Oligonucleotides, such as small interfering RNA (siRNA), are promising form of drugs for the treatment of a wide range of diseases. Oligonucleotides based active pharmaceutical ingredients (APIs) induce therapeutic effects by modulation of gene expression. Two decades ago, it was demonstrated that intoduction of a synthetic siRNA into the cytoplasm of the cell results in specific degradation/neutralization of complementary mRNA. This leads in to suppressed gene expression through a process called RNA interference (RNAi). However, the use of siRNA as API is challenging due to its limited bioavailability via systemic administration. In order to efficiently deliver siRNA into cells, it was necessary to develop a sophisticated platform. In 2018 U.S. Food and Drug Administration approved the first siRNA therapeutic, Onpattro (Patisiran), based on LNP. In master's thesis, we prepared LNPs with encapsulated siRNA, Patisiran. LNPs were prepared according to design of experiments principle with ethanol injection method. We used the same lipid components as the commercial product (Onpattro). The aim of our study was to examine the impact of process parameters on the formation of LNPs with encapsulaed Patisiran. We studied the effect of Patisiran concentration in the aqueous phase, lipid concentration in the ethanol phase, mixing speed, injection speed, injection time, pH, temperature and ionic strength on LNP size, LNP size distribution, zeta potential, concentration of non-encapsulated patisiran and thermal stability of LNP. The majority of the prepared LNPs were within the size range of up to 500 nm, homogeneous and had a zeta potential within the range of ± 30 mV. The melting temperature of the produced LNPs was nearly identical measured melting temperature of the drug Onpattro (Patisiran), indirectly indicating the comparability of the particles. We have demonstarated, by using different techniques, such as dynamic light scattering, differential dynamic calorimetry, ultraviolet-visible spectroscopy and cryo-electron microscopy, that some process parameters have a sagnificant statistic impact on the properties of LNPs with encapsulated siRNA. Our results showed that the concentration of Patisiran and the concentration of lipids in the ethanol phase affect the average size of LNPs. Higher concentration of the siRNA and higher ion strength lowered the concentration encapsulated Patisiran. Furthermore, higher ion strength increased the thermal enthalpy of LNPs. Understanding which process parameters affect the physico-chemical properties of LNP is crucial to ensure the reproducibility and robustness of their preparation process. The delivery of oligonucleotide therapeutics based on LNP technology enables the wider potential of this technology in a wide variety of future applications.
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