Cancer-Targeting Nanoparticles for Combinatorial Nucleic Acid Delivery

Abstract

Nucleic acids are a promising type of therapeutic for the treatment of a wide range of conditions, including cancer, but they also pose many delivery challenges. For efficient and safe delivery to cancer cells, nucleic acids must generally be packaged into a vehicle, such as a nanoparticle, that will allow them to be taken up by the target cells and then released in the appropriate cellular compartment to function. As with other types of therapeutics, delivery vehicles for nucleic acids must also be designed to avoid unwanted side effects; thus, the ability of such carriers to target their cargo to cancer cells is crucial. Classes of nucleic acids, hurdles that must be overcome for effective intracellular delivery, types of nonviral nanomaterials used as delivery vehicles, and the different strategies that can be employed to target nucleic acid delivery specifically to tumor cells are discussed. Additonally, nanoparticle designs that facilitate multiplexed delivery of combinations of nucleic acids are reviewed.

Keywords: cancer therapy; gene delivery; nanoparticles; nucleic acid; targeted delivery.

Figure 1.. Challenges of nucleic acid delivery to tumors.

Effective and specific delivery of nucleic acids to tumors requires encapsulation or condensation of the cargo into nanoparticles. Nanoparticles must then remain stable in circulation, evading clearance and avoiding aggregation with other particles, and then leave the circulation to accumulate at the tumor site. Once there, particles must enter cells, and various intracellular barriers must be overcome depending on the type of nucleic acid cargo being delivered.

Figure 2.. Shape effects of spherical vs filamentous micelles.

Filomicelles are self-assembled from diblock co-polymers (a) with nano-scale diameter and micro-scale length. The filomicelles extend in flow (b) and evade phagocytosis while spherical micelles in flow are internalized. When the micelles are injected systemically in mice, they persist in circulation for days, and longer micelles have a longer circulation half-life than shorter micelles. Filomicelles are efficiently internalized (d) by lung epithelial cells in static culture. Reproduced from Geng et al., “Shape effects of filaments versus spherical particles in flow and drug delivery,” Nature Nanotechnology 2:249–255, 2007,^[95] with permission from Springer Nature.

Figure 3.. Optimization parameters for cancer-specific nanocarriers.

Physical and chemical properties of delivery vehicles affect tumor accumulation, particle internalization and cargo delivery, and ultimately the therapeutic outcome. Classes of targeting moieties and their sizes are also summarized.

Figure 4.

A prostate cancer targeted multifunctional envelope-like nano device (MEND) (A) nanocarrier is synesized by siRNA self-assembly with two block co-polymers: sharp oligoarginine functionalized pH responsive Meo-PEG-b-P(DPA-co-GMA-Rn) and PSMA targeted ACUPA-PEG-b-PDPA. Schematic shows targteted intracellular siRNA delivery after IV administration of MENDs. This strategy enables efficient gene silencing and significantly slows LNCaP tumor growth (B) compared with control and non-targeted NPs. Representative images of tumor bearing mice on day 18 (C) and photographs of harvested LNCaP tumors afetr 30 days (D). Reprinted with permission from Xu, Xiaoding, et al., “Multifunctional envelope-type siRNA delivery nanoparticle platform for prostate cancer therapy,” ACS Nano 11(3): 2618–2627.^[158] Copyright (2017) American Chemical Society.

Figure 5.

Schematic (A) illustrating the selection process for prostate cancer-specific internalizing RNA aptamers. Nanoparticles coated with prostate cancer-specific internalizing aptamers are specifically taken up in target PC3 cells (B) to a higher degree than in non-target HeLa cells. Bare particles without aptamer are taken up at low levels in both target and non-target cells, so aptamer conjugation is necessary for target-specific uptake. Uptake is distributed throughout the cytosol of targeted calls (C). When particles are loaded with Docetaxel (D), the aptamer conjugated particles (Dtxl-NP-Apt) are significantly more potent non-targeted particles (Dtxl-NP) at killing target cells. Reprinted with permission from Xiao, Zeyu, et al., “Engineering of targeted nanoparticles for cancer therapy using internalizing aptamers isolated by cell-uptake selection.” ACS Nano 6(1): 696–704.^[171] Copyright (2012) American Chemical Society.

Figure 6

A vibrating mesh nebulizer (A) was used to prepare luciferase mRNA delivery vectors for aerosol administration. Nano-scale polyplexes were encapsulated in micron-sized droplets and administered to a whole-body chamber. Hyperbranched PBAE hDD90–118 polyplexes enabled high levels of luciferase delivery in the lungs (B) after 24 hours, and local delivery by inhallation resulted in highly specific delivery to lung tissue and negligible off-target luciferase (c) measured by bioluminescence. Particles maintained a similar size and morphology before and after nebulization, characterized by electron microscopy (D). Particles also have a narrow size distribution before and after nebulization (E). Reprinted with permission from Patel, Asha Kumari, et al., “Inhaled Nanoformulated mRNA Polyplexes for Protein Production in Lung Epithelium,” Advanced Materials (2019):e1805116,^[229] with permission from John Wiley and Sons.

