STAAR Lipids: A Novel Ionizable Lipid Synthetic Platform for Efficient mRNA Delivery In Vivo with Tunable Lung Targeting

doi:10.21203/rs.3.rs-5124244/v1

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STAAR Lipids: A Novel Ionizable Lipid Synthetic Platform for Efficient mRNA Delivery In Vivo with Tunable Lung Targeting

https://doi.org/10.21203/rs.3.rs-5124244/v1

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Ionizable lipids are an essential component of lipid nanoparticles (LNPs) for an efficient mRNA delivery. However, optimizing their chemical structures for high protein expression, efficient endosomal escape, and selective organ targeting remains challenging due to complex structure-activity relationships and multistep synthesis. In this study, we introduce a rapid, high-throughput platform for screening ionizable lipids using a two-step, scalable synthesis involving a one-pot 3-component click-like reaction. This method, herein known as the STAAR approach, standing for Sequential Thiolactone Amine Acrylate Reaction, allowed for the combinatorial synthesis and in vivo screening of 91 novel lipids, followed by a structure-activity study. This led to the development of CP-LC-0729, an ionizable lipid that significantly surpasses the benchmark in protein expression while showing no in vivo toxicity. Additionally, the STAAR lipid platform was further validated by incorporating a one-step strategy to yield a permanently cationic lipid which was tested following a fifth-lipid formulation strategy. The in vivo results showed a highly selective lung delivery with a 32-fold increase in protein expression, outperforming current endogenous targeting strategies. All these findings underscore the potential of lipid CP-LC-0729 and the STAAR lipid platform in advancing the efficiency and specificity of mRNA delivery systems, while also advancing the development of new ionizable lipids.

Biological sciences/Biotechnology/Nucleic-acid therapeutics

Biological sciences/Chemical biology/Lipids

The interest in mRNA technology as a promising platform for the treatment of diverse pathologies, including cancer, has increased exponentially since the COVID-19 pandemic, when two mRNA vaccine candidates (i.e. mRNA-1273 and BNT162b) were authorized to tackle SARS-CoV-2. Indeed, mRNA-based technology has shown great potential in applications such as gene editing therapies, vaccines against infectious diseases and protein supplementation therapies^1–3. In order to achieve protein expression in living organisms, mRNA molecules need a delivery platform to avoid nuclease degradation, promote cellular endocytosis and improve endosomal escape. Lipid nanoparticles (LNPs) demonstrate both advanced clinical status and effectiveness in overcoming delivery barriers for mRNA therapeutics, highlighting their importance as nonviral platforms^4,5.

LNPs commonly consist of mRNA, helper lipids (usually phospholipids), cholesterol, PEG-conjugated lipids and ionizable lipids. The role of ionizable lipids has been demonstrated as crucial in stabilizing and delivering mRNA^6,7. A fundamental chemical feature of ionizable lipids lies in the tertiary amino group which becomes charged in acidic pH but remains neutral at physiological conditions⁸. During the formulation process at acidic pH, their positive charge aids in condensing the mRNA and, upon internalization, as the endosomes undergo acidic maturation, they assist in membrane disruption, facilitating endosomal escape. At physiological pH, ionizable lipids exhibit a neutral charge, resulting in reduced toxicity levels and enhanced biocompatibility^9,10. Two substantial research challenges currently being addressed involve understanding and improving the efficiency of LNPs' endosomal escape (currently estimated to be around 2%) and achieving targeted in vivo delivery to specific cells or tissues for precision therapeutics^11,12.

Despite the extensive research and advances into ionizable lipid development, ionizable lipids typically consist of distinct common structural components: an ionizable headgroup and at least two hydrophobic alkyl tails tethered together through a linker, often containing degradable bonds such as esters. Ester bonds have been chosen for their ability to break down in vivo thanks to the activity of esterases, which improves their biocompatibility ^13,14. Moreover, ionizable lipids commonly include at least two hydrophobic tails, which can foster a cone-like shape, improving endosomal escape and mRNA delivery by promoting the formation of inverted hexagonal phases^12,15. Adjusting the bonds and chemical structures in ionizable lipids to achieve effective and safe in vivo delivery has sparked interest recently. However, three significant challenges persist regarding ionizable lipid design and synthesis: Firstly, despite some general characteristics previously outlined, precise structural design rules are still lacking regarding the relationship between their structural features and in vivo delivery⁹. Moreover, major bottlenecks in mRNA delivery, such as endosomal escape, could be addressed by gaining a better understanding of structure-activity relationships¹². Indeed, the most recurrent approach to novel ionizable lipid development is to conduct massive screenings of synthetic candidates based on combinatorial chemistry to identify those that are particularly effective in drug delivery^16–18.

Secondly, addressing the necessity for screening lipid candidates requires avoiding multi-step and complicated synthetic protocols, highlighting the importance of rapid and cost-effective synthetic strategies.^19,20 Numerous strategies have been explored so far, for example, aza-Michael addition with acrylates, ester formation, and epoxide opening¹⁶. However, obtaining the necessary building blocks typically involves several time-consuming syntheses, harsh conditions and purification steps, which can hinder structural optimization and once again the understanding of structure-activity relationships^14,21.

Thirdly, there is a growing interest in developing LNPs that can target certain tissues or organs through passive targeting¹¹. In this sense, one of the most successful strategies up to date is the inclusion of a fifth lipid in the composition of the LNPs. Siegwart and coworkers developed formulations by incorporating either cationic or anionic lipids, thereby enabling targeted delivery to the lungs or spleen, respectively²².

Here, we developed a rapid, high-throughput and cost-effective platform for screening ionizable lipids. We utilized a versatile multidimensional synthesis approach involving mild conditions and two tandem one-pot reactions: thiolactone ring opening and Michael addition. We then investigated the structure-activity relationships of hydrophobic tails and functional groups leading to the best ionizable lipid candidate. Finally, the combination of this candidate, CP-LC-0729, with a permanently cationic lipid, which can be accessed via a single synthetic step, demonstrated a very high selectivity to lungs with a remarkable 32-fold increase in protein expression compared to the benchmark. Altogether, these results highlight the potential of the STAAR platform in generating new promising lipid candidates for both hepatic and extrahepatic therapeutic applications.

