Gene delivery to the liver is a complex process and faces many challenges due to both biochemical and biophysical barriers that exist at multiple levels. Here are some of these barriers:

1. Extracellular Barriers: After administration, gene vectors need to navigate the circulatory system to reach the liver. They must evade the immune system, avoid sequestration by non-target organs like the spleen or lungs, and survive in the bloodstream long enough to reach the liver. Some non-viral vectors may also interact with serum proteins forming protein “corona” which could affect their targeting ability and immune profile.
2. Endothelial Barriers: The liver is a highly vascularized organ, and the fenestrated endothelium of liver sinusoids acts as a barrier for larger gene delivery vectors. Although these fenestrations can allow vectors less than 100 nm in size to pass, larger vectors may struggle.
3. Cellular Uptake: To enter liver cells (hepatocytes), vectors must cross the cellular membrane. This often involves binding to specific receptors on the cell surface, which can be a limiting factor if the receptors are not expressed at high levels or if they are not accessible due to the presence of the glycocalyx.
4. Endosomal Escape: Once inside the cell, the vectors often end up in endosomes. To prevent degradation in the lysosomes, the vector must escape from the endosome into the cytoplasm. This is a major challenge, particularly for non-viral vectors.
5. Nuclear Entry: For the transgene to be expressed, the DNA must enter the nucleus. This is another major barrier, particularly for non-dividing cells like hepatocytes. Viral vectors often have mechanisms to transport their DNA into the nucleus, but non-viral vectors may struggle with this step.
6. Transgene Expression: Once in the nucleus, the transgene needs to be effectively transcribed and translated into protein. Factors such as the choice of promoter, the presence of insulator sequences, and the integration site (for integrating vectors) can affect the level and duration of transgene expression.
7. Immune Responses: Lastly, both the vector and the transgene can induce immune responses, leading to the elimination of transfected cells and/or rapid clearance of the vector, thus limiting the effectiveness of the therapy.

Different strategies are being developed to overcome these barriers, such as designing vectors with improved targeting and endosomal escape capabilities, using tissue-specific promoters to drive transgene expression, and combining gene therapy with immunomodulatory treatments to reduce immune responses. However, it’s a delicate balance to increase transfection efficiency and expression while maintaining safety and minimizing off-target effects.