Core/shell lipid-based nanovectors have been proposed to deliver siRNA and miRNA. The research group of Huang gave a significant contribution to the development of this strategy by developing nanoparticles obtained by RNA complexation with polycation, then covered by a lipid shell. More in detail, in the first studies, RNA (previously mixed with calf thymus DNA at the 1:1 weight ratio) was condensed into the NP core by mixing with protamine, a highly positively-charged peptide already used as transfection reagent for nucleic acids. The high molecular weight DNA was reported to improve the core compaction, while the use of calf thymus DNA, compared to plasmid DNA, was preferred for the limited amounts of immunostimulating CpG motifs [
75]. The resulting RNA-containing complexes were covered with a lipid shell by mixing with cationic liposomes (DOTAP/chol) to provide liposome-polycation-DNA (LPD) nanoparticles. To reduce the particle size and to increase the stability in serum (thus preventing the NP aggregation), LPD NPs were mixed with DSPE-PEG by post-insertion method [
76]. It is worthy of note that PEGylation introduced steric hindrance to LPD, thus reducing about 80% of their delivery efficiency; the use of ligands, e.g., anisamide, linked to the DSPE-PEG restored the delivery efficacy of these NPs. Anisamide, a small molecule to target human lung cancer cells also allowed achieving significant improvement of LPD localization in lung cancer [
76]. In different animal models of cancer, siRNA, when encapsulated in LPD NPs, showed an increased circulation time compared to free siRNA; no differences were found between targeted and untargeted NPs [
75]. Interestingly, NPs were cleared from the blood significantly quicker in tumor-bearing than in healthy mice. Moreover, NPs increased the half-life of the siRNA in the blood, increased the area under the curve and decreasing the clearance [
76]. About 80%–70% of the injected siRNA accumulated into the tumor by using LPD NPs, without significant differences between targeted and untargeted NPs [
75,
76]. Interestingly, in the tumor tissue, the targeted NPs allowed an increased siRNA delivery into the cytoplasm, compared to untargeted NPs. This technology has also been used to successfully deliver a mixture of siRNAs against different targets to obtain simultaneous silencing of three oncogenes, with consequent significant inhibition of tumor growth and prolonged mice survival in a model of lung metastasis [
76]. Taking into account the capability of doxorubicin (DOX) to interact with double stranded DNA by non-covalent intercalation, DOX was also co-encapsulated with siRNA for combined therapy [
77]. The replacement of calf thymus DNA with hyaluronic acid (HA) into the core of the NPs (LPH NPs), allowed reducing the production of pro-inflammatory cytokines [
78]. The pro-apoptotic effect of siRNA-encapsulating NPs can be enhanced by replacing DOTAP with a newly-synthesized, non-glycerol-based trivalent cationic lipid, namely DSGLA [
79]. The authors also demonstrated that, when adding the DSPE-PEG to the NPs, a large percentage of lipids was stripped off by the DSPE-PEG and formed smaller particles (mean diameter lower than 100 nm). In particular, about 90% of the siRNA used in the formulation was associated to approximately 37.2% of the total lipid and 20.2% of the input DSPE-PEG. In the same study, the authors demonstrated that the arrangement of PEG in the brush mode on the NP surface prevented opsonization in serum and abolished the non-specific uptake by RES in the isolated liver [
80]. LPH NPs, targeted with the single-chain antibody fragment (scFv), have also been used to simultaneously deliver siRNA and miRNA (miR34a) [
77]. To increase the siRNA delivery, HA and protamine were replaced by calcium/phosphate (CaP) NPs to complex siRNA in the core of the lipid-coated NPs or LCP NPs [
75]. The authors hypothesize that LCPs NPs can enter by endocytosis and disassemble into the endosomes for the lower pH; this should induce an increase of the endosome intracellular pressure and swelling, with consequent release of the entrapped siRNA [
75]. This technology has been successfully used to simultaneously deliver siRNA against three different targets in different experimental models of tumor [
81,
82]. The technology proposed by Huang and colleagues certainly represent a valid alternative to lipid vesicles, although these two delivery systems were never compared in the same experimental conditions. As in the case of SNALPs, the core/shell NPs described above appear as a versatile delivery system, quite independent on the experimental condition of use. A scale-up process could evaluate if core/shell NPs could also be proposed for clinical use. Self-assembled anionic NPs encapsulating RNA have also been proposed. The anionic charge should reduce the toxicity and the rapid clearance from the circulation generally associated to the cationic NPs. Thus, NPs consisting in a mixture of peptide ligands and anionic, PEGylated liposomes were developed [
83]. These components, at the optimized molar charge ratios and in the well-established order of mixing, self-assemble into anionic NP with about 80%–91% packaging of siRNA in the absence of serum; in presence of serum, the quenching was only reduced by 1.2 to two-fold [
84]. The authors of these studies reported a higher stability of PEGylated anionic nanocomplexes, compared to their cationic counterpart. They also report a more ready decomplexation in presence of heparin, suggesting their potential to dissociate once into the cytoplasm. These NPs were successfully used to deliver the siRNA into the rat brain [
84]. The use of cationic formulation has been associate to toxicity at different levels, such as mitochondrial damage, interfering with blood coagulation cascade, induction of interferon response, promotion of cytokine production, and complementary activation [
85,
86]. Thus, neutral lipid-based nanoparticles (LNPs) have been also proposed to deliver RNA oligonucleotides with a more safe profile. These NPs has also been chemically modified with hyaluronan (Han) to confer stealth properties and to target CD44-overexpressing cancer cells. In detail, these NPs were prepared by the self-assembling of neutral phospholipids and cholesterol. The authors showed a silencing effect only after six days following the transfection, while the same effect was found after two days in the case of lipofectamine used for comparison purpose [
84]. The authors attributed this delay to the slow release of siRNA from the endosomes while, in the case of the cationic liposomes (e.g., lipofectamine), the endosomal escape should be enhanced. It is worthy of note that, in this work, in vivo safety data on this formulation has not been provided, especially in comparison with cationic nanovectors.