One-Pot Parallel Synthesis of Lipid Library via Thiolactone Ring

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Cite This: Bioconjugate Chem. XXXX, XXX, XXX−XXX

One-Pot Parallel Synthesis of Lipid Library via Thiolactone Ring Opening and Screening for Gene Delivery Mijanur R. Molla,*,†,# Alexander Böser,† Akshita Rana,† Karina Schwarz,† and Pavel A. Levkin*,† †

Institute of Toxicology and Genetics, Karlsruhe Institute of Technology, Hermann-von-Helmholtz-Platz 1, 76344 Eggenstein-Leopoldshafen, Baden Württemberg, Germany S Supporting Information *

ABSTRACT: Efficient delivery of nucleic acids into cells is of great interest in the field of cell biology and gene therapy. Despite a lot of research, transfection efficiency and structural diversity of gene-delivery vectors are still limited. A better understanding of the structure−function relationship of gene delivery vectors is also essential for the design of novel and intelligent delivery vectors, efficient in “difficult-to-transfect” cells and in vivo clinical applications. Most of the existing strategies for the synthesis of gene-delivery vectors require multiple steps and lengthy procedures. Here, we demonstrate a facile, three-component one-pot synthesis of a combinatorial library of 288 structurally diverse lipid-like molecules termed “lipidoids” via a thiolactone ring opening reaction. This strategy introduces the possibility to synthesize lipidoids with hydrophobic tails containing both unsaturated bonds and reducible disulfide groups. The whole synthesis and purification are convenient, extremely fast, and can be accomplished within a few hours. Screening of the produced lipidoids using HEK293T cells without addition of helper lipids resulted in identification of highly stable liposomes demonstrating ∼95% transfection efficiency with low toxicity.

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he discovery of RNA interference (RNAi)1 has opened a new avenue in the field of gene therapy,2,3 which has been of great interest for treating a wide variety of human diseases ranging from viral infections4 to hereditary disorders5 and cancer.6−8 By activating the RNAi pathway, siRNA can knock down a gene by targeting and cleaving complementary mRNA.9,10 Another commonly used nucleic acid is plasmid DNA (pDNA) harboring a gene of interest. Hence, cellular transfection is used for both loss-of-functions as well as gain-offunction genetic experimentation. There are two types of commonly used gene delivery vectors: viral vectors and synthetic vectors. Synthetic vectors offer greater flexibility, more facile manufacturing, and safer delivery compared to viral vectors.11,12 To date, delivery approaches of nucleic acids involve direct conjugation of carrier with RNA,13,14 complexation with cationic lipids,15−17 cationic polymers,18−20 and antibody fusion proteins.21,22 Among all of these approaches, lipid-based transfection reagents are well investigated synthetic materials for nucleic acid delivery. However, there are some challenges yet to be addressed: (i) despite years of research, there is still no clear understanding of the structure−function relationship in synthetic transfection reagents, and (ii) there are © XXXX American Chemical Society

cells (e.g., primary cells, blood-derived cells, stem cells, etc.) that become more and more important for biomedical applications, but are very difficult to transfect. A possible solution to these problems is to perform unbiased screenings of large libraries of lipid to gain better understanding of the structure−function relationship and to identify novel, robust, and highly efficient transfection reagents. There are several commercially available cationic lipids used as transfection reagents.23−26 However, these lipids are usually synthesized via multiple steps, involving various chemical protection−deprotection steps, use and removal of catalysts, purifications, and individual optimizations,3 thereby introducing limitations in the ability to construct a library of substantial size and variety. To address these problems, combinatorial synthesis27−30 was proposed to generate libraries of artificial lipids for cell transfection.31,32 Here, we design a combinatorial library of lipidoids for gene delivery applications. Until now, there have Received: January 4, 2018 Revised: February 15, 2018

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DOI: 10.1021/acs.bioconjchem.8b00007 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Bioconjugate Chemistry