Sequential Thiolactone Amine Acrylate Reaction (STAAR Lipids)

Similarly to other ionizable lipid structures, STAAR lipids consist of a polar head, a linker, and hydrophobic tails. Given that one of the aims of this study was precisely to examine how the various fragments of these lipids can influence their in vivo performance, a combinatorial chemistry approach was followed to screen a selection of lipids that included different moieties in their structure. For simplicity, each lipid was named as AiBjCk to account for the reagents used for their synthesis. In this nomenclature, Ai represents an amine (polar head); Bj indicates the linker, which consists of a thiolactone incorporating a hydrophobic tail, and Ck represents an acrylate with another hydrophobic tail.

The synthetic process of STAAR lipids is illustrated in Fig. 1a. Briefly, this two-step synthesis starts with the coupling of homocysteine thiolactone with a carboxylic acid to yield the corresponding linker intermediate (Bj). The second step involves a tricomponent one-pot sequential reaction which starts with a ring-opening addition of the intermediate (Bj) with an amine (Ai) followed by a Michael addition with an acrylate (Ck) to yield the lipid AiBjCk²³. Remarkably, this multicomponent reaction can be carried out at room temperature, without need for catalysts or inert atmosphere, and within a 2-hour reaction time. Moreover, the high purity (>80%) obtained make it an optimal method for rapid multidimensional screening of ionizable lipids, eliminating the need for further purification steps.

The general structure of the resulting STAAR lipids include two amide bonds (one originating from the reaction of the amine Ai and the thiolactone Bj and the other one connecting this thiolactone to its hydrophobic tail), a thioether bond (from Bj), and an ester group (from Ck). Consequently, any changes to be made in the hydrophobic tails can be achieved by modifying the structure of the thiolactone derivatives (Bj) and acrylates (Ck).

Two-phase screening strategy for ionizable lipids

To assess how different moieties affect the final structure and activity of the lipid, a two-phase screening strategy was devised in which one component of the lipid (Ai, Bj, or Ck) was initially kept constant while the other two were varied. This method enabled us to systematically evaluate the impact of each component on the lipid's performance. By fixing one fragment and varying the other two, the intention was to efficiently identify the most promising candidates, thereby streamlining the screening process and optimizing our approach. Thus, the screening process was divided into two optimization phases, with each phase focusing on the optimization of a different lipid moiety (Fig. 2a, 2b).

For this optimization phase, the effectiveness of each IL candidate was evaluated by formulating each lipid individually into LNPs using a pipette rapid mixing procedure²⁴. Thus, each LNP was composed of an ionizable lipid (AiBjCk), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), cholesterol, and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG-2000) (50: 10: 38.5: 1.5 molar ratios, respectively) and firefly luciferase mRNA (with a 6:1 molar N/P ratio Fig. 1b).

Also, due to the weak correlation between in vitro and in vivo LNP performance²⁵, a direct screening in vivo using mice as animal model was followed, for which a firefly luciferase mRNA dose of 0.05 mg/kg was administered intramuscularly (i.m.) and the luminiscence results were measured at 4h post-inoculation.

Screening Phase 1: Optimization of linker’s hydrophobic chain (Bj)

The first phase of the structure optimization, aimed to select the optimal Bj component by synthesizing 21 lipids in which the polar head was fixed (A1 was selected) while combining three types of thiolactones with different acrylates (Fig. 2c). This amine was chosen as its structure closely resembles that of the MC3 polar head, featuring a spacer of three methylene units between a tertiary amine and a carbonyl group²⁶. The hydrophobic chains linked to the thiolactone derivatives (Bj) were selected to have distinctly different structures: a linear chain (B1) with 18 carbon atoms, an unsaturated chain (B2), and a branched chain (B3).

The analysis of the protein expression (Fig. 2c) achieved for the LNPs for each individual lipid revealed that A1B2Ck lipids (with k ranging from 1 to 7) resulted in increased protein expression for in vivo mRNA delivery, regardless of the acrylate used (Ck). Moreover, unsaturated chains yielded higher expression than saturated ones. Notably, hydrophobic moieties C3 and C7 were particularly effective.

Screening Phase 2: Optimization of polar head (Ai) and second hydrophobic chain (Ck)

Based on the findings from the screening phase 1, B2 was selected as the foundation for the next screening step. This second phase aimed to optimize the other two moieties of the molecule: the polar head (Ai) and the second hydrophobic chain (derived from Ck). Once again, the use of the STAAR synthesis approach for this optimization process allowed for a swift exploration of these structural moieties thanks to the high purities achieved in the sequential reactions used in their synthesis. More specifically, 70 AiB2Ck novel lipids were synthesized, with i and k ranging from 2 to 11 and 1 to 7, respectively (Fig. 2d). Moreover, to validate the strategy employed, an assay was carried out to corroborate that there were no significant differences between the use of crude reaction and purified ionizable lipid in terms of protein expression (Supplementary Fig. 3).

Regarding the Ai moiety, diverse polar heads, including cyclic, linear, aromatic, and analogues with a variable number of ionizable N groups, underwent testing. Upon in vivo evaluation, cyclic and aromatic amines (A5, A6, A7, A8, A9) failed to demonstrate significant expression, the only exception being that of A11. In contrast, among the linear amines, A2 and A4 displayed the best results, with lipid A4B2C3 notably yielding the highest expression levels. Remarkably, the comparison of amines A1 and A4, which are differenced by just one single methylene unit, resulted in an almost 5-fold increase in expression for lipid A4B2C3 over A1B2C3, emphasizing the role of the number of methylenes between the tertiary amine and the primary amine of Ai in the lipid performance²⁷.

