Lipid nanoparticles (LNPs) have emerged as a groundbreaking delivery system for vaccines and therapeutic mRNAs. Ionizable lipids are the most pivotal component of LNPs due to their ability to electrostatically interact with mRNA, allowing its encapsulation while concurrently enabling its endosomal escape following cellular internalization. Thus, extensive research has been performed to optimize the ionizable lipid structure and to develop formulations that are well tolerated and allow efficient targeting of different organs that result in a high and sustained mRNA expression. However, one facet of the ionizable lipids’ structure has been mostly overlooked: the linker segment between the ionizable headgroup and their tails. Here, we screened a rationally designed library of ionizable lipids with different biodegradable linkers. We extensively characterized LNPs formulated using these ionizable lipids and elucidated how these minor structural changes in the ionizable lipids structure radically influenced the LNPs’ biodistribution in vivo. We showed how the use of amide and urea linkers can modulate the LNPs’ pKa, resulting in an improved specificity for lung transfection. Finally, we demonstrated how one of these lipids (lipid 35) that form LNPs entrapping a bacterial toxin [pseudomonas exotoxin A (mmPE)] in the form of an mRNA reduced tumor burden and significantly increased the survival of mice with lung metastasis.

Squamous cell carcinomas of the head and neck (HNSCC) originate in the upper aerodigestive tract, including the oral cavity, pharynx, and larynx. Current treatments of locally advanced HNSCC often lead to high treatment failure, and disease recurrence, resulting in poor survival rates. Advances in mRNA technologies and lipid nanoparticle (LNP) delivery systems led to several clinical trials involving LNP-CRISPR-Cas9 mRNA-based therapeutics. Despite these advances, achieving cell-type-specific extrahepatic mRNA delivery is still challenging. This study introduces a safe and effective intratumoral EGFR-targeted CRISPR-LNP delivery strategy for knocking out SOX2, which is a cancer-specific gene. To assess their therapeutic potential, it is shown that LNPs made from ionizable lipids with helper lipids co-encapsulating Cas9 mRNA and sgRNA targeting SOX2 (sgSOX2), lead to a ≈60% reduction in HNSCC cell viability in vitro. Next, using a xenograft HNSCC mouse model, targeted delivery of αEGFR- CRISPR-sgSOX2-LNPs to HNSCC cells resulted in a 90% inhibition of tumor growth and a 90% increase in survival for > 84 days, with tumor disappearance observed in 50% of the mice. These findings emphasize the potential of targeted mRNA-Cas9-LNPs in clinically accessible solid tumors, specifically in reaching tumor cells and inducing persistent therapeutic responses in tumors with high-recurrence rates like HNSCC.

Lipid Nanoparticles (LNPs) recently emerged as an invaluable RNA delivery platform. With many LNP-based therapeutics in the pre-clinical and clinical pipelines, there is extensive research dedicated to improving LNPs. These efforts focus mainly on the tolerability and transfectability of new ionizable lipids and RNAs, or modulating LNPs biodistribution with active targeting strategies. However, most formulations follow the well-established lipid proportions used in clinically approved products. Nevertheless, investigating the effects of LNPs composition on their biodistribution can expand the toolbox for particle design, leading to improved delivery strategies. Herein, a new LNPs (30-n-LNPs) formulation with increasing amounts of phospholipids is investigated as a possible mRNA delivery system for treating Inflammatory Bowel Diseases. Compared to LNPs with benchmark composition (b-LNPs), n-LNPs containing 30% distearoylphosphatidylcholine (DSPC) are well tolerated following intravenous administration and display natural targeting toward the inflamed colon in dextran sodium sulfate (DSS)-colitis bearing mice, while de-targeting clearing organs such as the liver and spleen. Using interleukin-10-encoding mRNA as therapeutic cargo, n-LNPs demonstrated a reduction of pathological burden in colitis-bearing mice. n-LNPs represent a starting point to further investigate the influence of LNPs composition on systemic biodistribution, ultimately opening new therapeutic modalities in different pathologies.