Figure 1. Three-component, one-pot synthesis of lipidoids using nucleophilic ring opening of thiolactones followed by the disulfide exchange reaction. Structures of (a) amines; (b) pyridyl disulfide derivatives; (c) thiolactone derivatives; (d) representative reaction scheme.

been only a few reports regarding synthesis of combinatorial libraries of lipids for nucleic acid delivery.29 For instance, Akincet al. developed two chemical methods based on azaMichael addition31,33,34 and amine addition to epoxide35 for delivery of RNAi therapeutics. Nevertheless, despite the simplicity, these synthetic strategies require high temperature, very long reaction time (several days), and laborious purifications. Recently, we described a two-step and a onestep method based on thiol−yne, thiol−ene photoclick reactions36,37 and direct alkylation of amines,38 respectively. Sheng et al. reported synthesis of sterol-based cationic lipids using copper-mediated39 azide−alkyne cycloaddition (CuAAC) click chemistry. In most cases, long synthetic procedures and complicated purification steps are the major drawback. In addition, most of the existing strategies for the combinatorial synthesis of lipidoids are two-component reactions requiring large variations in building blocks to produce a large library. Here, we developed an extremely simple, rapid, cost-effective, and highly efficient combinatorial synthetic methodology with easy purification. The method is based on nucleophilic ring opening of thiolactone (cyclic thioester) in a three-component, additive-free (catalyst, acid, or base), one-pot reaction. The reaction utilizes the latent functionality of thiolactone as previously demonstrated.40−45 Using this method we synthesized a library of 288 structurally diverse lipidoids from 26 (18 + 4 + 4) different building blocks (Figure 1). The produced library was used in a high-throughput cell transfection screen, which resulted in the identification of several lipidoids possessing up to 95% transfection efficiency in HEK293T cells, similar to that of commercially available transfection reagents, Lipofectamine 2000 (L2K) and ScreenFect A (SFA). There are several advantages of our design and synthetic strategy: (i) the in situ generation of a thiol by nucleophilic ring opening of thiolactone with amines, followed by a thiol− disulfide reaction, is a rapid, simple, versatile one-pot protocol, critical for parallel combinatorial syntheses; (ii) there are numerous commercially available structurally diverse amines that can be exploited as building blocks; (iii) since it is a threecomponent one-pot reaction, only a few chemical variations in building blocks can result in chemical libraries of extremely large diversity and size. For example, only ten different building

blocks in each of the three components would result in 1000 lipidoids. The extensive structure−activity data obtained from the method can significantly increase the probability of finding a potential candidate for efficient gene delivery.38 To generate the library, a parallel synthesis was performed using different thiolactone derivatives, pyridyl disulfide derivatives, and amines. In a typical synthesis, excess amine (5 or 6 equiv) was added to the mixture of thiolactone (1 equiv) and pyridyl disulfide derivatives (1.1 equiv) in CHCl3, and the reaction mixture was allowed to stir for 3 h at room temperature. The CHCl3 was evaporated and the crude product was washed with methanol to get pure product. The progress of the reaction was monitored spectroscopically as the generated byproduct 2-pyridothione was UV active.46 The kinetics of the reaction showed that the ring opening with primary amine and secondary amine takes 30 and 100 min, respectively, for the reaction to reach completion, in the case of small scale reactions (Figure S1). Using this one-pot synthetic methodology, a total of 288 lipidoids were synthesized within a few hours, which is the inimitable feature of this particular method of synthesizing nonviral transfection agents compared to most of the reported procedures.33−38 After purification, all the synthesized lipidoids were characterized by MALDI-TOF mass spectrometry and selected molecules by NMR spectroscopy. For the initial screening, at first, liposomes were prepared from the lipidoids using 200 mM sodium acetate buffer (pH = 5.0) at optimum conditions (see Supporting Information for detailed preparation procedure). In most of the reported procedures or in the case of commercially available transfection agents23−26 during the preparation of liposomes, phosphatidylethanolamine (DOPE) was used together with a lipid in order to increase the liposomal stability, aid fusion with cellular membranes, and boost endosomal escape of nucleic acid within cells.47 The use of DOPE or any additional lipid, however, makes those methods more complicated and expensive. Surprisingly, liposomes produced by using our lipidoids showed good stability and efficiency without the addition of DOPE. The prepared liposomes were characterized by dynamic light scattering (DLS), zeta potential analysis, and cryo-transmission electron microscopy (cryo-TEM). The liposomes as well as the lipoplexes showed sizes between 50 and 100 nm and long-term B