With regards to the hydrophobic tails (stemming from Ck), we confirmed the previously observed trend for Bj, with the butyloctanoate derived branched acrylate (C3) yielding the lipids with highest expression levels. Additionally, linear 18-carbon monounsaturated (C1) also showed promising results in vivo. We also noticed that when shorter acrylate chains were used, the in vivo expression decreased up to 60 times, irrespective of it being branched or linear, this including isooctyl (C7), ethylhexyl (C2), and linear octyl (C6). Moreover, intermediate length chains (C5 and C4, 12 and 14 carbons in length, respectively), led to higher expression compared to shorter chains, yet the expression remained three times lower than C3 and C1. This suggests that longer chains, featuring unsaturations or branches in the hydrophobic moiety Ck, might be necessary to achieve the desired electronic and steric balance in the STAAR lipids.

In relation to physicochemical parameters of LNPs, observed sizes ranged from 150 to 230 nm, with the majority falling beneath 190 nm. Polydispersity indexes mostly lay below 0.25, while ζ potentials ranged between 5 mV and -25 mV, with all lipids but four exhibiting a negative ζ potential. mRNA encapsulation rates varied from 20% to 100% (Supplementary Table 2). Notably, lipids with higher activity achieved encapsulation rates between 60% and 80% (with a direct pipetting method), hence suggesting this might represent the optimal encapsulation range for STAAR lipids LNPs in the screening phase. Moreover, we noted that even though employing an amine featuring two ionizable groups (A5) as lipid head results in high encapsulation rate, this does not reflect in its performance, as the in vivo activity was found to be 2 to 30 times lower compared to lipids containing amine A4, which contains only one ionizable group.

Fine-tuning of the ionizable lipid structure

After identifying A4B2C3 as the most effective ionizable lipid in the two-phase screening, further structural studies were conducted to assess the impact of hydrophobic chain lengths and functional groups and their roles in influencing lipid performance.

Assessment of the hydrophobic tails of lipid A4B2C3

Therefore, the first step in this optimization started with the variation of the hydrophobic chains to gain insights into structure-activity relationships. Following the same synthetic approach as in the screening phase, we synthesized and purified eight new ionizable lipids, hence introducing three distinct branched chain lengths via the Bj and Ck moieties (Fig. 3a). Having optimized the lipid structure in the initial phases, for these refining phases we relied on microfluidic techniques as a way to confirm the previous conclusions while keeping the formulation process precisely controlled. Nanoparticles were obtained with sizes ranging from 80 to 130 nm, with the majority being under 100 nm, and they exhibited high encapsulation efficiencies (90-99%, Supplementary Table 4). Thus, a purified batch of the optimized lipid A4B2C3 along with these new 8 lipids were formulated into LNPs along with two separate controls. Even though both controls used MC3 as ionizable lipid in the same molar and N/P ratios as the screened lipids, one of them included DOPE as helper lipid (same as the formulated STAAR lipids), namely MC3 DOPE, whereas the other one replicated the MC3 LNP benchmark by using DSPC. Remarkably, the protein expressions found for eight out of these nine STAAR lipids were comparable or higher than the MC3 (DOPE) LNP. Moreover, when comparing the in vivo results to those of the MC3 benchmark LNP²⁶ (Supplementary Table 3), all lipids except one showed higher expression than the benchmark. Among these, the result for A4B2C8 (known as CP-LC-0729) was particularly striking, as it demonstrated an expression value 6.4 times higher than the benchmark control (Fig. 3b). The exception was observed with lipid A4B4C3, characterized by the shortest hydrophobic chain combination, and for which the expression dropped to 0.034 times relative to the MC3 benchmark. The results suggest that a tail with a minimum carbon number may be necessary for achieving high protein expression, aligning with the trend observed during the two-phase screening, where shorter chains such as C2, C6, and C7 consistently resulted in lower protein levels.

Influence of functional groups in the structure-activity relationship of STAAR lipids

After exploring the impact of altering the hydrophobic chains and identifying lipid CP-LC-0729, which contains two branched chains (B2, C8), as the best candidate for high-performing in vivo activity, our focus then shifted to study the functional groups within the structure. As previously reported in literature, certain bonds, such as esters, play a pivotal role in endosomal escape and degradation pathways^28–31. Taking advantage of the chemical versatility of STAAR lipids, which allows for modular modification of functional groups, we investigated how the nature of specific bonds can influence the performance of the STAAR lipids. Aside from the ionizable amine in the polar head, all the synthesized lipids during the two-phase screening display an identical distribution of functional groups: two amide bonds (located in positions termed α and β), one ester bond and a thioether bond.

To explore the roles of amide and ester bonds in α and β positions,^32,33 we synthesized and purified structural variations of CP-LC-0729 as depicted in Fig. 3c. These products were subsequently formulated with the same N/P and lipid ratios used in the initial screenings, employing microfluidic mixing.

As a first step, we inverted the C-N carbonyl order of the β amide bond (CP-LC-0729-03) resulting in a threefold reduction of in vivo protein expression compared to CP-LC-0729 (Fig. 3c and 3d). Then, to further explore the role of the α amide bond, lipid CP-LC-0729-02 was synthesized, where the α amide is methylated, resulting in a 3.4-fold decrease of in vivo expression. In the next step, the amide bonds were partially or completely substituted with ester bonds in CP-LC-0729-01, CP-LC-0729-04 and CP-LC-0729-05. In this case, we noticed a significant decrease in effectiveness—by two orders of magnitude—when replacing both amides (α and β positions) with ester groups in CP-LC-0729-05 (Fig. 3d). This change resulted in the lowest performance among the proposed variations. As a middle-ground case, when combining an amide and an ester group, the in vivo performance was dependent on relative position of the groups, yielding intermediate outcomes. Thus, these results indicated that preserving the α amide bond (CP-LC-0729-04) led to better expression results than replacing it with an ester group (CP-LC-0729-05). Remarkably, the protein expression value of the methylated α amide lipid CP-LC-0729-02 was on par with CP-LC-0729-03 and CP-LC-0729-04.

