Downregulation of gene expression via siRNA has been an important research tool in in vitro studies.  Susceptibility of siRNAs to nuclease degradation, though, makes use of them in vivo problematic.  In 2005, researchers working with hepatitis B virus(HBV) in rodents, determined that certain modifications of the siRNA prevented degradation by nucleases within the plasma and lead to increased gene silencing compared to unmodified siRNA.  Modifications to the sense and antisense strands were made differentially.  With respect to both sense and antisense strands, 2-OH was substituted with 2-fluoro at all pyrimidine positions.  In addition, sense strands were modified at all purine positions with deoxyribose, antisense strands modified with 2-O-methyl at the same positions.  The 5 and 3 ends of the sense strand were capped with abasic inverted repeats, while a phosphorothioate linkage was incorporated at the 3 end of the antisense strand.
Although this research demonstrated a potential RNAi therapy using modified siRNA, the 90% reduction in HBV DNA in rodents resulted from a 30 mg/kg dosage with frequent administration.  Because this is not a viable dosing regime, this same group looked at the effects of encapsulating the siRNA in a PEGylated lipid bilayer, or SNALP.  Specifically, the lipid bilayer facilitates uptake into the cell and subsequent release from the endosome, the PEGylated outer layer providing stability during formulation due to the resulting hydrophilicity of the exterior.  According to this 2005 study, researchers obtained 90% reduction in HBV DNA with a 3 mg/kg/day dose of siRNA for three days, a dose substantially lower than the earlier study.  In addition, in contrast to unmodified or modified and non-encapsulated siRNA, administration of SNALP-delivered siRNA resulted in no detectable levels of interferons, such as IFN-a, or inflammatory cytokines associated with immunostimulation.  Even so, researchers acknowledged that more work was necessary in order to reach a feasible dose and dosing regime.
In 2006, researchers working on silencing of apolipoprotein B(ApoB) in non-human primates achieved 90% silencing with a single dose of 2.5 mg/kg of SNALP-delivered APOB-specific siRNA.  ApoB is a protein involved with the assembly and secretion of very-low-density lipoprotein(VLDL) and low-density lipoprotein(LDL), and it is expressed primarily in the liver and jejunum.  Both VLDL and LDL are important in cholesterol transport and its metabolism.  Not only was this degree of silencing observed very quickly, in about 24 hours post-administration, but the silencing effects maintained for over 22 days after only a single dose.  Researchers tested a 1 mg/kg single dose, too, obtaining a 68% silencing of the target gene, indicating dose-dependent silencing.  This dose-dependent silencing was evident not only on the degree of silencing but the duration of silencing, expression of the target gene recovering 72 hours post-administration.
Although SNALPs having a 100 nm diameter have been used effectively to target specific genes for silencing, there are a variety of systemic barriers that relate specifically to size.  For example, diffusion into solid tumors is impeded by large SNALPs and, similarly, inflamed cells having enhanced permeation and retention make it difficult for large SNALPs to enter.  In addition, reticuloendothelial elimination, blood–brain barrier size-selectivity and limitations of capillary fenestrae all necessitate a smaller SNALP in order to effectively deliver target-specific siRNA.  In 2012, scientists in Germany developed what they termed "mono-NALPs" using a fairly simple solvent exchange method involving progressive dilution of a 50% isopropanol solution.  What results is a very stable delivery system similar to traditional SNALPs, but one having only a diameter of 30 nm.  The mono-NALPs developed here, however, are inactive, but can become active carriers by implementing specific targeting and release mechanisms used by similar delivery systems.