Nucleus‐specific RNAi nanoplatform for targeted regulation of nuclear lncRNA function and effective cancer therapy

Abstract

In the context of cancer therapy, a recently identified therapeutic target is represented by the essential subtype of RNA transcripts ‐ the long noncoding RNAs (lncRNA). While this is the case, it is especially difficult to successfully regulate the expression of this subtype in vivo, particularly due to the protection granted by the nuclear envelope of nuclear lncRNAs. This study documents the development of a nucleus‐specific RNA interference (RNAi) nanoparticle (NP) platform for the targeted regulation of the nuclear lncRNA function, in order to effectuate successful cancer therapy. An NTPA (nucleus‐targeting peptide amphiphile) and an endosomal pH‐responsive polymer make up the novel RNAi nanoplatform in development, which is capable of complexing siRNA. The nanoplatform is capable of accumulating greatly in the tumor tissues and being internalized by tumor cells, following intravenous administration. The exposed complexes of the NTPA/siRNA may conveniently escape from the endosome with the pH‐triggered NP disassociation, following which it can target the nucleus by specifically interacting with the importin α/β heterodimer. In orthotopic and subcutaneous xenograft tumor models, this would result in a notable suppression of the expression of nuclear lncNEAT2 as well as greatly impede the growth of tumors in liver cancer.

Remodeling of Mitochondrial Metabolism by a Mitochondria‐Targeted RNAi Nanoplatform for Effective Cancer Therapy

“…In this formulation, the lipophilic triphenylphosphonium cation (TPP + ) group and oligoarginine sequence (RRRRRR) of MMPA could respectively accumulate in the mitochondrial matrix and penetrate the mitochondrial membrane, [ 24,25 ] while the polymer Meo‐PEG‐ b ‐PDPA with a p K a (≈6.34, Figure S4, Supporting Information) close to the endosomal pH (6.0–6.5) could spontaneously self‐assemble into well‐defined NPs in aqueous solution with hydrophilic PEG shells and hydrophobic PDPA cores. [ 26,27 ] During the formation of siRNA‐loaded NPs, the cationic peptide MMPA could form complexes with negatively charged siRNA with the hydrophobic alkyl chain of MMPA localized on surface of MMPA/siRNA complexes, which could be then embedded into the hydrophobic PDPA core of NPs via the co‐assembly with the amphiphilic polymer Meo‐PEG‐ b ‐PDPA (Scheme 1). [ 17,24,27 ] Through changing the feed compositions of MMPA and siRNA to adjust the N/P molar ratio (Figure S5, Supporting Information), the spherical siRNA‐loaded NPs formulated at an N/P molar ratio of 20/1 (denoted MT‐NPs(siATP6)) were chosen for the following experiments due to their relatively small size (≈85 n m , Figure A,B), high siRNA encapsulation efficiency (≈85%), moderate surface charge (≈9 mV), and good stability (Figures S5, S6, Supporting Information) compared to other NPs prepared at different N/P molar ratios.…”

“…[ 26,27 ] During the formation of siRNA‐loaded NPs, the cationic peptide MMPA could form complexes with negatively charged siRNA with the hydrophobic alkyl chain of MMPA localized on surface of MMPA/siRNA complexes, which could be then embedded into the hydrophobic PDPA core of NPs via the co‐assembly with the amphiphilic polymer Meo‐PEG‐ b ‐PDPA (Scheme 1). [ 17,24,27 ] Through changing the feed compositions of MMPA and siRNA to adjust the N/P molar ratio (Figure S5, Supporting Information), the spherical siRNA‐loaded NPs formulated at an N/P molar ratio of 20/1 (denoted MT‐NPs(siATP6)) were chosen for the following experiments due to their relatively small size (≈85 n m , Figure A,B), high siRNA encapsulation efficiency (≈85%), moderate surface charge (≈9 mV), and good stability (Figures S5, S6, Supporting Information) compared to other NPs prepared at different N/P molar ratios. For these NPs, the protonation of their hydrophobic PDPA cores at a pH close to the endosomal pH (e.g., pH 6.0) could induce the disassociation of nanostructure and exposure of MMPA/siRNA complexes (Figure 1C), leading to the decrease in the number of NPs (Figure 1D) and fast siRNA release (Figure 1E).…”

“…[26,27] During the formation of siRNA-loaded NPs, the cationic peptide MMPA could form complexes with negatively charged siRNA with the hydrophobic alkyl chain of MMPA localized on surface of MMPA/siRNA complexes, which could be then embedded into the hydrophobic PDPA core of NPs via the co-assembly with the amphiphilic polymer Meo-PEG-b-PDPA (Scheme 1). [17,24,27] Through changing the feed compositions of MMPA and siRNA to adjust the N/P molar ratio (Figure S5, Supporting Information), the spherical siRNA-loaded NPs formulated at an N/P molar ratio of 20/1 (denoted MT-NPs(siATP6)) were chosen for the following experiments due to their relatively small size (≈85 nm, Figure 1A,B), high siRNA encapsulation Scheme 1. Schematic illustration of the preparation of mitochondria-targeted RNAi nanoplatform A) and targeted regulation of mtDNA-encoded ATP6 expression for breast cancer (BCa) therapy B).…”

“…However, within the same time frame, a majority of siRNA molecules loaded in the NPs made with the polymer methoxyl-poly(ethylene glycol)-b-poly-(lactic-co-glycolic acid) (Meo-PEG-b-PLGA) and cationic peptide MMPA are restricted in the endosomes (Figure S7, Supporting Information), highlighting the importance of endosomal pH sensitivity to the endosomal escape of loaded siRNA. [26,27] In addition, when using the mixture of Cy5-labeled cationic peptide (C 17 H 35 -CONH-GRRRRRRGGGK(Cy5)G-OH, Figure S8, Supporting Information) and MMPA to prepare the siRNA-loaded NPs and then incubating with MDA-MB-231 cells, it can be observed that the siRNA molecules escaped from the endosomes are in form of MMPA/siRNA complexes, as indicated by the overlapped red (siRNA) and cyan (cationic peptide) fluorescence in Figure 1F. More importantly, these escaped MMPA/siRNA complexes could efficiently target the mitochondria and specifically deliver siRNA into the mitochondria (Figure 1G), which is proven by the co-localization between red (siRNA) and green fluorescence (mitochondria).…”

Optimization of lipid assisted polymeric nanoparticles for siRNA delivery and cancer immunotherapy

“…Given this limitation, there is an urgent need to develop alternative clinically available delivery systems for siRNA therapy. [9][10][11][12][13][14][15][16][17][18] Previously, we utilized a poly(ethylene glycol)-b-poly(D,Llactide) and cationic lipid combination to encapsulate siRNA, [19][20][21][22][23] employing a double emulsion-solvent evaporation technique. This approach yielded exceptional siRNA loading efficiency, exceeding 95%, owing to its nanoconfined loading mechanism.…”