Regarding the LNP physicochemical characterization, we noticed that altering the α amide bond led to lower LNP pKa. Specifically, pKa values were the lowest for CP-LC-0729-05 (pKa=5.48) and CP-LC-0729-01 (pKa =6.12). Additionally, a similar trend was observed when measuring ζ potential, as CP-LC-0729-05 (ζ=-18.0 mV) and CP-LC-0729-01 (ζ=-10.3 mV) displayed the lowest values of the series (Fig. 3e).

Additionally, to further evaluate the behaviour of lipid CP-LC-0729 in different formulations, an in vivo study using LNPs with different molar ratios of the four lipid components was conducted. The results, detailed in Supplementary Table 7, indicate that CP-LC-0729 performs well even when the molar ratios diverge from the ones used in more conventional formulations, highlighting its versatility.

Additional biological assays of the top-performing ionizable lipid CP-LC-0729 LNP.

In vivo safety profile of lipid CP-LC-0729

After selecting the best performing ionizable lipid candidate through the in vivo screening and subsequent optimization phases, different biological assays were carried out to evaluate the safety profile of the candidate, CP-LC-0729. Thus, in vivo toxicity assays on CP-LC-0729 LNPs were conducted to assess any potential organ damage by measuring liver enzymes (ALT, AST, ALP) and kidney function (UREA) at 24 and 48h after systemic injection at a high dose (2.5 mg/kg). When compared to the PBS buffer used as a control, CP-LC-0729 LNPs showed no significant impact on the levels of these enzymes (Fig. 4a). The change in mice body weight was monitored for 15 days post-inoculation, revealing no apparent differences compared to the negative control (PBS buffer) (Fig. 4b).

Evaluation of cell internalization of CP-LC-0729 LNPs

To gain deeper insights into the mechanisms of cell internalization, the endocytosis pathways of CP-LC-0729 LNP were examined in vitro in hepatic HepG2 cells. Four endocytic pathways were assessed by incubating the LNPs with specific inhibitors for each route before in vitro transfection¹⁹. The results show that the uptake of CP-LC-0729 LNPs strongly relies on lipid rafts, as indicated by inhibition with methyl-β-cyclodextrin. Other internalization pathways, such as macropinocytosis and clathrin-mediated endocytosis, showed a smaller contribution. Additionally, genistein did not inhibit uptake, indicating that caveolae-mediated endocytosis is not significant (Fig. 4d). We also conducted hemolysis assays to assess endosomal escape of CP-LC-0729 LNPs through their membrane-disrupting activity under both acidic (pH 6) and physiological (pH 7.4) conditions^19,34. These tests showed that CP-LC-0729 LNPs increased hemolysis in the acidic medium up to 90%, ten times higher than the MC3 (DOPE) LNP used as control. In contrast, at physiological pH, the hemolytic activity remained comparable to the control level (PBS) indicating no hemotoxicity. (Fig. 4c)

Extrahepatic organ targeting of CP-LC-0729 using permanently cationic lipids.

Recognizing the critical importance of targeting specific organs for effective therapeutic outcomes, we focused on achieving extrahepatic mRNA delivery to broaden the scope and versatility of ionizable lipid CP-LC-0729 and the STAAR screening platform. As reported by Siegwart and coworkers,^22,35,36 the addition of a fifth permanently charged lipid results in selective targeting of the lungs or spleen, attributed to changes in the protein corona and LNP surface charge. Herein we applied a simple approach for the synthesis of a permanently cationic lipid, building upon a STAAR ionizable lipid by means of a single additional step and later carried out a comparative study with what is reported in the literature following the fifth-lipid strategy. Hence, a novel cationic lipid was synthesized, denoted as (+) CP-LC-0729, as it is derived from the methylation of the tertiary amine of the top-performing ionizable lipid CP-LC-0729 (Fig. 5a). Subsequently, the performance of ionizable lipids MC3 and CP-LC-0729 was compared, in LNPs incorporating cationic lipids (+) CP-LC-0729 or the standard commercial alternative DOTAP as fifth lipids, in representative formulations with firefly luciferase mRNA (Fig. 5b)³⁷. As shown in Supplementary Table 5, the cationic lipid molar percentages were compared using LNPs incorporating different proportions of this fifth lipid (30%, 40% and 50% (mol/mol)). The experiments were conducted using microfluidics mixing for LNPs, followed by intravenous administration. LNPs were termed as [cationic lipid (percentage of cationic lipid in molar rate) ionizable lipid] LNP, for example, LNP using DOTAP as cationic lipid at 50% combined with MC3 as ionizable lipid was designated [DOTAP (50%) MC3] LNP.

Regarding the physiochemical properties, LNPs with (+) CP-LC-0729 showed similar values to CP-LC-0729 LNP, including particle size (60-85 nm) and 99 % mRNA encapsulation efficiency. However, we noticed positive surface ζ potentials in (+) CP-LC-0729 LNPs, which varied depending on the cationic lipid ratio used (Supplementary Table 6).

After inoculation, ex vivo assays revealed distinct variations in protein expression in the lungs, depending on the cationic lipid ratio. The [(+) CP-LC-0729 (50%) CP-LC-0729] lipid nanoparticles (LNPs) demonstrated remarkable selectivity for the lungs, achieving 88% selectivity. Even the [(+) CP-LC-0729 (40%) CP-LC-0729] and [(+) CP-LC-0729 (30%) CP-LC-0729] LNP exhibited strong selectivity, 88% and 82%, respectively (Supplementary Fig 4 and 5), far exceeding the 38% selectivity observed for the benchmark LNP, [DOTAP (50%) MC3].

Moreover, when [(+) CP-LC-0729] was formulated with MC3, the resulting expression levels were twofold lower than when it was combined with CP-LC-0729 (Fig. 5c). Even more remarkably is the fact [(+) CP-LC-0729 (50%) CP-LC-0729] LNP exhibited 32-fold higher luciferase expression compared to the benchmark [DOTAP (50%) MC3] LNP (Fig. 5c). These findings demonstrate that both the selectivity and protein expression of our CP-LC-0729 combinations are significantly superior to the benchmark [DOTAP (50%) MC3] formulation.

