Adoptive cell therapies that genetically engineer immune cells with chimeric antigen receptors (CARs) have shown limited success against solid tumors due to the immunosuppressive tumor microenvironment (TME) and logistical challenges of ex vivo cell manipulation. Here, we introduce an immune cell-tropic lipid nanoparticle (LNP) platform that enables systemic delivery of CAR-encoding mRNA for the in vivo generation of panCAR immune cells. A single intravenous injection of this LNP system efficiently and transiently engineers T cells, macrophages, dendritic cells, and NK cells across the spleen, bone marrow, and peripheral blood, yielding a synergistic, multilineage antitumor response. Using human epidermal growth factor receptor 2 (HER2) as a CAR target, we demonstrate that repeated administration of LNP formulated with HER2-CAR mRNA (LNP-panCAR HER2 ) effectively inhibits tumor growth and prolongs overall survival in three murine syngeneic xenograft tumor models, without causing obvious side effects. Immune profiling of treated tumors reveals a remodeled TME with a shift toward an immunostimulatory phenotype, characterized by reduced M2-like macrophages and an increased presence of effector T cell subsets. Our findings establish LNP-panCAR as a broadly applicable, off-the-shelf in vivo CAR cell therapy platform for solid tumor immunotherapy and beyond.

Chimeric antigen receptor (CAR) immune cell therapy has revolutionized the treatment of hematologic malignancies, yet conventional ex vivo manufacturing remains costly, complex, and logistically demanding. Emerging in vivo engineering strategies, especially those based on nonviral nanoparticles (NPs), offer a transformative alternative by enabling direct programming of immune cells within the body. Recent advances in NPs-mediated delivery of CAR constructs, including mRNA, circular RNA (circRNA), self-amplifying RNA (saRNA), DNA and gene editing tools, have demonstrated promising potential for achieving transient, tunable and repeatable expression with reduced genotoxicity. Immune cell targeting has been achieved by multiple targeting strategies for T cells, NK cells and macrophages, allowing precise control of CAR expression and functional activation in disease-relevant contexts. For T cells specifically, antibody-functionalized NPs categorized by antibody types including CD3, CD4, CD5, CD7 and CD8, demonstrate differential engagement of T cell subsets and influence their activation profiles across various therapeutic contexts. This review highlights key advances in the development of immune cell targeting NPs, discusses evolving CAR architectures and quality control considerations, and outlines future directions for integrating in vivo CAR platforms into broader immunotherapeutic landscapes. Nonviral NPs are poised to redefine CAR therapy by enabling scalable, off-the-shelf immune interventions for cancer, autoimmune, and fibrotic diseases.

Targeted systemic mRNA delivery to extrahepatic tissues remains a formidable challenge, especially in the absence of targeting ligands on lipid nanoparticles. In this study, we introduce a series of dimethylamino-based ionizable lipidoids (DMA-Lipidoids) engineered for selective mRNA delivery to the spleen. Using a combinatorial approach, we synthesized 48 chemically distinct lipidoids by pairing four DMA-containing amine heads with 12 newly designed hyperbranched tails. Remarkably, lipidoids with tails H228, H226x, H246x, and H446x demonstrated exceptional spleen-targeting efficiency. To refine the lipidoid design, we constructed and screened a secondary library of 36 lipidoids containing DMA analogues. Through this two-round screening process, we identified lipidoids with both high potency and spleen selectivity. The lead candidate, DMA4-H228, achieved precise delivery of ovalbumin mRNA to antigen-presenting cells (APCs), driving interferon-α (IFN α) production and APC activation. This robust immune response effectively inhibited tumor growth. Overall, these innovative DMA-lipidoids demonstrate strong spleen-targeting capabilities, offering a transformative platform for mRNA vaccine development.

Intravenous mRNA vaccines show promise for stimulating immune cells but face challenges in targeting APCs due to high liver accumulation. To address this, we developed ROS-responsive PBAE nanoparticles, 93-TKA10-19, designed to target splenic APCs, enhance mRNA expression, and induce a Th1-skewed immune response, advancing mRNA vaccine efficacy.