Figure 7

DOTAP-cholesterol liposomes loaded with transcriptionally targeted eAFP-VISA-BikDD or non-targeted CMV-BikDD were I.V. injected in orthotopic ML-1 tumor-bearing mice. Both particles significantly reduced tumor burden (a) and representative photos are shown from 1 week after the last treatment. Mouse survival (b) was significant on treatment groups, and the transcriptionally targeted DNA therapy extended survival significantly compared with the non-targeted DNA. Tissue samples from mice in (a) were fixed and stained for apoptosis using a TUNEL assay (c). The percentage of apoptotic cells were quantified in random fields from both tumor and healthy liver. While the targeted and non-trageted therapies induced similar numbers of apoptotic cells in the tumor, the transcriptionally targeted DNA induced less apoptosis in the healthy liver tissue. Reprinted with permission from Li, L. Y., et al., “Targeted hepatocellular carcinoma proapoptotic BikDD gene therapy,” Oncogene 30(15):1773, 2011,^[262] with permission from Springer Nature.

Figure 8.. Types of nanomaterials used for nucleic acid delivery.

Broad classes of materials and nanostructures used as nucleic acid delivery vehicles are summarized, including lipid-based nanoparticles (A), cationic polymer-based nanoparticles (B), nanoparticles based on other polymer types (C), inorganic nanoparticles (D), and nanostructures that use DNA itself as a structural component (E). Part E adapted from Doye et al., “Coarse-graining DNA for simulations of DNA nanotechnology,” Physical Chemistry Chemical Physics 15(47):20381–20772, 2013,^[369] with permission from the Royal Society of Chemistry.

Figure 9.

A liposomal formulation (NOV340) facilitated co-delivery of miR-34a and let-7b. After multiple injections (a), tumor lesions in the left lobe of lungs in animals treated with one or both miRNAs were fewer and smaller in number as seen by hematoxylin and eosin staining (b) and quantified in (c). Tumor sizes were lower in animals treated with both miRNAs compared to each individual miRNA (d), and tumor proliferation was lowe after treatment (e-f). Survival was also extended by treatment with miR-34a. Figure reprinted by permission from Springer Nature: Kasinski et al., “A combinatorial microRNA therapeutics approach to suppressing non-small cell lung cancer,” Oncogene 34(27):3547–3555, 2015.^[303]

Figure 10.

A cationic and bioreducible PBAE was used to form nanoparticles with each of two miRNAs (miR-148a and miR-296–5p) or a combination of both in order to prevent the growth and tumorigenicity of stem-like GBM cells (A). After intratumoral injection of nanoparticles, the combination of both miRNAs was more effective than either individual sequence in reducing tumor size (B) and causing necrosis of tumor tissue (C). Combination miRNA delivery also significantly extended survival (D). Figure adapted with permission from Lopez-Bertoni et al., “Bioreducible Polymeric Nanoparticles Containing Multiplexed Cancer Stem Cell Regulating miRNAs Inhibit Glioblastoma Growth and Prolong Survival,” Nano Letters 18(7):4086–4094.^[270] Copyright 2018 American Chemical Society.

Figure 11.

Lipid-polymer hybrid nanoparticles were used to co-deliver an miRNA (miR-34a) and siRNA (siKras) to lung tumor. The combination of both decreased the number of cancer cells in vitro (A) and significantly slowed tumor growth in vivo (B). The RNA combination also increased the number of CC3⁺ apoptotic cells in the tumor (C), and combining miRNA and siRNA with cisplatin further improved animal survival over any of the component treatments alone (D), indicating that combining these modalities can have an additive effect on tumors. Figure reproduced from Xue et al., “Small RNA combination therapy for lung cancer,” Proceedings of the National Academy of Sciences of the United States of America, 111(34):E3553-E3561.^[327] Copyright 2014 National Academy of Sciences.

Figure 12.

Mesoporous silica nanoparticles allow the co-delivery of multiple cargos and functionalization with a range of materials. In this example, MSNs were functionalized with thiol-reactive groups. The pores were loaded with chemotherapeutics doxorubicin or cisplatin, and the thiol-reactive groups were used to load two siRNA sequences and a tumor-targeting peptide sequence to the surface. Figure reprinted with permission of Taylor & Francis, Ltd, from Taratula et al., “Innovative strategy for treatment of lung cancer: targeted nanotechnology-based inhalation co-delivery of anticancer drugs and siRNA,” Journal of Drug Targeting 19(10):900–914, 2011.^[350]

Figure 13.

The high surface area of graphene quantum dots allowed them to be functionalized with two probes to inhibit miR-21 and survivin as well as polymers PLA and PEG. The resulting GQDs were biocompatible in addition to retaining favorable optical properties. Figure reprinted with permission from Dong et al., “Multifunctional Poly(l-lactide)–Polyethylene Glycol-Grafted Graphene Quantum Dots for Intracellular MicroRNA Imaging and Combined Specific-Gene-Targeting Agents Delivery for Improved Therapeutics,” ACS Applied Materials and Interfaces, 7(20):11015–11023.^[367] Copyright 2015, American Chemical Society.

Figure 14.

Intertwining DNA-RNA nanocapsules (iDR-NCs) were fabricated into a multimodal delivery vehicle. A CpG-rich DNA sequence and Stat3 shRNA sequence were amplified by rolling circle replication or transcription, respectively, forming microflowers (MFs) (A). The MFs were shrunk, or condensed, into iDR-NCs using PEG grafted to a cationic, hydrophobic polypeptide (PPT-g-PEG), which was also used to load tumor antigens into the NCs (B). These iDR-NCs could then be delivered to APCs in lymph nodes as a vaccine to promote gene knockdown, immunostimulation, and a tumor-specific response (C). Figure is reproduced with permission from Zhu et al., “Intertwining DNA-RNA nanocapsules loaded with tumor neoantigens as synergistic nanovaccines for cancer immunotherapy,” Nature Communications 8:1482 (2017).^[372]