(622a) Development of Synexomes: Next Generation Lipid-Based Drug Delivery

The field of liposomal drug delivery has been burgeoning over the past several decades. However, the efficacy of this drug delivery system is limited by its susceptibility to the human immune system^1,2. The goal of this research project is to generate completely synthetic lipid nanoparticles (SynExomes) that are produced using a microfluidic system and can persist longer in the human body by resisting phagocytosis, especially upon encountering opsonins and macrophages.

The overall goal of this research project is to generate completely synthetic lipid nanoparticles (SynExomes) that are produced using a microfluidic system and can persist longer in the human body by resisting phagocytosis, especially upon encountering opsonins and macrophages. This goal will be achieved by pursuing three subordinate objectives: i) Modulating the composition of lipid bilayer and conjugating poly-(carboxybetaine) (PCB) to the lipid bilayer to prevent opsonin adsorption on the liposomal surface ii) Surface modification of liposomes by attachment of CD47 protein to delay phagocytosis iii) Determining the impact of the above modifications on SynExome half-life in vivo. The proof-of-concept efficacy of this platform will be demonstrated in an infected chronic diabetic wound model where presence of macrophages, opsonins, and inflammation is highly elevated and localized. Here, we describe the first step of the process – synthesis of PCB and its conjugation to the lipid 1,2-distrearoyl-sn-glycero-3-phosphoethanolamine (DSPE). We also demonstrate the proof-of-concept functionality of the microfluidic platform for the production of lipid nanoparticles (LNPs).

Methods

DSPE-PCB synthesis involves four distinct steps:

Monomer synthesis: Synthesis of carboxybetaine tertiary butyl ester monomer was performed as described by Cao et al³. 2-(dimethylamino)ethyl methacrylate and tert-butyl bromoacetate were dissolved in acetonitrile and reacted under nitrogen protection for 24 h at 50 °C. The monomer was precipitated out of the reaction mixture using excess diethyl ether, dried and stored in a vial under nitrogen protection at -20°C.

Polymer synthesis: Synthesis of NHS-PCB-tBu polymer was performed using a reversible addition-fragmentation chain transfer (RAFT) polymerization reaction⁴. Optimized amounts of the monomer, (RAFT agent), and AIBN were dissolved in N,N-dimethylformamide (DMF). The reaction mixture was placed in an ice bath and nitrogen gas was purged into the reaction liquid. After one hour, the purging was stopped, and polymerization reaction was carried out at 70 °C. The unreacted starting material was separated from the polymer product by dialysis.

DSPE-PCB-tBu conjugation: NHS-PCB-tBu polymer and DSPE lipid were dissolved in a mixture of chloroform, DMF and triethylamine and the conjugation reaction was carried out at room temperature for five days⁵. The product fraction containing DSPE-PCB-tBu, unconjugated PCB-tBu and unconjugated DSPE were precipitated out from the remainder of the reaction mix using excess diethyl ether. Acetonitrile extraction was of the desired product (DSPE-PCB-tBu and unconjugated PCB-tBu) was carried out and the precipitates were dried overnight using hi-vac.

Hydrolysis of DSPE-PCB-tBu: The precipitates containing DSPE-PCB-tBu and unconjugated PCB-tBu were hydrolyzed in trifluoroacetic acid for 4 hours at room temperature. The resulting DSPE-PCB and unconjugated PCB were then precipitated out and unconjugated PCB-tBu was separated from the desired product by diafiltration.

Microfluidic platform evaluation for LNP synthesis: 2.5 mM lipid solution containing 35 mol% cholesterol and 65 mol% DPPC in ethanol was used to make LNPs using a commercially available microfluidic cartridge (NanoAssemblr, Precision Nanosytems). The LNPs were then diluted 1:1 in PBS, dialyzed in PBS to remove ethanol, and filtered using 0.2 µm filter. Dynamic Light Scattering (DLS) and transmission electron microscopy (TEM) were used to determine LNP size and polydispersity.

Significance

The synthetic nature and homogeneity in size of SynExomes achieved through a microfluidic production platform circumvents the issue of variability in size. Use of PCB in the SynExome formulation does not require the use of significant amounts of cholesterol in the bilayer to stabilize it. Therefore, it does not restrict the lipid choices, as in case of PEGylated liposomes. Similarly, a first of its kind CD47 ligand conjugation to lipid through PCB could make these particles more resistant to phagocytosis than conventional liposomes.

References

1. Dams, E. T., P. Laverman, W. J. Oyen, G. Storm, G. L. Scherphof, J. W. van Der Meer, F. H. Corstens, and O. C. Boerman. 2000. “Accelerated Blood Clearance and Altered Biodistribution of Repeated Injections of Sterically Stabilized Liposomes.” The Journal of Pharmacology and Experimental Therapeutics292 (3): 1071–79. https://www.ncbi.nlm.nih.gov/pubmed/10688625.
2. Wassef, Nabila M., Gary R. Matyas, and Carl R. Alving. 1991. “Complement-Dependent Phagocytosis of Liposomes by Macrophages: Suppressive Effects of ‘stealth’ Lipids.” Biochemical and Biophysical Research Communications. https://doi.org/10.1016/s0006-291x(05)80266-9.
3. Shaoyi Jiang, Zhiqiang Cao, Hong Xue, Lei Zhang. 2013. Self-assembled particles from zwitterionic polymers and related methods. USPTO 8617592B2. US Patent, filed May 3, 2012, and issued December 31, 2013.
4. Cao, Zhiqiang, Lei Zhang, and Shaoyi Jiang. 2012. “Superhydrophilic Zwitterionic Polymers Stabilize Liposomes.” Langmuir: The ACS Journal of Surfaces and Colloids28 (31): 11625–32. https://doi.org/10.1021/la302433a.
5. Rajendrakumar, Santhosh Kalash, Ning-Chu Chang, Adityanarayan Mohapatra, Saji Uthaman, Byeong-Il Lee, Wei-Bor Tsai, and In-Kyu Park. 2018. “A Lipophilic IR-780 Dye-Encapsulated Zwitterionic Polymer-Lipid Micellar Nanoparticle for Enhanced Photothermal Therapy and NIR-Based Fluorescence Imaging in a Cervical Tumor Mouse Model.” International Journal of Molecular Sciences19 (4). https://doi.org/10.3390/ijms19041189.