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Figure 2. Overview of the transfection efficiencies obtained during an initial screening in HEK293T cells. (a) Color-coded representation of transfection efficiencies of all 288 lipidoids based on the building blocks they consist of (for the explanation of abbreviations see Figure 1). (b) Color-coded categorization of the transfection efficiencies. (c) Tabular summary of transfection efficiencies. (d) Chemical structure of the three bestperforming lipidoids.

liposomes of T18U-PY16-A1 were found to be stable for at least 180 days, showing very similar transfection efficiency even after 180 days (Figure S2). In an initial screen, 73% of all lipidoids from the synthesized library showed transfection efficiency in HEK293T cells. About 27% of lipidoids could not be used in the initial screening due to precipitation in the acetate buffer during liposome preparation (Figure 2c). Notably, 46 lipidoids showed transfection efficiencies higher than 40%, and among them, three (T18U-PY16-A1, T18U-PY12-A8, and T14-PY12-A8) (see Figure 2d for structure) exhibited efficiencies equal to or greater than that of SFA and L2K (Figure S3, Table S2). The transfection table (Figure 2a,b) shows that overall enhanced delivery performance (more than 60% efficiency) was achieved for lipidoids with either one unsaturated tail or one shorter tail (carbon number 12 or 14) or both short tails. The flow cytometry results of the cell control, positive control, and three best performing lipidoids are shown in Figure S4. The following conclusions can be drawn from the initial screening results. Among the three best performing lipidoids, the first lipidoid (T18U-PY16-A1) has unsaturated tail T18U, the second one (T18U-PY12-A8) has both unsaturated and shortest tail (PY12-number of carbon atom = 12), and the third lipidoid (T14-PY12-A8) has the shortest tail, PY12. The better performance of unsaturated hydrophobic tails might be attributed to the restricted chain flexibility, permitting enhanced interaction with lipids from the cell membrane. A similar phenomenon is reported in the literature, where lipids with unsaturated tails have higher transfection efficiency than their saturated analogues due to increased membrane fluidity or efficient membrane fusion and endosomal escape.48−50

stability over the period of 180 days. Lipoplexes were produced by complexation of liposomes with eGFP plasmid DNA (pDNA) (see SI for detail). The acidic buffer was used in the process to protonate the lipidoid headgroup in order to initiate the complexation with negatively charged pDNA. Subsequently, we compared size and zeta potentials of selected liposomes and lipoplexes (Table S1). In vitro cell transfection was performed using a one-step method, where freshly trypsinized and resuspended HEK293T cells were added directly to the lipoplexes, followed by transfer to culture wells. For the screening, 75 ng of eGFP-pDNA was used per well in a 96-well plate and each lipidoid was analyzed at 3 different ratios in triplicate (lipidoid to pDNA = 36:1, 24:1, and 12:1 in wt/wt). To form lipoplexes, 2.7 μg (36 × 75 ng), 1.8 μg (24 × 75 ng), or 0.9 μg (12 × 75 ng) of lipidoids in the form of liposomes were mixed with 75 ng of pDNA separately to obtain ratios of 36:1, 24:1, and 12:1, respectively, followed by addition to the cells. After overnight incubation, nuclei of adherent cells were stained with Hoechst, and imaged with an automated fluorescence microscope. Flow cytometry was used to calculate the percentage of transfected cells (transfection efficiency) as well as to quantify viable cells by PI (propidium iodide) staining. Two commercially available transfection agents, ScreenFectA (SFA) and Lipofectamine 2000 (L2K), were used as positive controls. In all the cases, transfection efficiencies for the 36:1 ratio were found to be higher compared to 24:1 and 12:1. The efficiency increased with increasing the amount of lipidoids. In order to evaluate the stability of the produced liposomes, we chose the top performing lipidoid (T18U-PY16-A1) and repeated transfection experiments using the same batch of liposomes over 180 days. Although the helper lipid DOPE was not used in the liposome preparation, C