Locked nucleic acids are a third-generation antisense oligonucleotides with high binding affinity. A major limitation is the high dosages they require to achieve efficacy which may induce unwanted adverse effects. Here, we report the use of Lipid-based nanoparticles to deliver locked nucleic acids for treating intestinal inflammation in mice. Eight formulations with novel ionizable lipids were screened for stability and toxicity. Particles were loaded with splice-switcher sequence, enabling a precise assessment of potency in vitro. Three lead candidates were tested in vivo, demonstrating a 30-fold dose reduction compared to the unformulated oligonucleotides. The most potent formulation, encapsulating a sequence against Tumor necrosis factor alpha, was evaluated in a mouse model of colitis. Treatment reduced disease severity and inflammatory cytokines, with good safety. These findings support the use of lipid nanoparticles for the precise delivery of locked nucleic acids and highlight their promise for future therapies.

Ionotropic gelation is widely used to fabricate targeting nanoparticles (NPs) with polysaccharides, leveraging their recognition by specific lectins. Despite the fabrication scheme simply involves self-assembly of differently charged components in a straightforward manner, the identification of a potent combinatory formulation is usually limited by structural diversity in compound collections and trivial screen process, imposing crucial challenges for efficient formulation design and optimization. Herein, we report a diversity-oriented combinatory formulation screen scheme to identify potent gene delivery cargo in the context of precision cardiac therapy. Distinct categories of cationic compounds are tested to construct RNA delivery system with an ionic polysaccharide framework, utilizing a high-throughput microfluidics workstation coupled with streamlined NPs characterization system in an automatic, step-wise manner. Sequential computational aided interpretation provides insights in formulation optimization in a broader scenario, highlighting the usefulness of compound library diversity. As a result, the out-of-bag NPs, termed as GluCARDIA NPs, are utilized for loading therapeutic RNA to ameliorate cardiac reperfusion damages and promote the long-term prognosis. Overall, this work presents a generalizable formulation design strategy for polysaccharides, offering design principles for combinatory formulation screen and insights for efficient formulation identification and optimization.

Locked nucleic acids (LNAs) are a subtype of antisense oligonucleotides (ASOs) that are characterized by a bridge within the sugar moiety. LNAs owe their robustness to this chemical modification, which as the name suggests, locks it in one conformation. This perspective includes two components: a general overview on ASOs from one side and on delivery issues focusing on lipid nanoparticles (LNPs) on the other side. Throughout, a screening of the ongoing clinical trials involving ASOs is given, as well as a take on the versatility and challenges of using LNAs. Finally, we highlight the potential of LNPs as carriers for the successful delivery of LNAs.

Background and rationale: Cancer therapy have evolved remarkably over the past decade, providing new strategies to inhibit cancer cell growth using immune modulation, with or without gene therapy. Specifically, suicide gene therapies and immunotoxins have been investigated for the treatment of tumors by direct cancer cell cytotoxicity. Recent advances in mRNA delivery also demonstrated the potential of mRNA-based vaccines and immune-modulators for cancer therapeutics by utilizing nanocarriers for mRNA delivery. Methods: We designed a bacterial toxin-encoding modified mRNA, delivered by lipid nanoparticles into a B16-melanoma mouse model. Results: We showed that local administration of LNPs entrapping a modified mRNA that encodes for a bacterial toxin, induced significant anti-tumor effects and improved overall survival of treated mice. Conclusions: We propose mmRNA-loaded LNPs as a new class of anti-tumoral, toxin-based therapy.

Messenger RNA (mRNA) lipid nanoparticle (LNP) vaccines have emerged as an effective vaccination strategy. Although currently applied toward viral pathogens, data concerning the platform’s effectiveness against bacterial pathogens are limited. Here, we developed an effective mRNA-LNP vaccine against a lethal bacterial pathogen by optimizing mRNA payload guanine and cytosine content and antigen design. We designed a nucleoside-modified mRNA-LNP vaccine based on the bacterial F1 capsule antigen, a major protective component of Yersinia pestis, the etiological agent of plague. Plague is a rapidly deteriorating contagious disease that has killed millions of people during the history of humankind. Now, the disease is treated effectively with antibiotics; however, in the case of a multiple-antibiotic-resistant strain outbreak, alternative countermeasures are required. Our mRNA-LNP vaccine elicited humoral and cellular immunological responses in C57BL/6 mice and conferred rapid, full protection against lethal Y. pestis infection after a single dose. These data open avenues for urgently needed effective antibacterial vaccines.