Chimeric antigen receptor (CAR) T cell therapy has shown promise in treating hematologic malignancies, but it still faces challenges, including high costs, a time-consuming manufacturing process, and the necessity of lymphodepletion. Here, we generate circular RNAs (circRNAs) encoding CAR proteins, referred to as circRNACAR, which mediates remarkable tumor killing in human primary T cells. We demonstrate that circRNACAR, delivered with immunocyte-tropic lipid nanoparticles (LNPs), can form in vivo panCAR cells (CAR-T, CAR-natural killer [NK], and CAR-macrophage), significantly inhibit tumor growth, and reshape the tumor microenvironment in mice. Importantly, combining in vivo panCAR with circRNA-based vaccines encoding the corresponding HER2 antigens exhibits synergistically enhanced anti-tumor immunity. Notably, circRNACAR can in return boost the level of vaccination-elicited HER2-specific antibodies, mediating effective killing of tumor cells by macrophages. In combination with vaccination, in vivo panCAR demonstrates a synergistic enhancement of anti-tumor immunity across various mouse models, thereby establishing a framework for the synergistic in vivo panCAR-VAC immunotherapy.

The accelerated advent of mRNA-based therapeutics and vaccines, highlighted by the battle against SARS-CoV-2, underscores the urgency to refine lipid nanoparticles (LNPs) for efficient mRNA delivery. In this work, we introduce a novel series of ionizable lipids characterized by double ethanolamine head groups, significantly amplifying mRNA binding affinity. A succinct three-component formulation is subsequently delineated, obviating the conventional dependency on phospholipids inherent in traditional four-component LNPs. Intriguingly, this formulation enables particle formation under neutral pH conditions, a notable departure from the acidic milieu traditionally required, attributable to the enhanced nonionic interactions predominating in mRNA encapsulation. The resultant particles exhibit exceptional stability, superior mRNA encapsulation efficiency, and maintain robust delivery efficacy. When deployed as a vaccine platform, the formulation elicited pronounced humoral and T-cell immune responses, concurrently exhibiting a favorable toxicity profile with a reduced induction of pro-inflammatory cytokines such as IL-6. Our exploration suggests that by fine-tuning the non-electrostatic interactions between the ionizable lipid and mRNA, the dynamics of particle formation can be considerably divergent from the prevailing paradigms of mRNA-LNP formation, hinting at a broader horizon for lipid optimization within the realm of mRNA delivery systems.

Traditional Chinese herbal (TCH) medicines have emerged as a prospective and affordable method to treat various diseases with a broad range of biological activity; however, traditional preparations, like decoctions, are often associated with low bioavailability, thus resulting in limited efficacy against cancer. The drawbacks of active TCH components, including instability, poor permeability, high hydrophilicity or hydrophobicity, undesirable pharmacokinetic profiles, and off-target toxicity, also exist. Most TCH medicines are thus limited to a clinical alternative for the treatment of chronic diseases. A liposomal delivery system is the most common class of FDA-approved nanomedicines, which has improved pharmacokinetics, enhanced targetability, and reduced side effects. Therefore, we anticipate that liposomal delivery technology will help concentrate drugs inside tumors, and fully release the therapeutic potential and reduce the side effects of TCH medicines. The review provides a brief overview of several representative TCH components and related liposome delivery strategies for enhanced cancer therapy. Current challenges associated with liposomal targeting of TCH medicines are also discussed for interested researchers.

Significance The current application of messenger RNA (mRNA)-based technology has largely been confined to liver diseases because of the lack of a specific and efficient extrahepatic in vivo systemic mRNA delivery system. Here, we have developed a library of N-series lipid nanoparticles (LNPs) that could specifically regulate the protein composition of protein corona on the surface of LNPs, which allows specific delivery of mRNA to the lung. We further demonstrated that our lung-targeting LNP could effectively deliver mouse tuberous sclerosis complex 2 ( Tsc2 ) mRNA into TSC2-null cells and restore its function, resulting in enhanced control of tumor burden in a preclinical model of lymphangioleiomyomatosis, a destructive lung disease caused by loss-of-function mutations in the Tsc2 gene.

The targeted delivery of messenger RNA (mRNA) to desired organs remains a great challenge for in vivo applications of mRNA technology. For mRNA vaccines, the targeted delivery to the lymph node (LN) is predicted to reduce side effects and increase the immune response. In this study, we explored an endogenously LN-targeting lipid nanoparticle (LNP) without the modification of any active targeting ligands for developing an mRNA cancer vaccine. The LNP named 113-O12B showed increased and specific expression in the LN compared with LNP formulated with ALC-0315, a synthetic lipid used in the COVID-19 vaccine Comirnaty. The targeted delivery of mRNA to the LN increased the CD8 + T cell response to the encoded full-length ovalbumin (OVA) model antigen. As a result, the protective and therapeutic effect of the OVA-encoding mRNA vaccine on the OVA-antigen–bearing B16F10 melanoma model was also improved. Moreover, 113-O12B encapsulated with TRP-2 peptide (TRP2 180–188 )–encoding mRNA also exhibited excellent tumor inhibition, with the complete response of 40% in the regular B16F10 tumor model when combined with anti–programmed death-1 (PD-1) therapy, revealing broad application of 113-O12B from protein to peptide antigens. All the treated mice showed long-term immune memory, hindering the occurrence of tumor metastatic nodules in the lung in the rechallenging experiments that followed. The enhanced antitumor efficacy of the LN-targeting LNP system shows great potential as a universal platform for the next generation of mRNA vaccines.

