Nanoparticles as a Multifunctional Nanoplatform for Boosting Nonsmall Cell Lung Cancer siRNA Therapy
Stimuli-Responsive and Highly Penetrable Nanoparticles as a Multifunctional Nanoplatform for Boosting Nonsmall Cell Lung Cancer siRNA Therapy
In cancer therapy, it is acknowledged that large-size nanoparticles stay in the circulation system for a long time, but their permeability to tumor tissues is poor. To address the conflicting need for prolonging circulation time and favorable tumor tissue penetration ability, a charge conversional multifunctional nanoplatform was strategically designed to improve the efficacy of small interfering RNA (siRNA) therapy against nonsmall cell lung cancer (NSCLC). The development of nanodrug delivery systems (NDDSs) was constructed by loading siRNA on polyamidoamine (PAMAM) dendrimers to build small-sized PAM/siRNA via electrostatic interaction and then capped with a pH-triggered copolymer poly(ethylene glycol) methyl ether (mPEG)-poly-l-lysine (PLL)-2,3-dimethylmaleic anhydride (DMA) (shorted as PLM) under physiological conditions. While in the tumor microenvironment, the acidic reaction of the PLM copolymer changes from negative charge to positive charge due to the cleavable amide bond between mPEG-PLL and DMA, leading to large-size nanoparticles (NPs) with a negative charge that turns into a positive charge and small NPs with a high tumor-penetrating ability. All of the in vitro and in vivo studies validated that PLM/PAM/siRNA NPs possess desirable features including excellent biocompatibility, a prolonged circulation time, significant pH sensitivity, high tumor tissue penetration ability, and sufficient endo-/lysosomal escape. Taken together, all results suggest tremendous potential of the gene therapy based on the stimuli-sensitive PLM/PAM/siRNA NPs, providing a profound application prospective treatment strategy in cancer gene therapy.
Boosting siRNA Therapy for Nonsmall Cell Lung Cancer (NSCLC) with pH-Responsive Charge-Conversional Nanoparticles (PLM/PAM/siRNA NPs)-a
a (A) PLM/PAM/siRNA was prepared with siRNA and PAMAM dendrimers via electrostatic interaction and then capped with a pH-sensitive copolymer PLM at pH 7.4. (B) PLM/PAM/siRNA NPs boasted a long circulation time, enhanced tumor penetration, efficient cell uptake, and sufficient endo-/lysosomal escape in the effective treatment of A549 cells in tumor-bearing nude mice.
Small interfering RNA (siRNA) performed tremendous potential for disease progression by the specific silencing of genetic targets. A diverse set of challenges (e.g., rapid enzymatic digestion, poor cellular uptake, and lysosomal degradation) hamper the clinical application of siRNA due to its negative charge characteristic and large molecular size.To address these hurdles, a series of carriers (such as meso-porous silica, gold nanoshells, and polymers are utilized to pack siRNA. To overcome the rapid enzymatic digestion of siRNA, high doses of nanodrug delivery systems (NDDSs) are applied, which induces unavoidable systemic toxicity. In addition, some researchers have decorated polyethylene glycol (PEG) on the surface of various carriers to shield the positive charge and increase circulation time (decreasing the recognition of the reticuloendothelial system (RES)).Nevertheless, the shortcoming of this procedure is that PEGylation invariablymakes the NDDS too stable to cause sufficient tumor tissue penetrability and better cellular uptake. Taken together, it is contradictory to make siRNA more stable in the circulation system with better cellular uptake. So, there is an urgent need for some methods to jointly solve those contradictory problems.
Optimization of PLM/PAM/siRNA formulation. (A) EE% verification with an increase of the N/P ratio of PAM/siRNA. (B) EE% verification with an increase of the molar ratio (copolymer/siRNA, mol/mol) of PLM/PAM/siRNA and PLS/PAM/siRNA groups. (C) Particle size and ζ-potential verification with an increase of the molar ratio for PLM/PAM/siRNA NPs. (D) Agarose gel electrophoresis analysis of different formulations with different N/P ratios or different molar ratios. Free siRNA was invoked as a control group. (E) Heparin decomplexation assay was carried out. (F) In vitro serum stability using agarose gel electrophoresis analysis.
Still, it should be noted that there are also a diverse set of barriers because of heterogeneous structures and unique tumor microenvironments (elevated tumor interstitial fluid pressure (IFP) and dense tumor extracellular matrix (ECM)) that prevent the NDDS from penetrating deeper into tumor tissue. As is known, the physicochemical characteristics (size, surface charge, and particle shape) play critical roles in tumor penetration capability. Herein, a series of attempts are applied to regulate size, charge, or shape to increase the penetration capability of nanoparticles (NPs). It is acknowledged that one of the most important ways to address the hurdles of siRNA delivery and tumor penetration capability is through stimuli-responsive NPs by endogenous stimuli (e.g., low pH, high concentration of matrix metalloproteinases (MMPs)) and external stimuli (e.g., light).