The potential of microtubule-associated protein targets for cancer therapeutics remains largely unexplored due to the lack of target-specific agents. Here, we explored the therapeutic potential of targeting cytoskeleton-associated protein 5 (CKAP5), an important microtubule-associated protein, with CKAP5-targeting siRNAs encapsulated in lipid nanoparticles (LNPs). Our screening of 20 solid cancer cell lines demonstrated selective vulnerability of genetically unstable cancer cell lines in response to CKAP5 silencing. We identified a highly responsive chemo-resistant ovarian cancer cell line, in which CKAP5 silencing led to significant loss in EB1 dynamics during mitosis. Last, we demonstrated the therapeutic potential in an in vivo ovarian cancer model, showing 80% survival rate of siCKAP5 LNPs-treated animals. Together, our results highlight the importance of CKAP5 as a therapeutic target for genetically unstable ovarian cancer and warrants further investigation into its mechanistic aspects.

Ionizable lipid-based nanoparticles (LNPs) are the most advanced non-viral drug delivery systems for RNA therapeutics and vaccines. However, cell type-specific, extrahepatic mRNA delivery is still a major hurdle, hampering the development of novel therapeutic modalities. Herein, a novel ionizable lipid library is synthesized by modifying hydrophobic tail chains and linkers. Combined with other helper lipids and utilizing a microfluidic mixing approach, stable LNPs are formed. Using Luciferase-mRNA, mCherry mRNA, and Cre mRNA together with a TdTomato animal model, superior lipids forming LNPs for potent cell-type specific mRNA delivery are identified. In vitro assays concluded that combining branched ester tail chains with hydroxylamine linker negatively affects mRNA delivery efficiency. In vivo studies identify Lipid 23 as a liver-trophic, superior mRNA delivery lipid and Lipid 16 as a potent cell type-specific ionizable lipid for the CD11bhi macrophage population without an additional targeting moiety. Finally, in vivo mRNA delivery efficiency and toxicity of these LNPs are compared with SM-102-based LNP (Moderna's LNP formulation) and are shown to be cell-specific compared to SM-102-based LNPs. Overall, this study suggests that a structural combination of tail and linker can drive a novel functionality of LNPs in vivo.

Multiple myeloma (MM) is a cancer of differentiated plasma cells that occurs in the bone marrow (BM). Despite the recent advancements in drug development, most patients with MM eventually relapse and the disease remains incurable. RNA therapy delivered via lipid nanoparticles (LNPs) has the potential to be a promising cancer treatment, however, its clinical implementation is limited due to inefficient delivery to non-hepatic tissues. Here, targeted (t)LNPs designed for delivery of RNA payload to MM cells are presented. The tLNPs consist of a novel ionizable lipid and are coated with an anti-CD38 antibody (αCD38-tLNPs). To explore their therapeutic potential, it is demonstrated that LNPs encapsulating small interference RNA (siRNA) against cytoskeleton-associated protein 5 (CKAP5) lead to a ≈90% decrease in cell viability of MM cells in vitro. Next, a new xenograft MM mouse model is employed, which clinically resembles the human disease and demonstrates efficient homing of MM cells to the BM. Specific delivery of αCD38-tLNPs to BM-residing and disseminated MM cells and the improvement in therapeutic outcome of MM-bearing mice treated with αCD38-tLNPs-siRNA-CKAP5 are shown. These results underscore the potential of RNA therapeutics for treatment of MM and the importance of developing effective targeted delivery systems and reliable preclinical models.