With the emerging advances in utilizing nanocarriers for biomedical applications, a molecular-level understanding of the in vivo fate of nanocarriers is necessary. After administration into human fluids, nanocarriers can attract proteins onto their surfaces, forming an assembled adsorption layer called protein corona (PC). The formed PC can influence the physicochemical properties and subsequently determine nanocarriers' biological behaviors. Therefore, an in-depth understanding of the features and effects of the PC on the nanocarriers' surface is the first and most important step towards controlling their in vivo fate. This review introduces fundamental knowledge such as the definition, formation, composition, conformation, and characterization of the PC, emphasizing the in vivo environmental factors that control the PC formation. The effect of PC on the physicochemical properties and thus biological behaviors of nanocarriers was then presented and thoroughly discussed. Finally, we proposed the design strategies available for engineering PC onto nanocarriers to manipulate them with the desired surface properties and achieve the best biomedical outcomes.

Loss-of-function mutations in Angiopoietin-like 3 (Angptl3) are associated with lowered blood lipid levels, making Angptl3 an attractive therapeutic target for the treatment of human lipoprotein metabolism disorders. In this study, we developed a lipid nanoparticle delivery platform carrying Cas9 messenger RNA (mRNA) and guide RNA for CRISPR-Cas9-based genome editing of Angptl3 in vivo. This system mediated specific and efficient Angptl3 gene knockdown in the liver of wild-type C57BL/6 mice, resulting in profound reductions in serum ANGPTL3 protein, low density lipoprotein cholesterol, and triglyceride levels. Our delivery platform is significantly more efficient than the FDA-approved MC-3 LNP, the current gold standard. No evidence of off-target mutagenesis was detected at any of the nine top-predicted sites, and no evidence of toxicity was detected in the liver. Importantly, the therapeutic effect of genome editing was stable for at least 100 d after a single dose administration. This study highlights the potential of LNP-mediated delivery as a specific, effective, and safe platform for Cas9-based therapeutics.

CONSPECTUS: Since the U.S. Food and Drug Administration (FDA) granted emergency use authorization for two mRNA vaccines against SARS-CoV-2, mRNA-based technology has attracted broad attention from the scientific community to investors. When delivered intracellularly, mRNA has the ability to produce various therapeutic proteins, enabling the treatment of a variety of illnesses, including but not limited to infectious diseases, cancers, and genetic diseases. Accordingly, mRNA holds significant therapeutic potential and provides a promising means to target historically hard-to-treat diseases. Current clinical efforts harnessing mRNA-based technology are focused on vaccination, cancer immunotherapy, protein replacement therapy, and genome editing. The clinical translation of mRNA-based technology has been made possible by leveraging nanoparticle delivery methods. However, the application of mRNA for therapeutic purposes is still challenged by the need for specific, efficient, and safe delivery systems. This Account highlights key advances in designing and developing combinatorial synthetic lipid nanoparticles (LNPs) with distinct chemical structures and properties for in vitro and in vivo intracellular mRNA delivery. LNPs represent the most advanced nonviral nanoparticle delivery systems that have been extensively investigated for nucleic acid delivery. The aforementioned COVID-19 mRNA vaccines and one LNP-based small interfering RNA (siRNA) drug (ONPATTRO) have received clinical approval from the FDA, highlighting the success of synthetic ionizable lipids for in vivo nucleic acid delivery. In this Account, we first summarize the research efforts from our group on the development of bioreducible and biodegradable LNPs by leveraging the combinatorial chemistry strategy, such as the Michael addition reaction, which allows us to easily generate a large set of lipidoids with diverse chemical structures. Next, we discuss the utilization of a library screening strategy to identify optimal LNPs for targeted mRNA delivery and showcase the applications of the optimized LNPs in cell engineering and genome editing. Finally, we outline key challenges to the clinical translation of mRNA-based therapies and propose an outlook for future directions of the chemical design and optimization of LNPs to improve the safety and specificity of mRNA drugs. We hope this Account provides insight into the rational design of LNPs for facilitating the development of mRNA therapeutics, a transformative technology that promises to revolutionize future medicine.

