br NP vectors have emerged
NP vectors have emerged as a promising alternative to viral systems for targeted in vivo delivery of nucleic HA 1077 therapies. Stable in vivo knockdown using siRNA delivery systems has been achieved and several compounds have received FDA approval44. However, the efficiency of nucleic acid delivery is poor and facilitating endosomal escape remains challenging. NPs incorporating pH-sensitive cationic
lipid carriers, such as DLinDMA (1,2-dilinoleyloxy-3-dimethyaminopropane), have been used to facilitate membrane fusion-mediated endosomal escape whereby the acidic environment of the late endosome triggers fusion of the NP surface with the endosomal membrane and subsequent release of the NP contents into the cytoplasm45,46. Newer pH-sensitive cationic lipids, such as DLin-MC3-DMA, can increase the in vivo ED50 (minimum dose of siRNA to produce the desired biological effect in 50% of subjects in vivo) by a factor of almost 10047. However, these remain relatively inefficient with >97% of the payload being lost in the late endosome. Sato and colleagues recently evaluated the effects of modulating the hydrophobicity of the head and tail regions of the unsaturated pH-responsive lipid YSK12-C4 on intracellular delivery and endosomal escape of lipid NPs containing siRNAs targeting clotting factor VII48. They assessed efficiency of endosomal escape by quantifying the fraction of siRNA loaded into the RNA-induced silencing complex (RISC). Lipid NPs with CL4H6 lipids (containing bulky hydrophobic tails) were significantly more efficient carriers than traditional DLin-MC3-DMA carriers. Approximately 4.2% of the CL4H6 siRNA payload was successfully loaded to the RISC complex which translated to an approximate 50% reduction in ED50. It is worth noting that a significantly higher proportion of the siRNA payload entered the cytosol than was loaded to the RISC but continued interaction between the nucleic acid payload and degraded lipid carrier limited subsequent processing. Studies such as these demonstrate that engineering of novel lipid carriers may represent a viable method to improve the efficiency and efficacy of in vivo NP-based nucleic acid delivery.
Another method of endosomal escape involves the use of endosmotic or proton-buffering polymers such as poly(ethylenimine) (PEI) to stimulate a proton-sponge effect49,50. The exact mechanisms by which proton-buffering nanocomplexes stimulate endosomal escape has not been clearly established but is believed to involve osmotic flux across the outer endosomal leaflet leading to membrane destabilization, permeability, or polymer-supported pore formation51. Regardless of the exact mechanism, recent preclinical studies have demonstrated that endosmotic carriers can efficiently delivery cargo to the cytoplasm intact and separate from the nanocarrier enabling efficient in vivo delivery of functional nucleic acid therapies. A schematic representation of endosomal escape mechanisms with engineered NPs is shown in Figure 2. r> 3. Utilizing NPs to Enhance Cancer Immunotherapy
3.1 Enhancing Antigen Presentation
One of the initiating events in generating tumor-specific (adaptive) immunity is the identification and uptake of tumor neoantigens by professional APCs including dendritic cells (DCs). Activated DCs must then present these antigens along with co-stimulatory molecules to effector T-cells. This process can be disrupted in several ways. First, neoantigen processing and presentation is a relatively inefficient process. Only a small fraction of the antigens presented on major histone compatibility (MHC) complex 1 by tumors are unique neoantigens that can be distinguished from normal cellular proteins and are expressed in sufficiently high concentrations to be targeted by T-cells52. Some immunoresistant histologies are believed to have relatively low neoantigen burdens and/or mechanisms to actively downregulate neoantigen expression or shield them from APCs and T-cells. Ablative therapies like radiation can stimulate the release of neoantigens but recognition of these antigens by APCs appears to be poor and, in the absence of co-stimulatory signals, can induce T-cell anergy. Second, many immunosuppressive cell populations in the TME secrete inhibitory cytokines and ligands to prevent DC maturation and presentation to effector cells53. Identifying novel ways of improving antigen processing and presentation in APCs has been a focus of intensive research in cancer immunotherapy.
3.1.1 Co-delivery of Exogenous Antigens and Immune Adjuvants to APCs
A major focus of research for immunotherapeutic nanomedicines has been the targeted delivery of engineered neoantigents to APCs in an effort to stimulate tumor vaccination. NP carriers are ideally-suited to this task for several reasons. First, they efficiently protect neoantigens for in vivo delivery. Second, owing to their “virus-like” physical properties, they are efficiently recognized and taken up by APC and accumulate in lymphoid tissues. Third, immunogenic stimulation requires co-activation by a second signal. Recognition of neoantigens by APCs in the absence of a stimulatory signal is not only ineffective, but can promote immune tolerance. NPs can be engineered with specific tumor peptides and immune adjuvants (like CpG-ODN) to ensure co-delivery of both signals to activated APCs54. Alternatively, the NP itself can be composed of potent immune adjuvants such as PC7A or PEI55,56. Several preclinical studies have demonstrated proof-of-principle for this approach using engineered NPs with established tumor neoantigens including OVA and gp10057-59. As one example, an A1 lipoprotein nanodisc used to codeliver tumor neoantigens and CpG to mice bearing MC38 or B16F10 tumors stimulated approximately 90% complete tumor regression compared to 25-38% regression with systemic delivery of CpG and tumor antigens when combined with dual checkpoint inhibition60. It is also possible to incorporate nucleic acid therapy into immune adjuvant and neoantigen vaccine strategies. Zhu et al. generated nucleic acid nanocarriers stabilized by PEG-grafted polypeptides for the simultaneous delivery of CpG, stat3 siRNA, and the somatic tumor neoantigen Adpgk found in syngeneic MC38 colorectal tumors61. Three weeks after immunization, only 1.1% of peripheral CD8+ T-cells had Adpgk-specific receptors in CpG treated mice compared to 9.5% of NP vaccine-treated mice. Inorganic, protein-based (albumin), and microparticle emulsion systems have also been engineered as cancer vaccines62-67.