Donor-bridge-acceptor framework has been studied widely for their detailed photophysical properties with the aim of efficient long range charge transfer. Carbazole attached with electron acceptors has been used for charge transfer properties, thermally activated delayed fluorescence, exciton dynamics etc. In this study, we synthesized donor–acceptor dyads of seconday amine (carbazole and diphenyl amine) attached to perylene through nitrogen, with phenyl (P-Ph-N-CBZ and P-Ph-N-BP) and without phenyl (P-N-CBZ and P-N-BP). Further their detailed photophysical and electroluminescent properties are discussed. P-N-BP is known and is used as standard in this work. DFT studies show nearly orthogonal geometry of P-N-CBZ with the dihedral angle of ∼ 71° between the planes of perylene and carbazole. For P-Ph-N-BP and P-Ph-N-CBZ, the perylene and amines are adopting almost co-planar geometries. Emission spectra of P-Ph-N-CBZ and P-N-CBZ in dichloromethane are structural with clear vibronic features while for P-Ph-N-BP and P-N-BP, the emission is structureless and broad indicating prominent electronic coupling between molecular orbitals of donor and acceptor in the latter case. Fluorescence quantum yields of phenyl-linked dyads are found to be higher than directly linked perylene-amine derivatives. High quantum yield (0.79) in non-polar hexane and polar DMSO (0.66) is observed for P-Ph-N-CBZ. It is interesting to note that the fluorescence lifetimes of these dyads are increasing while fluorescence quantum yields are decreasing with increasing polar solvents, suggesting that the emission has a significant contribution from the 1CT state. Dipole moments estimated from DFT studies and photophysical methods are higher for phenyl-linked dyads than those directly attached which is also consistent with their solvatochromism properties. Weaker electronic coupling in phenyl-linked dyads resulted due to the long distance between donor and acceptor in phenyl-linked dyads. Furthermore application in OLEDs was shown for P-Ph-N-CBZ and P-Ph-N-BP. OLEDs of P-Ph-N-BP showed a peak emission at 568 nm resulting in an amber device. A high luminance of 4.3 × 103 Cd/m2 at a current density of 100 mA/cm2 and a maximum EQE of 4.2% with a low turn on volatage of ∼ 4 V was obtained for these devices. Thus, our results show that weaker charge transfer takes place in perylene based donor–acceptor dyads resulting in bright OLED devices.

Chemo-immunotherapy is a combination of “standard-of-care” chemotherapy with immunotherapy and it is considered the most advanced therapeutic modality for various types of cancers. However, many cancer patients still poorly respond to current regimen of chemo-immunotherapy and suggest nanotherapeutics as a boosting agent. Recently, heme oxygenase-1 (HO1) is shown to act as an immunotherapeutic molecule in tumor myeloid cells, in addition to general chemoresistance function in cancer cells suggesting that HO1-targeted therapeutics can become a novel, optimal strategy for boosting chemo-immunotherapy in the clinic. Currently the available HO1-inhibitors demonstrate serious adverse effects in clinical use. Herein, tumor myeloid cell- and cancer cell-dual targeted HO1-inhibiting lipid nanotherapeutic boost (T-iLNTB) is developed using RNAi-loaded lipid nanoparticles. T-iLNTB-mediated HO1-inhibition sensitizes cancer cells to “standard-of-care” chemotherapeutics by increasing immunogenic cell death, and directly reprograms tumor myeloid cells with distinguished phenotype. Furthermore, tumor myeloid cell reprogramming by T-iLNTB induces CD8+ cytotoxic T cell recruitment, which drives “Cold-to-Hot” transition and correlates with improved responsiveness to immune checkpoint inhibitor in combination therapy. Finally, ex vivo study proves that HO1-inhibition directly affects tumor macrophage differentiation. This study demonstrates the potential of T-iLNTB as a novel therapeutic modality for boosting chemo-immunotherapy.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been identified as the causal agent of COVID-19 and stands at the center of the current global human pandemic, with death toll exceeding one million. The urgent need for a vaccine has led to the development of various immunization approaches. mRNA vaccines represent a cell-free, simple, and rapid platform for immunization, and therefore have been employed in recent studies toward the development of a SARS-CoV-2 vaccine. Herein, we present the design of an mRNA vaccine, based on lipid nanoparticles (LNPs)-encapsulated SARS-CoV-2 human Fc-conjugated receptor-binding domain (RBD-hFc). Several ionizable lipids have been evaluated in vivo in a luciferase (luc) mRNA reporter assay, and two leading LNPs formulations have been chosen for the subsequent RBD-hFc mRNA vaccine strategy. Intramuscular administration of LNP RBD-hFc mRNA elicited robust humoral response, a high level of neutralizing antibodies and a Th1-biased cellular response in BALB/c mice. The data in the current study demonstrate the potential of these lipids as promising candidates for LNP-based mRNA vaccines in general and for a COVID19 vaccine in particular.