Poly(ethylene glycol)-b-polypeptide block copolymer micelles, with excellent safety, are one of the most clinically studied nanocarriers for anticancer drug delivery. Notably, self-assembled nanosystems based on hydrophobic polypeptides showing typically a low drug loading and burst drug release are limited to preclinical studies. Here, we report that poly(ethylene glycol)-b-poly(alpha-aminopalmitic acid) (PEG-b-PAPA) block copolymer could be easily prepared with tailored M-n through ring-opening polymerization of alpha-aminopalmitic acid N-carboxyanhydride (APA-NCA). Interestingly, PEG-b-PAPA copolymers exhibited superb solubility in common organic solvents (including CHCl3, CH2C6 and THF), while stable nanomicelles were formed in phosphate buffer, with a small size of 59 nm and a low critical micelle concentration of 2.38 mg/L. These polylipopeptide micelles (Lipep-Ms) allowed facile loading of a potent anticancer drug, docetaxel (DTX), likely due to the existence of a strong interaction between the lipophilic drug and polylipopeptide in the core. Notably, cRGD-peptide-functionalized Lipep-Ms (cRGD-Lipep-Ms) were also obtained with similar biophysical characteristics. The in vitro studies showed efficient cellular uptake of DTX-loaded cRGD-Lipep-Ms by B16F10 cells and fast intracellular drug release due to the enzymatic degradation of PAPA blocks in endo/lysosome, leading to a pronounced anticancer effect (IC50 = 0.15 mu g DTX equiv/mL). The in vivo therapy studies showed that DTX-cRGD-Lipep-Ms exhibited superior tumor growth inhibition of B16F10 melanoma, improved survival rate, and little side effects as compared to free DTX. These polylipopeptide micelles appear as a promising and robust nanoplatform for anticancer drug delivery.

In situ vaccination is a promising strategy for cancer immunotherapy owing to its convenience and the ability to induce numerous tumor antigens. However, the advancement of in situ vaccination techniques has been hindered by low cross-presentation of tumor antigens and the immunosuppressive tumor microenvironment. To balance the safety and efficacy of in situ vaccination, we designed a lipidoid nanoparticle (LNP) to achieve simultaneously enhancing cross-presentation and STING activation. From combinatorial library screening, we identified 93-O17S-F, which promotes both the cross-presentation of tumor antigens and the intracellular delivery of cGAMP (STING agonist). Intratumor injection of 93-O17S-F/cGAMP in combination with pretreatment with doxorubicin exhibited excellent antitumor efficacy, with 35% of mice exhibiting total recovery from a primary B16F10 tumor and 71% of mice with a complete recovery from a subsequent challenge, indicating the induction of an immune memory against the tumor. This study provides a promising strategy for in situ cancer vaccination.

AbstractEngineering T lymphocytes is an emerging approach in a variety of biomedical applications. However, delivering large biologics to primary T lymphocytes directly in vivo is technically challenging due to the low transfection efficacy. Herein, we investigated a library of synthetic lipid‐like molecules (lipidoids) for their capability of delivering mRNA into primary T lymphocytes both ex vivo and in vivo. We initially screened a library with a large structural variety of lipidoids ex vivo and identified imidazole‐containing lipidoids that are particularly potent in T lymphocytes transfection. We further optimized lipidoid structures by constructing and screening a detailed lipidoid library containing imidazole or imidazole analogues to perform a structure–activity correlation analysis. Using the lead lipidoid as a delivery vehicle for Cre mRNA in vivo through intravenous injection, we achieved 8.2 % gene recombination in mouse T lymphocytes.

PLGA-based nanoparticles are the most studied for cancer therapy. Insufficient stability and sustained drug release, however, often lead to reduced targetability and antitumor efficacy in vivo. In this work, we report on cRGD-installed reduction-responsive cross-linked nanotherapeutics based on a star PLGA-lipoic acid conjugate (cRGD-sPLGA XNPs) for potent and targeted chemotherapy of B16F10 melanoma in mice. cRGD-sPLGA XNPs exhibited nearly quantitative encapsulation of doxorubicin (DOX), giving DOX-cRGD-sPLGA XNPs with 13.2 wt % DOX and a small size of 91.0 ± 0.6 nm. DOX-cRGD-sPLGA XNPs with a cRGD surface density of 48% exhibited the best cellular uptake in αvβ3 overexpressing B16F10 cells and delivered DOX into the cell nuclei after 6 h of incubation, in contrast to nontargeted DOX-sPLGA XNPs that delivered DOX mainly in the cytoplasm. Cell viability experiments showed that DOX-cRGD-sPLGA XNPs had about 2-fold better inhibitory activity in B16F10 cells than nontargeted DOX-sPLGA XNPs. Interestingly, DOX-cRGD-sPLGA XNPs achieved a great melanoma accumulation of 10.96% ID/g and significantly better suppression of B16F10 melanoma than DOX-sPLGA XNPs and Lipo-DOX. DOX-cRGD-sPLGA XNPs brought about marked improvement of the survival rate of B16F10 melanoma-bearing mice at 20 mg of DOX equiv/kg. Smart nanotherapeutics based on the star PLGA-lipoic acid conjugate have emerged as an appealing nanoplatform for targeted tumor therapy.