Additionally, [(+) CP-LC-0729 (50%) CP-LC-0729] LNP was formulated with a small percentage of the fluorescent probe DiR (dialkylcarbocyanine, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine iodide) to track fluorescence and determine LNP biodistribution. Interestingly, while the majority of LNPs were directed to the liver, luciferase expression was observed predominantly in the lungs (Supplementary Fig.6).

Even though a broad number of ionizable lipids have been discovered^30,38–41, there is still a need for simple, cost-effective methods that are quick, involve few steps, and employ mild conditions to create large ionizable lipid libraries. Here, we introduce a new three-component synthetic approach that allows for the rapid synthesis of STAAR ionizable lipids in two hours. This method streamlines lipid screening by avoiding cumbersome and complex synthetic requirements such as inert gas, high temperatures, or purification steps. With purities over 80%, this method simplifies the synthesis of new ionizable lipids in 1.5 mL tubes, reducing the reliance on expert organic synthesis knowledge, which allowed the rapid screening of nearly 100 novel lipids and facilitated the development of lipid CP-LC-0729.

Using the STAAR ionizable lipid AiBjCk synthesis approach, independent modifications of moieties A, B, and C were successfully carried out, leading to the development of multiple promising candidates. The two-phase in vivo screening strategy proved to be highly effective in optimizing these components, particularly by focusing on the Bj and Ck moieties in distinct phases. This methodological approach not only reduced the number of required experimental animals but also enhanced the efficiency of the screening process.

In the initial phase, it was observed that the branched derivative B2 significantly outperformed other Bj thiolactone derivatives, indicating the critical role of branching in improving protein expression. This observation aligns with existing literature, which suggests that branched structures often confer enhanced performance in lipid-based delivery systems.³⁴

The subsequent phase highlighted the profound impact of the C moiety, where branched and unsaturated Ck chains, especially C3, demonstrated substantial improvements in protein expression. The role of hydrophobic tail structures, as evidenced by the effectiveness of C3, reinforces the importance of hydrophobic interactions in lipid functionality, in contrast to the poor protein expression observed with shorter hydrophobic chains like C2, C6, and C7. This outcome is particularly relevant when considering the design of lipids aimed at optimizing therapeutic delivery, where the balance between hydrophilicity and hydrophobicity is crucial.

Additionally, findings on the Ai moiety revealed the sensitivity of lipid performance to minor structural changes, such as the spacer length in the polar head. The superior performance of A4 over A1 (five times more protein expression), despite the minimal difference of a single methylene unit, underscores the delicate balance required in the design of effective ionizable lipids. This result not only contributes to the understanding of structure-activity relationships but also provides a clear pathway for further refinement of lipid designs.

Following the discovery of lipid A4B2C3 as the top-performing candidate in the initial two-phase screening approach, the research focused on exploring the impact of further modifications to the lipid structure, specifically targeting the hydrophobic branched chains. The purpose of this subsequent stage was to deepen the understanding of how variations in branch length and saturation could influence protein expression, thereby refining the structure-activity relationship for improved delivery efficiency.

To achieve this, eight additional branched ionizable lipids were synthesized, all containing the A4 polar head but differing in the hydrophobic tail structure. This step was crucial for assessing the extent to which these structural modifications could enhance the overall performance of the lipid. The results were striking: seven out of the eight tested lipids showed a total flux that was at least double that of the MC3 benchmark. The exception was A4B4C3, which showed low protein expression, suggesting a minimum branch length requirement for structure optimization, which is consistent with previous results found in literature¹⁹. This demonstrated that fine-tuning the hydrophobic branched chains can significantly boost delivery efficiency.

In particular, A4B2C8 (CP-LC-0729) showed a total flux value 6.4 times higher than the MC3 benchmark. This indicates that not only was the screening strategy successful, but it also provided critical insights into how specific structural changes can lead to markedly better delivery outcomes. The findings from this stage suggest that optimizing lipid structures, particularly through careful adjustment of the hydrophobic components, can lead to the development of more efficient and targeted delivery systems.

Having seen the influence of the hydrophobic chains on the performance of the lipids, the following step in the structure optimization was directed to the role of functional groups, particularly focusing on the two amide groups. Hence, five variants of CP-LC-0729 were synthesized in which the α and β amide groups in the lipid structure were sequentially modified. Remarkably, the activity studies showed that altering the order of carbonyl bond in the β amide (N-CO to CO-N) resulted in a threefold decrease in expression. This change may be attributed to the shift in the relative position of the β amide hydrogen bond, which can potentially affect the intermolecular force landscape and packing of the molecule⁴².

Significantly, substituting the two amide groups with ester bonds, thereby incorporating a total of three ester groups into the chemical structure, led to a profound decrease in protein expression by two orders of magnitude (CP-LC-0729-05). This contrasts with the fact that other commercial ionizable lipids such as ALC-0315, SM-102, or MC3 only feature ester groups. Moreover, the LNP pKa decreased by more than one unit, and the ζ potential was the lowest amongst the six functional group variation lipids. Interestingly, when lipids contain only one amide, their expression is four and a half times higher when located in the α position (CP-LC-0729-04) compared to the β position (CP-LC-0729-01). Additionally, in the case of the methylated α amide (CP-LC-0729-02), which no longer acts as a hydrogen bond donor, the expression decreased threefold compared to the non-methylated counterpart, suggesting that hydrogen bonds might play a key role. We hypothesize that the observed difference may arise from the α amide proximity to the polar head, potentially enhancing its availability to interact with mRNA via hydrogen bonding or intermolecular interactions between ionizable lipids. The optimal combination, as evidenced by our findings, involves amides located at both the α and β sites, reflecting in the chemical structure of lipid CP-LC-0729.

