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.
Hannah J Vaughan, Camila G Zamboni, Kathryn M Luly, Ling Li, Kathleen L Gabrielson, Laboni F Hassan, Nicholas P Radant, Pranshu Bhardwaj, Florin M Selaru, Martin G Pomper, Jordan J Green
Hepatocellular carcinoma (HCC) has limited treatment options, with modest survival after systemic chemotherapy or transarterial chemoembolization (TACE). Gene therapies hold promise for treating HCC, but delivery remains a critical hurdle. While poly(beta-amino ester) (PBAE) nanoparticles have shown efficacy in transfecting HCC cells, their delivery via locoregional routes—specifically intra-arterial injection—had not been investigated, despite. ### In Vitro Transfection (N1-S1 Rat HCC Cells) | Parameter | Result | |---------------|------------| | Transfection efficiency (all PBAEs) | >50% GFP+ cells at various doses and w/w ratios | | Cell viability | >70% for.
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.
CAR T cell therapy is FDA/EMA-approved for B cell malignancies and multiple myeloma, but manufacturing relies on viral vectors—associated with safety concerns, high cost, and production challenges—or electroporation, which can be highly cytotoxic. Nanosystems may offer a safer, cost-effective alternative, but T cells are difficult to transfect, so rational design of lipid- and polymer-based carriers is urgently needed. Si-PDMAEMA-pDNA achieved 46% transfection in Jurkat cells and 44% in primary human T cells; pDNA/PEI-based systems reached 51% in Jurkat and 60% in primary human T cells. - pIAE + anti-CD3 + PiggyBac transposon.
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
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.
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.
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.
Sérgio Scalzo, Anderson K. Santos, Heloisa A. S. Ferreira, Pedro A. Costa, Pedro H. D. M. Prazeres, Natalia J. A. Da Silva, Lays C. Guimarães, Mário De Morais E Silva, Marco T. R. Rodrigues Alves, Celso T. R. Viana, Itamar C. G. Jesus, Alice P. Rodrigues, Alexander Birbrair, Anderson O. Lobo, Freder
Cardiomyocytes are hard-to-transfect cells, and gene therapy for cardiovascular disease is limited by insufficient delivery to cardiac tissue, nucleic acid degradation, and safety concerns with viral vectors. A safe, effective non-viral platform for pDNA delivery to cardiomyocytes is needed. LNP4 was the top performer: ~1.3-fold higher GFP fluorescence than the second-best LNP8 and ~10-fold higher than the lowest performer LNP6. - LNP4 achieved >60% transfection efficiency at day 2 and >80% at day 4 in.
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. -.
Sajid Iqbal, Alessandro Francisco Martins, Muhammad Sohail, Jingjing Zhao, Qi Deng, Muhan Li, Zhongxi Zhao
Polymeric gene delivery vectors require detailed physicochemical characterization to ensure reproducible formulation and efficient transfection. PBAE-447 is a promising biodegradable cationic polymer, but its simple nanoparticles can degrade or aggregate in aqueous/physiological conditions. This study characterizes PBAE-447 and evaluates PEGylation and lyophilization to improve stability and transfection. Polymer characterization: Mn 5,354; Mw 9,575; MP 4,934; PDI 1.7. Complete end-capping confirmed by ¹H NMR. - Solubility/buffering/swelling: PBAE-447 completely soluble at 10 µg/µL in NaAc; buffering capacity required.
mRNA is an attractive platform for cancer immunotherapy, but its instability, susceptibility to RNase degradation, and inefficient endosomal escape limit therapeutic applications. Lipid-based nanocarriers are non-viral vectors that can protect mRNA, improve transfection, and deliver it to intracellular compartments suitable for translation. Optimal pKa for ionizable lipids: 6.2–6.5 for intravenous mRNA delivery; 6.6–6.9 for intramuscular mRNA delivery. - Modifying lipid-to-mRNA ratio can shift lipoplex charge: anionic lipoplexes target spleen, cationic.