Proteolysis-targeting chimaera (PROTAC) technology is an emerging approach for achieving targeted degradation of a protein of interest (POI) intracellularly. However, the cell permeability of PROTACs is limited by their high molecular weight and total polar surface area. Moreover, the activation of the proteasome-mediated degradation by PROTAC requires the formation of a ternary (three-component) complex, composed of the PROTAC, the POIs, and E3-ligases related proteins (E3Ps). Simplifying the three-component system to two-component system could theoretically increase the efficiency of the formation of ternary complex and enhance the protein degradation efficiency. Herein, we demonstrate that pre-fusion of PROTACs with E3Ps (called "pre-fused PROTACs") before administration could transform the original PROTAC system to two-component system. After delivery by lipid nanoparticles, the degradation of POI by pre-fused PROTACs was dramatically increased and accelerated compared with standard PROTACs. Moreover, we demonstrated that this approach could be generalized to another hydrophobic tag (HyT) degrader by demonstrating the improved targeted protein degradation after pre-fusion the HyT degrader with heat shock protein 70 (HSP70).

Poly( d , l ‐lactide‐ co ‐glycolide) (PLGA)‐based nanotherapeutics are intensively employed for the treatment of various diseases, though they frequently suffer from stability, selectivity, and drug release problems. Here, a facile fabrication of coating‐sheddable CD44‐targeted PLGA nanoparticles using photoclick‐crosslinkable hyaluronic acid‐ graft ‐tetrazole (HA‐ g ‐Tet) as a surfactant followed by photoclick reaction with l ‐cystine dimethacrylamide (MA‐Cys‐MA) (X‐PHS@NPs) for targeted delivery of docetaxel (DTX) to breast tumors in vivo is reported. X‐PHS@NPs exhibit strong blue fluorescence and a size of 109 nm. DTX‐loaded X‐PHS@NPs (DTX‐X‐PHS@NPs), while stable at physiological conditions (pH 7.4, 37 °C), release about 76.6% DTX under 10 m glutathione conditions. DTX‐X‐PHS@NPs are potent toward CD44‐overexpressing MCF‐7 human breast cancer cells with a low IC 50 of 0.27 µg mL −1 . Interestingly, X‐PHS@NPs reveal excellent tumor penetration ability and DTX‐X‐PHS@NPs induce significantly more effective inhibition of MCF‐7 human breast tumors in nude mice than free DTX. These coating‐sheddable CD44‐targeted PLGA nanoparticles provide an interesting platform for cancer chemotherapy.

Stimuli-responsive polymer-drug conjugates (PDCs) provide promising approaches in anticancer treatment. Here, we report the synthesis and biological evaluation of PDCs made of the highly potent antimitotic agent monomethyl auristatin E conjugated to dendritic polyglycerol and dendritic polyglycerol sulfate via a reductively cleavable, self-immolative disulfide linker. Cell viability assays with the human cancer cell lines A549 (lung carcinoma) and HeLa (cervix carcinoma) revealed that the drug's cytotoxicity was reduced by conjugation to the polymers, with the sulfated conjugates being more effective than the non-sulfated ones. Kinetic studies using real-time cell analysis indicated a retarded drug release from the polymers, with a much later cytotoxic response after treatment with the non-sulfated conjugates due to less cellular uptake, as confirmed by flow cytometry and confocal laser scanning microscopy. In contrast, the non-cleavable dPGS-MMAE conjugate that was synthesized for comparison was not cytotoxic under the same conditions. Overall, reductively cleavable dPGS-SS-MMAE conjugates showed promising results in vitro and good tolerability in vivo. Further in vivo studies are planned.

Genome-editing technologies hold tremendous potential for treating genetic diseases. However, the efficient and safe delivery of genome-editing elements to the location of interest, and the achievement of specific targeted gene correction without off-target side effect remains a big challenge. In this perspective, we highlight recent developments and discuss the challenges of non-viral nanoparticles for the delivery of genome-editing tools. Finally, we will propose promising strategies to improve the delivery efficacy and advance the clinical translation of gene-editing technology.