Thus, the modular synthesis of STAAR ionizable lipids has allowed us to discover how variations in these groups, their arrangement, and hydrophobic chains distinctly influence mRNA delivery. This method represents a powerful tool, enabling the straightforward modification of hydrophobic chains and functional groups at precise locations. This versatility sets it apart from other ionizable lipid libraries previously described in the literature¹⁶. The STAAR synthesis methodology lays the groundwork for developing lipid variants with tailored properties and deeper structure-relationship studies, thereby expanding the potential of lipid screening for novel RNA therapeutic applications. Indeed, our team is currently exploring new lipid candidates using this versatile approach with exciting preliminary results.

Following the screening and optimization phases, which led to the identification of the highly promising lipid structure CP-LC-0729, we proceeded to assess both the in vivo safety profile and uptake mechanism of this compound. Thus, the analysis of hepatic and kidney related enzymes along with the mice body weigh change displayed similar results compared to the negative control (PBS buffer). While a more comprehensive assessment, including testing across different animal models, is necessary to establish a complete safety profile of this lipid, our preliminary results indicate excellent tolerability following systemic administration, with no signs of toxicity observed under the conditions studied. In addition to these promising in vivo results, we conducted in vitro studies to investigate the uptake pathways, showing lipid rafts as the main contributing mechanism. Regarding endosomal escape properties, hemolysis assays (pH=6) showed that CP-LC-0729 LNPs exhibited 10-fold higher membrane-disruption activity compared to MC3(DOPE) LNPs. Remarkably, at pH 7.4, CP-LC-0729 LNPs exhibited low hemolytic activity, comparable to the PBS negative control. This suggests a potential enhanced endosomal escape mechanism of CP-LC-0729 LNPs at acidic pH, while maintaining hemocompatibility at physiological conditions, a result that is in accordance with the fact that lipid CP-LC-0729 shows a higher in vivo performance than MC3 while displaying no signs of toxicity⁴³.

With regards to extrahepatic delivery in mRNA therapeutics, previous studies have shown that the addition of cationic lipids to LNP formulations can modify the surface properties of LNPs, influencing the formation of the protein corona and consequently altering in vivo targeting¹¹. In our study, we explored a streamlined approach to modify the STAAR ionizable lipid CP-LC-0729 into its cationic form (+) CP-LC-0729, using a single synthetic step. Notably, ex vivo assays revealed that the [(+) CP-LC-0729 (50%) CP-LC-0729] formulation resulted in a 32-fold increase in lung protein expression compared to the benchmark [DOTAP (50%) MC3] LNPs, while also achieving lung selectivity of 88%.

These results underscore the strong potential of CP-LC-0729, which, as an ionizable lipid, consistently outperforms MC3 across formulations. The ability to achieve significantly higher expression and selectivity highlights the advantages of this lipid system for lung-targeted delivery. Furthermore, the optimal results observed when combining (+) CP-LC-0729 with CP-LC-0729 may suggest that structural similarity between the ionizable and cationic forms may improve lipid packing and enhance overall LNP performance. However, more studies are needed to confirm this hypothesis.

Ongoing studies aim to further explore these mechanisms, but the marked improvement in both selectivity and protein expression positions CP-LC-0729 as a highly promising alternative to other traditional ionizable lipids such as MC3 both for hepatic and extrahepatic delivery.

It is important to note the limitations associated with the findings of this study. First, some branched and unsaturated acrylates are not commercially available, requiring an additional synthesis step. Second, STAAR ionizable lipids containing functional group variations such as an ester in the α site, required extended synthesis procedures. Lastly, acknowledging the vast array of possibilities unlocked by the STAAR platform, ongoing studies aim to expand the scope of evaluated structures, optimize LNP formulation ratios and explore the interchangeability of the head and lipid tails around positions A, B, and C.

In conclusion, the STAAR platform enables the synthesis of ionizable lipids in a high-throughput, one-pot setup, offering cost-efficiency, modular structure design and mild reaction conditions with shortened times which can easily be scaled up. Through the combinatorial synthesis and in vivo screening of 91 molecules, we identified several effective STAAR lipid candidates during two optimization phases. We then explored key structural characteristics governing their activity through structure-activity relationship studies, including hydrophobic tails and functional groups, identifying lipid CP-LC-0729 as the top-performing candidate and laying the groundwork for discovering new effective STAAR lipids in the future. Compared to the benchmark MC3, ionizable lipid CP-LC-0729 exhibited 6.4 times higher protein expression and showed no signs of in vivo toxicity, positioning it as a promising, readily scalable candidate for clinical advancement. We further validated the efficacy of the STAAR platform by introducing a one-step strategy for developing cationic lipids. The use of lipid CP-LC-0729, in combination with its permanently cationic counterpart, produced lipid nanoparticles (LNPs) that achieved a remarkable 32-fold increase in lung protein expression compared to the benchmark SORT LNP (MC3/DOTAP), with 88% organ selectivity. This result once again confirms the potential of CP-LC-0729, while also expanding its application to therapies requiring targeted delivery beyond the liver.

Materials

All chemical reagents were purchased from Sigma Aldrich (Burligton, Massachusetts, USA), Tokyo Chemical Industry (TCI, Tokyo, Japan), Fluorochem Ltd (Hadfield, UK) and Ambeed (Arligton Heights, Illinois, USA). 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) were purchased from Avanti Polar Lipids (Alabaster, Alabama, USA). 1,2- dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG- PEG 2000) was purchased from Cayman Chemicals (Ann Arbor, Michigan, USA). Cholesterol was purchased from Sigma Aldrich (Burligton, Massachusetts, USA). DLin-MC3-DMA (MC3) was purchased from Broadpharm (San Diego, California, USA). DOTAP was purchased from BOC Science (New York, USA).

mRNA Synthesis

Firefly luciferase mRNA was synthesized following a previous reported method⁴⁴. mRNA complete sequence is available in Supplementary Fig.1.

General method for the synthesis of STAAR ionizable lipids

STAAR lipids were synthesized using a tandem one-pot reaction. First, thiolactone derivatives (0,15 mmol, 1 equiv.) and acrylate (0,15 mmol, 1 equiv.) were dissolved in 300 ul tetrahydrofuran (THF) at RT, followed by the addition of amine (0,15 mmol, 1 equiv.). Two hours later, THF was removed under vacuum and the resulting crudes were used for initial screening. All crudes were characterized by mass spectrometry and their purity was assessed by HPLC coupled with a light scattering detector (ELSD) (Supplementary Table 1).