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Figure 3. Effect of the total number of tail-atoms (TNTA) on the transfection efficiency. (a) Chemical structures of two lipidoids and clarification how their corresponding TNTA is calculated. (b) Percentage of the number of lipidoids with transfection efficiencies (TFE) higher than 40% (N40) and the same TNTA (NTNTA)(green bars) and the number of lipidoids with transfection efficiencies higher than 60% (N60) bearing the same TNTA (red bars).

structures among the building blocks for the headgroups: the amine building block has to consist of a primary amine with structure R-(CH2)2-NH2, whereas R consists of (i) at least one tertiary amine, (ii) aliphatic or cyclic structures (5 or 6membered rings), (iii) no aromatic functionalities, and (iv) the presence of nitrogen heteroatom for protonation. Note that headgroups of several lipidoids in this library have more than one protonizable nitrogen center, which theoretically should lead to high surface charges even after lipoplex formation by DNA-complexation and, therefore, enhanced transfection efficiency. This trend, however, is not observed in our library. It is known that features such as the particle size, surface charge, and morphology of lipoplexes play very important roles in transfection. Smaller particles with higher positive surface charge were shown to possess higher transfection efficiency.36 To investigate the role of size and surface charge in detail, four lipidoids from the top based on performance (T18U-PY16-A1, T18U-PY12-A8, T14-PY12-A8, and T14-PY16-A13) were selected. All four lipidoids have 3 different head groups (A1, A8, and A13) and 4 different tails (T18U-PY16, T18U-PY12, T14-PY12, and T14-PY16). We then constructed a smaller sublibrary of (3 × 4 = 12) 12 lipidoids using these 3 head groups and 4 tails in a combinatorial approach (Figure 4a). It is interesting to note that simple combination of the top building blocks (i.e., altering head groups and tails) did not show any increase in efficiency and, on the contrary, the efficiency dropped by 60−80% (Figure 4b). The size and surface charge of the corresponding liposomes were compared and correlated to the transfection efficiency. All 12 liposomes showed comparable sizes between 60 and 160 nm as well as positive zeta potentials in the range of 25−100 mV (Table S1). This observation alone could not explain the lower transfection efficiency of some lipidoids from the sublibrary although they contain either one of the headgroups or one of the tail groups of the top performing lipidoids. To get more insight into the transfection results, we examined size and surface charge of the corresponding lipoplexes. However, only four of these lipoplexes showed high transfection efficiencies in cell experiments, whereas the other lipoplexes showed low efficiencies in the range of 10−40%. The common features of the four efficient lipoplexes are small sizes (60−100 nm) and positive zeta-potentials (35−70 mV) (Figure 4c,d). The residual eight

