac fibrosis, conventional CAR T cells persist for months to years and could cause chronic off-target toxicity by continuously attacking fibroblasts throughout the body, impairing wound healing.[reference:0][reference:1] A method to. ### LNP Characterization & In Vitro CAR Expression | Parameter | Result | |---------------|------------| | LNP hydrodynamic diameter | ~80 nm[reference:49] | | Polydispersity index | 0.02-0.06[reference:50] | | mRNA.
Hamideh Parhiz, Jacob S. Brenner, Priyal N. Patel, Tyler E. Papp, Hamna Shahnawaz, Qin Li, Ruiqi Shi, Marco E. Zamora, Amir Yadegari, Oscar A. Marcos-Contreras, Ambika Natesan, Norbert Pardi, Vladimir V. Shuvaev, Raisa Kiseleva, Jacob W. Myerson, Thomas Uhler, Rachel S. Riley, Xuexiang Han, Michael
Nucleoside-modified mRNA lipid nanoparticle (modmRNA-LNP) technology is clinically successful, but its behavior in pre-existing inflammatory or immune-challenged conditions is poorly characterized. This gap raises the risk of adverse effects when modmRNA-LNPs are administered to patients with ongoing inflammation. IE with LPS + modmRNA-LNP: Serum IL-6 increased 14-fold vs LPS alone and 94-fold vs modmRNA-LNP in naive mice. Liver MIP-2 increased 11-fold vs LPS alone and 52-fold vs modmRNA-LNP in naive mice. Liver MIP-2 mRNA.
Kelsey L. Swingle, Margaret M. Billingsley, Sourav K. Bose, Brandon White, Rohan Palanki, Apeksha Dave, Savan K. Patel, Ningqiang Gong, Alex G. Hamilton, Mohamad-Gabriel Alameh, Drew Weissman, William H. Peranteau, Michael J. Mitchell
Congenital protein deficiencies often begin irreversible pathology in utero, but postnatal treatments are limited. In utero mRNA delivery could enable protein replacement before disease onset, yet LNP stability in fetal amniotic fluid and how it affects mRNA delivery had not been previously investigated. LNP A12 was most stable in mouse amniotic fluid; A1 was least stable. A12 had significantly higher bound protein content than A1 (p < 0.0021). - Percent change in PDI inversely correlated with in vitro luciferase.
Yulia Eygieris, Mohit Gupta, Jeonghwan Kim, Gaurav Sahay
LNPs enabled the COVID-19 mRNA vaccines and the first siRNA-LNP drug, but there is still no one-size-fits-all LNP for every RNA therapeutic. A better chemistry-level understanding of LNP self-assembly, component roles, and whole-particle properties is needed to rationally design safer and more efficient RNA delivery systems. ## 3. Hypothesis / Central Thesis LNP performance is governed by the coordinated chemistry of ionizable lipids, sterols Ionizable lipids typically have tertiary amine headgroups and biodegradable ester linkers; cpKa ~9–10.5, LNP pKa ~6–7, and cLogD ~10–14 are associated with potency. - Cholesterol analogues can strongly.
Martijn J.W. Evers, Wenjuan Du, Qiangbing Yang, Sander A.A. Kooijmans, Aryan Vink, Mies Van Steenbergen, Pieter Vader, Saskia C.A. De Jager, Sabine A. Fuchs, Enrico Mastrobattista, Joost P.G. Sluijter, Zhiyong Lei, Raymond Schiffelers
Modified mRNA (modRNA) is a promising cardiac regeneration therapeutic, but naked mRNA is large, negatively charged, and rapidly degraded. It is unknown whether systemically administered lipid nanoparticles (LNPs) can functionally deliver mRNA to ischemic/damaged myocardium after myocardial infarction. LNPs were <100 nm with PDI <0.2 and 95–99% mRNA encapsulation. - After ischemia-reperfusion, fluorescent LNPs accumulated in the infarcted area of the heart at 4 h and 24 h, but not in sham or control hearts. -.
Annabelle Biscans, Socheata Ly, Nicholas Mchugh, David A. Cooper, Anastasia Khvorova
Lipid-conjugated siRNAs can reach extrahepatic tissues, but silencing efficacy remains lower than in liver largely because only ~1–2% of internalized siRNA escapes endosomes into the cytoplasm. Ionizable lipids enhance endosomal escape in lipid nanoparticles (LNPs), but direct covalent conjugation of an ionizable lipid to siRNA had not been investigated. DLin-MC3-DMA conjugation retained RISC activity in vitro: IC50 values were 322 pM (unconjugated), 365 pM (cholesterol), and 481 pM (DLin-MC3-DMA). - Endosomal escape was enhanced: DLin-MC3-DMA-siRNA increased Gal8+.
Savan K. Patel, Margaret M. Billingsley, Caitlin Frazee, Xuexiang Han, Kelsey L. Swingle, Jingya Qin, Mohamad-Gabriel Alameh, Karin Wang, Drew Weissman, Michael J. Mitchell
mRNA delivery to T cells could enable ex vivo and in vivo T cell engineering, but LNPs still face poor extrahepatic delivery, endosomal recycling, and limited T cell transfection. Cholesterol analogs such as hydroxycholesterols may alter NPC1 recognition and endosomal trafficking, providing a route to improve T cell mRNA delivery. In primary human T cells, A1-25 and A1-50 improved mRNA delivery by 1.8-fold and 2.0-fold, respectively, vs S2. - In Jurkat cells, A1-25, A1-50, and B1-50 increased luciferase expression 2.1-fold, 1.9-fold, and.