The COVID-19 pandemic led to development of mRNA vaccines, which became a leading anti-SARS-CoV-2 immunization platform. Preclinical studies are limited to infection-prone animals such as hamsters and monkeys in which protective efficacy of vaccines cannot be fully appreciated. We recently reported a SARS-CoV-2 human Fc-conjugated receptor-binding domain (RBD-hFc) mRNA vaccine delivered via lipid nanoparticles (LNPs). BALB/c mice demonstrated specific immunologic responses following RBD-hFc mRNA vaccination. Now, we evaluated the protective effect of this RBD-hFc mRNA vaccine by employing the K18 human angiotensin-converting enzyme 2 (K18-hACE2) mouse model. Administration of an RBD-hFc mRNA vaccine to K18-hACE2 mice resulted in robust humoral responses comprising binding and neutralizing antibodies. In correlation with this response, 70% of vaccinated mice withstood a lethal SARS-CoV-2 dose, while all control animals succumbed to infection. To the best of our knowledge, this is the first nonreplicating mRNA vaccine study reporting protection of K18-hACE2 against a lethal SARS-CoV-2 infection.

Here, we have demonstrated that the presence of a carbonyl group at C7 position prevents the olefin metathesis of endo -norbornene derivatives due to complexation of the metal alkylidene. Time-dependent NMR studies showed the presence of new proton signals in the metal alkylidene region, which indicate the formation of metal complex with the carbonyl group of the substrate. These observations were further proved by ESI-MS analysis. Whereas computational studies showed that the catalyst was interacting with the C7 carbonyl group and aligned perpendicular to that of norbornene olefin. These endo -keto norbornene derivatives were reduced to hydroxyl derivatives dia­stereoselectively. Ring-rearrangement metathesis (RRM) of these hydroxyl derivatives, produced the [6/5/6], and [5/6/5] carbo-tricyclic cores of natural products in one step. Whereas the RRM of O -allyl derivatives, delivered the oxa-tricyclic compounds in a single step with excellent yields.

Lipid nanoparticles (LNPs) are the most advanced nonviral platforms for small interfering RNA (siRNA) delivery that are clinically approved. These LNPs, based on ionizable lipids, are found in the liver and are now gaining much attention in the field of RNA therapeutics. The previous generation of ionizable lipids varies in linker moieties, which greatly influences in vivo gene silencing efficiency. Here novel ionizable amino lipids based on the linker moieties such as hydrazine, hydroxylamine, and ethanolamine are designed and synthesized. These lipids are formulated into LNPs and screened for their efficiency to deliver siRNAs into leukocytes, which are among the hardest to transfect cell types. Two potent lipids based on their in vitro gene silencing efficiencies are also identified. These lipids are further evaluated for their biodistribution profile, efficient gene silencing, liver toxicity, and potential immune activation in mice. A robust gene silencing is also found in primary lymphocytes when one of these lipids is formulated into LNPs with a pan leukocyte selective targeting agent (β7 integrin). Taken together, these lipids have the potential to open new avenues in delivering RNAs into leukocytes.