Recently, in our group and in several other groups it was demonstrated that shorter lipid chains, such as C12 or C14, could enhance transfection efficiency.36,51,52 Here we also observed the same phenomenon for T14-PY12-A8. For more in-depth analysis of the structure−function relationship between the tail structures and the transfection efficiency we compared all lipidoid tail structures and found that there is a correlation between transfection efficiency and the total number of tail atoms (“TNTA”). Figure 3a clarifies the calculation of TNTA values. The lipidoid T14-PY16-A13 has a TNTA of 35, and the lipidoid T18U−P12-A3 has a TNTA of 35U. The descriptor U indicates the unsaturated bond in the thiolactone derived tail (Figure 3a). Figure 3b shows the percentage of the lipidoids (green bars) with transfection efficiencies higher than 40% (N40) for the lipidoids with the same TNTA (NTNTA). Here, a clear correlation between TNTA value (high-41, 39, and low-33) and transfection efficiencies (higher than 40%) is observed. More importantly, this correlation can be drawn regardless of the headgroup present in the lipidoid structure: for example, lipidoids with a TNTA of 37 are found to be very inefficient in the screening despite the fact that some of those lipidoids also consisted of head groups like A1 or A17, which often showed high efficiencies in other lipidoids of the screening. The number of lipidoids with transfection efficiencies higher than 60% (N60) (red bars) is shown in Figure 3b. Here, a similar trend is observed: a high TNTA, especially in combination with an unsaturated tail, as well as a low TNTA give rise to high transfection efficiencies. Note that a TNTA of 33 or 35/35U has shown a high number of lipidoids with high transfection efficiencies. TNTA values of the 3 best performing lipidoids T18U-PY16-A1, T18U-PY12-A8, and T14-PY12-A8 are 39U, 35U, and 31, respectively. In terms of the headgroup,16 the three top-performing lipidoids (T18U-PY16-A1, T18U-PY12-A8, and T14-PY12-A8) have head groups based on 2-(4-methyl-piperazin-1-yl)-ethylamine (A8) and on N,N-diethylethylenediamine (A1). There are reports in the literature where the diethylamine headgroup showed enhanced transfection, which we also observed for our top-performing lipidoid. By analyzing and comparing the structural composition of all the headgroups that are present in effective lipidoids, we could conclude the following common D


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Figure 4. (a) Sublibrary of (3 × 4 = 12) 12 lipidoids composed in a combinatorial way using 3 headgroups and 4 tails from four top performing lipidoids. Detailed experiments of those 12 lipidoids: (b) transfection efficiencies, (c) hydrodynamic diameter of lipoplex, (d) zeta potential of lipoplex, (e) fluorescence microscope images of in vitro transfection of pDNA into HEK-293T cells, (f) cryo-TEM images of three selected liposomes: top left, T14-PY16-A13; top right, T18-PY16-A1; bottom, T14-PY12-A8.

lipoplexes showed greatly enhanced sizes (130−1300 nm) as well as only negative zeta potentials (−20 mV to −80 mV). Interestingly, the size of the efficient lipoplexes remained the same even after 1 h (Figure S5). The very small particle size in combination with positive surface charge, which facilitates interaction with negatively charged cell membrane, might explain the observed higher transfection efficiency.53 The fluorescence microscopy images of transfected HEK293T cells using these 12 lipidoids are shown in Figure 4e, where the above-mentioned trend in transfection is clearly visible. We have also investigated the morphology of three liposomes from the library by the cryo-transmission electron microscope (cryo-TEM). TEM images of those selected liposome samples revealed formation of unilamellar or multilamellar vesicles (Figure 4f). Since the 12 analyzed liposomes shared strong structural similarities, at this point, it is hard to define a relationship between the structure of the lipidoid and favorable

characteristics (small size, positive surface charge) after lipoplex formation. These results confirm that, in addition to the chemical structure of lipids, particle size and surface charge of lipoplex are also important parameters in the context of cell transfection. We would like to emphasize that other factors like stability of lipoplexes, membrane fusibility, and lipid/DNA ratio are also very important parameters for transfection. In order to gain more understanding of the transfection behavior, we performed various in vitro transfection experiments with the top 11 lipidoids (efficiency more than 60%) identified from the initial screening. In order to sensitize transfection and compare results with commercial positive controls, we used a mixture of 10% GFP encoding pDNA and 90% empty vector (LacZ) for the transfection. This experiment demonstrated that T18U-PY16-A1 (highest performing lipidoid) has 35% higher transfection efficiency compared to the positive control (Figure S6). Next, we analyzed two more cell E


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efficient gene delivery vectors. We believe that knowledge about the basic structure−function relationship obtained in these studies might help in designing superior nonviral gene or drug delivery systems for both. Further structural analysis in detail and in vivo application are under investigation in our laboratory.