Rakan El-Mayta, Zijing Zhang, Alex G. Hamilton, Michael J. Mitchell
CAR T cell therapies have achieved clinical success but face limitations including restriction to autologous cell sources (to avoid HLA mismatch and GvHD), cumbersome manufacturing, high costs, and poor efficacy in solid tumours. NK cells have emerged as promising alternatives because they can kill cancer cells without HLA matching, can be derived from allogeneic sources, and have the potential to become "off-the-shelf" therapeutics. However, NK cells are resistant to genetic engineering and have limited proliferation and persistence, creating a need for improved delivery technologies to maximize their therapeutic potential. --- - CAR T cell dominance: ~96.4% of 520 active CAR-based cell therapy trials globally are CAR T-cell-based; CAR-NK trials represent a very small fraction. - Retroviral transduction efficiency: 60–90% for NK-92 cells; ~50% for primary NK cells. - Electroporation: mRNA electroporation achieved 80–90% transfection in
Qurrat Ul Ain, Estefania V.R. Campos, Ariel Huynh, Dominik Witzigmann, Sarah Hedtrich
Gene therapies are powerful tools to prevent, treat, and cure human diseases, but their application to skin diseases has received little attention despite the easy accessibility of skin and urgent medical need. The main obstacle is the unique barrier properties of human skin, which severely limit absorption of biomacromolecules and efficient delivery of nucleic acid payloads. This review discusses current approaches, successes, and failures of cutaneous gene therapy and provides guidance for next-generation concepts, with delivery strategies as the major translational obstacle. --- - Skin barrier: Human skin efficiently absorbs only small molecules (MW < ~800 Da) with moderate lipophilicity (logP 1–3), making biomacromolecule delivery challenging. - Clinical trial landscape: Of 1052 gene therapy trials, only 23 focus on skin conditions; ~70% of ongoing gene therapy trials are viral vector-based. - LNP optimization: Zwitterionic lipid ce
Gene therapy requires safe, precise, efficient, and cost-effective delivery. Viral vectors are efficient but face manufacturing, cost, and safety challenges; non-viral vectors are safer and more scalable but often have low delivery efficiency. Magnetic nanoparticles (MNPs) have emerged as a promising strategy to enhance viral and non-viral gene delivery under an external magnetic field, while also enabling magnetic targeting, MRI tracking, and magnetic hyperthermia. --- - Magnetofection speed: MNP-based gene carriers can be attracted to cell surfaces in a few minutes, compared with hours for regular transfection. - Transfection enhancement: EMF enhanced lipid/SPIO-mediated transfection sixfold. PEI/Si@MNP with Dox and P-gp shRNA showed magnetically targeted delivery under 0.42 T for 12 h. - In vivo neuronal transfection: NeuroMag delivered EYFP-channelrhodopsin to rat visual cortex neurons with 72.66% and 86.63% expression at 3 and 30 d
Arun R. K. Kumar, Yufeng Shou, Brian Chan, Krishna L., Andy Tay
This review examines how viral vectors, electroporation, microfluidic systems, nanoparticles and high-aspect-ratio nanostructures can improve the efficiency, viability and scalability of transfecting primary immune cells for applications such as CAR-T, CAR-NK and gene editing. It highlights the trade-offs between delivery efficiency, cell fitness, cargo flexibility, manufacturing complexity and clinical translation.
Dapeng Zhang, Elena N. Atochina-Vasserman, Devendra S. Maurya, Ning Huang, Qi Xiao, Nathan Ona, Matthew Liu, Hamna Shahnawaz, Houping Ni, Kyunghee Kim, Margaret M. Billingsley, Darrin J. Pochan, Michael J. Mitchell, Drew Weissman, Virgil Percec
Four-component lipid nanoparticles (LNPs) represent the leading non-viral vectors for mRNA delivery, but they have limitations including: (1) segregation of the neutral ionizable lipid as droplets in the LNP core, reducing transfection efficiency to ~1-2%; (2) the "PEG dilemma" where PEGylation increases circulation time but decreases cellular uptake and endosomal escape; and (3) instability at temperatures above -70°C. A one-component system. ### IAJD Libraries & DNP Formation | Parameter | Result | |---------------|------------| | Total IAJDs synthesized | 54 (6 libraries) | | In vitro active DNPs | 44/54 (81%) | | In vivo active DNPs | 31/54 (57%) | | DNPs.
Hamideh Parhiz, Jacob S. Brenner, Et Al. (full Author List Not Included In The Supplied Excerpt)
T cells are notoriously resistant to exogenous mRNA transfection, limiting in vivo T cell engineering for immunotherapy, CAR T generation, and HIV cure. A safe, specific, and efficient in vivo mRNA delivery platform for CD4+ T cells was needed. In vitro: anti-CD4/mRNA-LNPs bound specifically to human CD4+ T cells; ~80% of CD3+CD8− splenocytes became ZsGreen1+ with anti-CD4/Cre mRNA-LNPs, even at the lowest dose. - In vivo biodistribution: spleen uptake was.
Emily Harris, Devon Zimmerman, Eric Warga, Anil Bamezai, Jacob Elmer
Retroviral gene delivery for T cell therapies is expensive, semi-randomly integrating, and variable between patients. Non-viral alternatives are needed, but T cells are notoriously hard to transfect, especially primary T cells. Lipofectamine LTX was the best vehicle in Jurkat cells; optimized conditions in X-VIVO media yielded 63.0 ± 10.9% EGFP+ Jurkat cells vs. 23.1 ± 5.5% in RPMI. - Primary CD3+ T cells reached only 8.1 ± 0.8% EGFP+ under.
Tresa Lopez-Royo, Victor Sebastian, Laura Moreno-Martinez, Laura Uson, Cristina Yus, Teresa Alejo, Pilar Zaragoza, Rosario Osta, Manuel Arruebo, Raquel Manzano
CRISPR/Cas gene-editing components are often encoded on large plasmids (9–19 kb), which are difficult to encapsulate and transfect. Most PLGA nanoparticle (NP) gene-delivery studies use small nucleic acids (siRNA) or small plasmids (<6 kb), and the structural integrity and functional performance of large plasmids in PLGA NPs remain poorly characterized. There is a need to compare synthesis methods for encapsulating large plasmids without. pDNA stability: Batch ultrasound double emulsion completely degraded the 9.4 kb plasmid (standard) or left only 8.80% supercoiled (modified). Microfluidics-assisted double emulsion avoided degradation but produced only.
Rodgers, T., Muzzio, N., Watson, C., & Romero, G. (2021).
Gene-editing tools are large plasmid constructs that cannot spontaneously enter mammalian cells, and viral vectors are limited by cargo size, immunogenicity, and manufacturing challenges. PBAE nanoparticles are promising nonviral carriers, but unmodified PEG-PDHA nanoparticles release encapsulated plasmid DNA too rapidly for efficient intracellular delivery of large gene-editing cargo such as piggyBac transposon. Encapsulation and release: Optimal polymer/plasmid molar ratio = 0.36. Unmodified PEG-PDHA NPs released essentially all PBCAG within 2 h. Layer-by-layer NPs showed no detectable release within 24 h but transfection was.