For this perspective, their group developed a novel charge conversional and highly penetrable NDDS based on a pH-sensitive copolymer PLM and polyamidoamine (PAMAM) dendrimers as a multifunctional nanoplatform for boosting nonsmall cell lung cancer (NSCLC) RNAi therapy to overcome siRNA delivery challenges (short systemic circulation, in vivo stability, poor cellular uptake and endo-/lysosomal escape) and deeper penetration capability. 2,3-dimethylmaleic anhydride (DMA) grafted poly(ethylene glycol) methyl ether (mPEG)-poly-l-lysine (mPEG-PLL-DMA) was applied as an outer layer with a negative charge under a physiological environment. The inner core is PAMAM dendrimers (shorted as PAM) formed via electrostatic interaction for siRNA encapsulation. More importantly, polo-like kinase 1 (PLK1), a proto-oncogene, was applied to regulate cell mitosis to improve the therapeutic efficacy of tumor, which performs a negative correlation between the expression of PLK1 protein and the survival rate of tumor patients. Therefore, PLK1-siRNA was utilized to treat A549 tumors in this work.
In vitro and in vivo penetration assays. (A) CLSM examination of the PLM/PAM/siRNA and PLS/PAM/siRNA NPs distribution in A549 3D tumor spheroids at pH 7.4 and 6.5, respectively. Green fluorescence represents FAM-siRNA. The scale bar is 150 μm. (B) Penetration of free siRNA, PLM/PAM/siRNA, and PLS/PAM/siRNA NPs in vivo A549 tumor. Green and blue fluorescence indicated FAM-siRNA and the cell nuclei, respectively. Scale bar is 100 μm.
Taken together, it takes extraordinary intelligence to design charge switchable and highly penetrable NPs (PLM/PAM/siRNA), and the procedures was performed with the following features (i) Hydrophilic mPEG chains could prolong the blood circulation time and hence enhance tumor accumulation via the enhanced permeability and retention (EPR) effect; (ii) Tumor microenvironments (TMEs) pH-triggered rapid charge reversal of PLM copolymer leads to the detachment of PLM from the PLM/PAM/siRNA NPs due to a weakly acidic tumor microenvironment (pH 6.5–6.9). Accordingly, the large-sized NPs with a negative charge (PLM/PAM/siRNA NPs) instantaneously transformed into positive charge and small NPs (PAM/siRNA NPs) with a high tumor accumulation and tumor-penetrating ability. (iii) Subsequently, the positively charged small NPs (PAM/siRNA NPs) were easily absorbed and effectively achieved quick endo-/lysosomal escape thanks to the proton sponge of PAMAM dendrimers, which results in a rapid dissociation of the NPs, enhancing the tumor penetration capability and efficient cell uptake, enhancing cytosolic siRNA transport, and accomplishing cancer therapy. To validate these hypotheses above, they systematically evaluated the above nanoplatform for promoting anticancer efficacy. The PLM/PAM/siRNA NPs illustrated excellent physiochemical properties and sufficient endo-/lysosomal escape in vitro. More importantly, in vivo assays demonstrated that PLM/PAM/siRNA NPs possessed a prolonged circulation time, charge switchable, significant pH sensitivity characteristic, and high tumor tissue penetration ability, leading to effectively inhibiting the A549 tumor via tumor-associated gene PLK1 silencing.
(A) Luminescence images of A549 tumor-bearing nude mice following treatment with different formulations at 2, 12, and 24 h, respectively (n = 3). (B) Fluorescence images of excised major organs and tumors at 24 h after intravenous injection. (C) Quantified luminescence levels of ex vivo tumors at 24 h postinjection.
(A) Schematic illustration showing the timeline of the efficacy study. (B) Tumor volume changes. (C) Body weight changes. (D) Tumor weight study. (E) Mean tumor inhibition rate in A549 cell-bearing BALB/c nude mice. Data are presented as mean ± SD (n = 6). (F) Survival curves of A549 tumor-bearing nude mice after the treatment (n = 10).
Western blotting assay of PLM/PAM/siRNA NPs in A549 cell-bearing nude mice following 48 h of incubation (100 nM siRNA) to examine (A) ex vivo PLK1 expression and (B) PLK1 expression was also quantified. (C) Tumor tissues were used to prepare paraffin-embedded slides for H&E, TUNEL, PLK1, and Ki-67 studies. Scale bar represented 100 μm.
Stimuli-Responsive and Highly Penetrable Nanoparticles as a Multifunctional Nanoplatform for Boosting Nonsmall Cell Lung Cancer siRNA Therapy Menghao Shi, Yu Wang, Xiufeng Zhao, Jiulong Zhang, Haiyang Hu, Mingxi Qiao, Xiuli Zhao, and Dawei Chen ACS Biomaterials Science & Engineering 2021 7 (7), 3141-3155 DOI: 10.1021/acsbiomaterials.1c00582