Purification and characterization of ionizable lipid A4B2C8 (CP-LC-0729)

To purify the top-performing lipid, namely CP-LC-0729, its crude product was separated using a CombiFlash NextGen 300+ with gradient elution from 100% of dichloromethane to 50% of 80/20/1 DCM/MeOH/ NH₄OH (aq). CP-LC-0729 was characterized by mass spectrometry (MS) and nuclear magnetic resonance spectroscopy (NMR). MS-QDa: theoretical [M+H]⁺ = 740.63, experimental [M+H]⁺=740.85; ¹H NMR (400 MHz, CDCl₃) δ: 6.76 (m, 1H); 6.37 (d, J= 7.9 Hz, 1H); 4.60 (q, J= 7.1 Hz , 1H); 3.98 (d, J= 5.8 Hz, 2H); 3.36 (m, 2H); 2.78 (t, J= 6.8 Hz, 2H); 2.59 (m, 4H); 2.48 (t, J= 5.9 Hz, 2H); 2.28 (s, 6H); 2.06 (m, 2H); 1.96 (m, 1H); 1.58 (m, 3H); 1.41 (m, 2H); 1.25 (m, 44H); 0.87 (m, 12H).

STAAR cationic lipid (+) CP-LC-0729 synthesis

Lipid CP-LC-0729 (149.5 mg, 0.20 mmol) was dissolved in toluene (0.7 ml) under Argon atmosphere. Then, dimethylsulfate (22.9 ul, 0.24 mmol) was added and one hour later the solvent was evaporated under reduced pressure. Afterwards, the crude reaction was purified using a CombiFlash NextGen 300+ with gradient elution from 100% of DCM to 40% of MeOH to afford the pure product (70%).

MS-QDa: theoretical [M+H]⁺ = 754.66, experimental [M+H]⁺= 754.84;

¹H NMR (400 MHz, DMSO-d₆) δ: 8.38 (m, 1 H); 8.17 (m, 1H); 4.29 (m, 1H); 3.92 (d, J= 5.6 Hz, 2H); 3.49 (m, 2H); 3.33 (m, 2H); 3.09 (s, 9H); 2.68 (m, 2H); 2.60 (m, 2H); 2.41 (m, 1H); 2.19 (m, 1H); 1.80 (m, 2H); 1.57 (m, 1H); 1.48 (m, 2H); 1.36-1.04 (m, 46H); 0.89 (m, 12H).

Nanoparticle Formulation:

General Procedure

For all LNP formulations, mRNA was dissolved in a pH 4, 10 mM sodium citrate solution and combined with the lipid mixtures, which comprised a combination of STAAR ionizable lipids, DOPE, cholesterol, and DMG-PEG2000, at an N/P ratio of 6. For the specific experiments including a fifth cationic lipid, these lipidic and N/P ratios were modified as detailed in Supplementary Table 5.

In the initial screening process (optimization phases 1 and 2), a direct pipetting mixing method was used whereas a microfluidics equipment was employed for the subsequent phases and for the extrahepatic targeting formulations with the permanently cationic lipids. Hence, briefly, the methods followed in each of these stages is as follows:

Direct Pipetting Mixing (Lipid Screening Phases 1 and 2): The ethanolic lipid phase and the aqueous mRNA solution were mixed at an N/P ratio of 6 (mol/mol) by pipetting the aqueous solution into the organic phase with vigorous pipetting for about 10 seconds. The resulting LNPs were diluted 1:1 with a pH 8 buffered solution.²⁴

Microfluidic Mixing (Fine-tuning of lipid structure and cationic lipid formulations): Both the ethanolic lipid phase and the aqueous mRNA solution were mixed using a NanoAssemblr^® Ignite™ (Precision NanoSystems) device with a flow rate ratio (FRR) of 3:1 and a total flow rate (TFR) of 12 ml/min. The resulting LNPs were dialyzed overnight against a pH 8 Tris buffer solution containing 15% sucrose. The final LNP solution was adjusted to a concentration of 100 µg/ml of mRNA, filtered through a 0.22 µm filter, and stored at 4ºC.

Characterization

¹H-RMN were measured using a Bruker 400 MHz NMR spectrometer. HPLC-ELSD-MS was performed on a Waters Alliance equipped with Waters ELSD 2424 and Waters Acquity QDa detectors. The hydrodynamic size, zeta potential and polydispersity index (PDI) physicochemical parameters of LNPs were measured using a Malvern Zetasizer^® Advance Lab Blue Label Equipment. The mRNA encapsulation efficiency and the pKa of LNP were obtained using Quant-IT^® Ribogreen following the manufacturer’s instructions and a 6-(p-toluidinyl)naphthalene-2-sulfonic acid (TNS) assay, respectively⁴⁵. Characterization data is available in supplementary information.

Animals and cell lines

All animal experiments were conducted in agreement with European and national directives for protection of experimental animals. Experimental procedures were approved by the Ethics Committee for Animal Experiments of University of Zaragoza (PI07/23).

8-10 weeks old female BALB/cAnNRj mice were purchased from Janvier Labs. All mice were housed and maintained in specific pathogen–free conditions in the facilities of Centro de Investigaciones Biomédicas de Aragón (Zaragoza, Spain; reference ES 50 297 0012 01). Animals underwent a one-week acclimation period upon arrival at the research facilities. Housing conditions were controlled, maintaining a room temperature of 20-24 °C with 50%-70% humidity, a light intensity of 60 lux and a light-dark cycle of 12 hours. Food and water were provided ad libitum.

HepG2 cells were obtained from the American Type Culture Collection (ATCC). Cells were cultured on RPMI 1640 (Gibco, 31870074), supplemented with 10% Fetal Bovine Serum (Sigma, F7524), 1% Penicillin-Streptomycin Solution (Gibco, 15140122) and 2 mM Glutamax (Fisher, 35050038).