Dendralenes are exquisite molecules as they exhibit enormous potential for the rapid generation of architecturally esoteric scaffolds when subjected to tandem Diels-Alder reactions, but their synthesis is a tall order. In conjunction with diversity-oriented synthesis, [3]dendralenes satisfy the potential demand for simultaneous and efficient synthesis of intricate collections of molecules that exhibit a range of activities for lead generation in drug discovery. This account chronicles our roller-coaster journey and systematic approach beginning from the synthesis of extremely unstable, non-isolable [3]dendralenes through moderately stable examples and then finally, highly functionalized, stable [3]dendralenes via sequential methylenation (using dimethylsulfonium methylide) and Horner-Wadsworth-Emmons olefination. It also describes the study of the attributes affecting their stability and reactivity with various dienophiles. In addition, it reports on how these dendralenes, upon astute maneuvering, can be engaged in a diene-transmissive Diels-Alder (DTDA) sequence, thus harnessing their full potential by construction of a small but diverse library of complex frameworks in a quick and efficient manner, with step and atom economy. 1 Introduction 2 Literature Methods for [3]Dendralene Synthesis 3 Our Tryst with [3]Dendralenes 3.1 Serendipitous Olefination with the Corey-Chaykovsky Ylide 3.2 Synthesis of [3]Dendralenes and Reactivity/Stability Studies 3.3 Diels-Alder Reactions of [3]Dendralenes 3.4 Tuning of [3]Dendralenes for the DTDA Reactions 3.5 Diversity-Oriented Synthesis with [3]Dendralenes 3.6 Rationalization of Factors Governing the Stability and Reactivity of [3]Dendralenes 4 Conclusions.

This is the first comprehensive study that details the synthesis of stable acyclic trisubstituted [3]dendralenes and deciphers their structural requisite for a successful diene transmissive Diels-Alder (DTDA) reaction by employing two different dienophiles and eventually generating a small repository of complex molecules, thus exemplifying how substituted [3]dendralenes could be deployed in diversity-oriented synthesis with high selectivities. A balance of reactivity and stability was struck by prudent selection of the position and nature of functional groups on these [3]dendralenes. Upon tandem Diels-Alder reactions with several symmetrical as well as unsymmetrical dienophiles, these dendralenes afforded diversity-oriented quick access to many polycyclic complex motifs possessing several functional groups and multiple stereogenic centers. Thus, the full potential of the dendralenes could be harnessed. The reactions proceeded under mild conditions with step and atom economy and were highly regio- and stereoselective besides being excellent yielding. The DTDA sequence resulted in the generation of four new carbon-carbon bonds, two new rings, and 3-7 stereogenic centers. The key feature of the method is that we could rapidly generate complexity along with functional and structural diversity from a trivial acyclic substrate with no stereogenic centers.

A novel route to functionalized [3]dendralenes with an allylsilane as integral component has been developed. The key step involves a dimethylsulfonium methylide mediated olefination of appropriately substituted silylmethylethenylidene phosphonoacetate followed by Horner–Wadsworth–Emmons reaction with aromatic aldehydes. The tri- and tetra substituted [3]dendralenes are stable towards self Diels–Alder cyclodimerization which can be attributed to stereoelectronic effect of the silylmethyl group favoring for an unreactive conformation.

Acyclic tetrasubstituted [3]dendralenes stable towards D-A cyclodimerization were prepared by the double olefination of substituted dienyl phosphonates with dimethylsulfonium methylide, followed by H-W-E olefination of the formed butadienylphosphonoacetates with aldehydes. The X-ray structure of a [3]dendralene with a 4-position benzyl substitution revealed that an electron-rich diene exists in the s-trans conformation, leading to the stability. This structural preference of dendralenes leads to an unusual regioselectivity in the D-A reaction, wherein a deactivated diene participated in the process and displayed endo selectivity. The steric bulk of the groups at the 2 and 4 positions govern the regioselectivity and dominates the electronic effects. For the dendralenes possessing a 4-methyl substituent, the electronically rich diene participated in the D-A reaction. These D-A adducts failed to undergo further cycloaddition owing to steric crowding, which resulted in gauche conformation of the diene, as evidenced by X-ray structures of the adducts.