Azadeh Kheirolomoom, Aris J. Kare, Elizabeth S. Ingham, Ramasamy Paulmurugan, Elise R. Robinson, Mo Baikoghli, Mohammed Inayathullah, Jai W. Seo, James Wang, Brett Z. Fite, Bo Wu, Spencer K. Tumble, Marina N. Raie, R. Holland Cheng, Lisa Nichols, Alexander D. Borowsky, Katherine W. Ferrara
Ex vivo T-cell engineering is effective but complex, costly, and difficult to scale. A method to transfect T cells directly in situ could simplify T-cell immunotherapy, but T-cell targeting may also trigger activation, depletion, cytokine release, and phenotypic changes that must be understood. In vitro: >80% of Jurkat cells expressed mCherry with 16% aCD3-LNPs; ~97% became CD69⁺; aCD3 coating caused T-cell depletion and CD3e internalization. - In vivo 24 h: aCD3-LNPs transfected ~2–4% of splenic T cells and.
Hanwen Zhang, Xuexiang Han, Mohamad-Gabriel Alameh, Sarah J. Shepherd, Marshall S. Padilla, Lulu Xue, Kamila Butowska, Drew Weissman, Michael J. Mitchell
LNPs can trigger innate immune responses and inflammation, which can suppress mRNA translation and cause adverse effects. There is a need for LNP formulations that reduce LNP-induced inflammation while maintaining or improving mRNA delivery. C9D1 and C10D0 had similar size, PDI, and >90% mRNA encapsulation; Dex substitution did not impair in vitro transfection or increase cytotoxicity. - Higher Dex substitution (C7D3, C5D5, C3D7, C0D10) reduced.
Yihua Pei, Yanjie Bao, Cristiano Sacchetti, Juthamart Brady, Kyra Gillard, Hailong Yu, Scott Roberts, Kumar Rajappan, Steven P. Tanis, Carlos G. Perez-Garcia, Padmanabh Chivukula, Priya P. Karmali
Systemic LNP delivery predominantly targets the liver, while extrahepatic delivery—especially to lung airway epithelium for diseases such as cystic fibrosis—remains challenging. DOTAP is a common cationic lipid for lung gene delivery but is racemic, pseudo-glyceryl, and slowly biodegradable. Novel readily hydrolysable DOTAP analogues are needed for safer and more effective inhaled/airway mRNA delivery. DOTAP+ and L1–L4 LNPs: <100 nm, >95% mRNA encapsulation, high mRNA purity; DOTAP− had ~71% encapsulation. - Freeze-thaw: all formulations retained size, PDI, and encapsulation within ~10% of initial values. -.
mRNA therapeutics have fueled hope to combat incurable diseases, but insufficient understanding of mRNA instability, immunogenicity, and delivery has impeded progress. The review argues that mRNA-based drugs—especially after the COVID-19 vaccine success—can become powerful, versatile tools, and that mRNA optimization and delivery systems are the key bottlenecks to solve. COVID-19 mRNA vaccines: ~90% effectiveness for full vaccination, 80% for partial; BNT162b2 95% efficacy in phase III; mRNA-1273 94.1% efficacy; Omicron-neutralizing antibodies largely undetectable in most recipients,.
Immunotherapy has improved outcomes in cancer and infectious disease, but off-target effects, systemic toxicities, and variable efficacy remain limiting. Nanoscale engineering offers ways to manipulate immune cell functions—enhancing immunity against cancers and pathogens, controlling the site of immune response, and promoting tolerance—by tuning nanoparticle size, shape, charge, and surface chemistry. CL4H6 lipid nanoparticles silenced STAT3 and HIF-1α in tumor-associated macrophages by 37% and 48%, respectively, increased M1 markers, reduced tumor size, and altered cytokine profiles in a B16-F10 model. -.
Randall A. Meyer, Sarah Y. Neshat, Jordan J. Green, Jose Luis Santos, And Anthony D. Tuesca
mRNA gene therapy has broad therapeutic potential (protein replacement, vaccines, regenerative medicine, oncology), but clinical translation is limited by inadequate understanding of how to target specific organs or cell types for protein expression. Reports on material properties and administration routes are disparate, preventing a global understanding of how these factors contribute to organ targeting for mRNA delivery. Liver targeting: LNPs intrinsically target liver via ApoE-mediated uptake through LDLR. Optimized ionizable lipids increased liver luciferase expression 18-fold. Oxidized cholesterol variants improved protein expression.
Mingyuan Li, Yuan Li, Shiqin Li, Lin Jia, Haomeng Wang, Meng Li, Jie Deng, Ali Zhu, Liqiao Ma, Weihong Li, Peng Yu, And Tao Zhu
The COVID-19 pandemic has greatly accelerated the application of mRNA technology, demonstrating its unique advantages over traditional biopharmaceutical and vaccine technology. However, mRNA instability in human physiological environments and inefficient in vivo delivery remain major barriers. mRNA chemical modifications and nano delivery systems are two key factors for in vivo applications. There is a need to summarize challenges for clinical. COVID-19 mRNA vaccine efficacy: BNT162b2 (Pfizer/BioNTech) 95% effective at 30 μg dose; mRNA-1273 (Moderna) 94.5% effective at 100 μg dose; both provide immunogenicity for at least 119 days after first vaccination. -.
Viral vectors dominate gene therapy but suffer from immunogenicity, insertional mutagenesis risk, cargo-size limits, and manufacturing challenges. Non-viral nanoparticles are safer, more economical, and easier to scale, but their clinical translation has been limited by inefficient delivery, especially for DNA and gene-editing cargoes that must reach the nucleus. There is a need to review non-viral nanomaterial design, delivery barriers, and. Viral vector dominance: Approximately 70% of gene therapy clinical trials to date have used viruses. - LNP gene editing in liver: A single administration of CRISPR-Cas9 LNPs achieved >97% reduction in target protein.