Cellular internalization inhibition

HepG2 cells were seeded in Delta-treated 96-well plates at a density of 10.000 cells/well in complete DMEM and incubated overnight at 37 ºC and 5 % CO₂. Then, cells were pre-treated for 1h with endocytosis inhibitors: amiloride (1 mM), chlorpromazine (15 µM), genistein (150 µM) and methyl-B-cyclodextrin (1.25 mM). After the inhibition treatment, cells were transfected with LNPs encapsulating luciferase mRNA (200 ng mRNA/well) and formulated with the different ionizable lipids. After 4h incubation the cell medium was replaced with fresh complete DMEM and incubated overnight. Cells were lysed with PBS-Triton 0.1 %, cell lysate was transferred to an opaque 96-well white plate and d-Luciferin (GoldBio LUCK-100 resuspended in 100 mM Tris-HCl pH 7.8, 5 mM MgCl₂, 250 μM CoA, 150 μM ATP buffer) was added to each well reaching a final concentration of 150 μg/mL. Luminescence was measured after 5 minutes of incubation at room temperature in a FLUOstar Omega plate reader (BMG Labtech). The efficiency of endocytosis was subsequently evaluated comparing luminescence signal of the untreated wells, and the wells treated with each inhibitor for each LNP.

Hemolysis assay

Erythrocytes were isolated from fresh heparinized human blood sample. A volume of 2 mL of blood was washed three times with neutral PBS (pH 7.4) by centrifugation at 800g for 5 minutes. Afterwards, the sample was resuspended in a total volume of 1 mL either in neutral or acid PBS (pH 6). Isolated erythrocytes were diluted one hundred times in PBS with the corresponding pH. Erythrocytes were incubated with LNPs at a final mRNA concentration of 2.5 ug/ml at 37ºC for 1 hour. Finally, the erythrocytes were centrifuged at 800g for 5 minutes and 60 μl of the supernatant was transferred to a 96-well plate. 0.1% Triton X-100 was included as a positive control for membrane disruption. Absorbance was measured at 540 nm in a FLUOstar Omega plate reader (BMG Labtech).⁴⁶

In vivo safety Evaluation

Animals were inoculated intravenously with 2.5 mg/kg of luciferase-encoding mRNA formulated in the indicated LNPs as described above. Throughout the experiment, mice were periodically weighed and monitored for observable clinical signs of toxicity.

Blood samples were collected from the submandibular vein on days 1 and 2 post-inoculation and serum samples were obtained for biochemical analysis. Samples were centrifuged at 8000 rcf at 4°C for 10 minutes and supernatants were collected. Biochemical analyses were performed using Cobas c-311 (Roche Diagnostics) according to manufacturer instructions.

In vivo imaging

For intramuscular inoculation, animals were injected with 0.05 mg/kg of the indicated Luciferase-encoding mRNA-LNPs into the right thigh muscle. LNPs were diluted in Tris buffer containing 15 % sucrose in a final volume of 30 µL, and administered using a 30G insulin syringe.

For intravenous inoculation, animals were injected into the tail vein with 0.2 mg/kg of the indicated Luciferase-encoding mRNA-LNPs. LNPs were diluted in Tris buffer containing 15 % sucrose in a final volume of 250 µL and administered using a 27G needle.

At 4 hours post-inoculation, mice were anesthetized by inhalation of Isoflurane (IsoVet) 4% using an animal inhalation chamber. The maintenance of the anesthesia was sustained at 1.5% isoflurane. D-luciferin (12507, Quimigen) was intraperitoneally injected at 150 mg/kg diluted in PBS. Luminescence images were acquired 20 minutes after luciferin inoculation.

For ex vivo luminescence studies, mice were euthanized 20 minutes after luciferin injection and liver, spleen, lungs, heart, kidneys and intestine were aseptically removed.

In vivo and ex vivo luminescence images as well as fluorescence images of DiR-labeled LNPs were acquired using the IVIS Lumina XRMS Imaging System, following manufacturer's instructions.

Data availability

The data supporting the conclusions of this research are accessible within the article and its supplementary files. The source data is included in this paper.

Statistical analysis

Data are presented as mean ± SD or mean. Student’s t-test, one-way or two-way analysis of variance (ANOVA) followed by Tukey test was applied for comparison between two groups or among multiple groups using GraphPad Prism 10.0, respectively.

Acknowledgements

This study was supported by Gobierno de Aragón (Spain) through project IDMF/2021/0009 (Nuevas tecnologías para el diseño y obtención de vacunas de ARN en Aragón). AP is funded by Industrial Doctorate programme (Spain) grant number DIN2021-011799 financed by the Ministerio de Ciencia e Innovación MCIN/AEI / 10.13039/501100011033. Authors would like to acknowledge the use of Servicios Científico Técnicos del CIBA (IACS-Universidad de Zaragoza) and Servicio de análisis bioquímicos de CIMA (Universidad de Navarra).

Author contributions

Conceptualization: AP, JH, JGW, JMO; Lipid molecular design and synthetic methodology: JH, AP, JGW; Lipid synthesis and characterization experiments: AP, JH, BB; LNP formulation and characterization: AT, TA, DDM; mRNA synthesis: DC; Biological and biochemical assays: AL, AGL, EM; Supervision: JGW, JMO, EPH; Writing the original draft: JH, AP, JGW; Manuscript review: all authors.

Competing interests

AP, JH, DDM, AT, JMO and JGW are inventors on patents related to this publication. All the authors declare to be employees of Certest Biotec.

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Yes there is potential Competing Interest. Álvaro Peña, Juan Heredero, Diego de Miguel, Alfonso Toro, Juan Martínez-Oliván and Javier Giménez-Warren are inventors on patents related to this publication. All the authors declare to be employees of Certest Biotec.

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STAAR Lipids: A Novel Ionizable Lipid Synthetic Platform for Efficient mRNA Delivery In Vivo with Tunable Lung Targeting

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