lines, HeLa cells and a more difficult-to-transfect cell line, HepG2 (liver hepatocellular carcinoma). In HeLa cells, all three best performing lipidoids showed high transfection comparable to SFA (Figure S7), but in HepG2 cells, though the efficiencies for all lipidoids were not very high, a significant increase in efficiency was observed for some lipidoids compared to the positive control (Figure S8). T18-PY12-A3 and T16-PY12-A1 showed almost three times higher transfection efficiency than the positive control. This can be attributed to their common structural features (shortest tail: C12, headgroup A1 and A3 have all the required features stated in above discussion) with three best performing lipidoids in the initial screening in HEK293T cells. Biocompatibility and low cytotoxicity of transfection agents are very important criteria for both in vitro and in vivo delivery of nucleic acids. To evaluate the cytotoxicity of lipidoids, propidium iodide (PI) was used to stain necrotic cells and the viability was measured by flow cytometry analysis. In the case of HEK-293T cell lines, all three best performing lipidoids (T18U-PY16-A1, T18U-PY12-A8, and T14-PY12-A8) showed viability at ∼85% or above in a concentration range at which the experiments were performed (45 μg/mL to 270 μg/mL) (Figure S9). For HepG2 cells two best performing lipidoids T18-PY12-A3 and T16-PY12-A1 showed cell viability ∼70% and ∼30% at highest concentration (270 μg/mL) (Figure S10). For HeLa cells, all three best performing lipidoids showed moderate cell viability (∼70%) (Figure S11). In summary, the utility of a one-pot combinatorial approach for the synthesis of a chemically diverse set of lipid-like materials within a couple of hours is described. This accelerated and versatile synthetic protocol enables an efficient parallel synthesis of large libraries of nonviral transfection agents for delivery of plasmid DNA. Notably, from the screening of the produced library, about 5% of all 288 synthesized lipidoids displayed very high transfection efficiency (more than 60% efficiency), and among them, ∼1% of lipidoids showed efficiencies above 90%. Most importantly, they share common structural features, which suggest certain design criteria for creating future intracellular delivery agents. The structural features are (i) amide linkage; (ii) two alkyl tails; (iii) at least one of the tails either unsaturated or short in length (C12 or C14); (iv) head groups with one or more protonizable tertiary amine, cyclic (5 or 6 member) or acyclic but nonaromatic. In addition, the total number of tail-atoms (TNTA) had to be either high (e.g., 39−41) or low (e.g., 33−35) to achieve high transfection efficiency. Apart from the structural requirement, there are other important parameters such as size and surface charge, which play an important role in transfection. The analysis of the structure−activity relationship revealed that hydrodynamic diameter and z-potential of the lipoplex need to be smaller (50−100 nm range) and positive (+50 to +70 mV), respectively. Hence, all these parameters need to be considered together when designing novel transfection vectors. Moreover, without the use of any helper lipid or cholesterol during liposome preparation the highest performing lipidoid, T18UPY16-A1, showed high liposome stability, ∼95% transfection efficiency, and very low toxicity. This result is as good as the commercially available transfection agents, where phospholipids are often used as helper lipid. In particular, three key factors, (i) very short reaction time, (ii) easy fine-tuning of the lipidoid structure, and (iii) additional lipid or cholesterol-free liposome preparation, make our method very convenient, versatile, and cost-effective, and, hence, suitable for the identification of novel

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.bioconjchem.8b00007. Materials and methods; synthesis and analysis including schemes and spectra (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Mijanur R. Molla: 0000-0002-8057-683X Pavel A. Levkin: 0000-0002-5975-948X Present Address #

University of Calcutta, Department of Chemistry, 92 APC Road, Kolkata, India-700009 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the Humboldt Foundation for providing MRM’s fellowship, Dr. Markus Drechsler from University of Bayreuth, Germany for the cryo TEM measurement. We also acknowledge Dr. Gerald Brenner-Weiß (IFG, KIT) for providing access to MALDI-ToF mass spectrometry analysis. PAL thanks ERC2013-Starting Ggant (337077-DropCellArray) for the financial support.

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REFERENCES

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