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Glioblastoma (GBM) is an aggressive central nervous system cancer with a dismal prognosis. The standard of care involves surgical resection followed by radiotherapy and chemotherapy, ...but five-year survival is only 5.6% despite these measures. Novel therapeutic approaches, such as immunotherapies, targeted therapies, and gene therapies, have been explored to attempt to extend survival for patients. Nanoparticles have been receiving increasing attention as promising vehicles for non-viral nucleic acid delivery in the context of GBM, though delivery is often limited by low blood–brain barrier permeability, particle instability, and low trafficking to target brain structures and cells. In this review, nanoparticle design considerations and new advances to overcome nucleic acid delivery challenges to treat brain cancer are summarized and discussed.
Nanoparticle-mediated siRNA delivery is a complex process that requires transport across numerous extracellular and intracellular barriers. As such, the development of nanoparticles for efficient ...delivery would benefit from an understanding of how parameters associated with these barriers relate to the physicochemical properties of nanoparticles. Here, we use a multiparametric approach for the evaluation of lipid nanoparticles (LNPs) to identify relationships between structure, biological function, and biological activity. Our results indicate that evaluation of multiple parameters associated with barriers to delivery such as siRNA entrapment, pK ₐ, LNP stability, and cell uptake as a collective may serve as a useful prescreening tool for the advancement of LNPs in vivo. This multiparametric approach complements the use of in vitro efficacy results alone for prescreening and improves in vitro–in vivo translation by minimizing false negatives. For the LNPs used in this work, the evaluation of multiple parameters enabled the identification of LNP pK ₐ as one of the key determinants of LNP function and activity both in vitro and in vivo. It is anticipated that this type of analysis can aid in the identification of meaningful structure–function–activity relationships, improve the in vitro screening process of nanoparticles before in vivo use, and facilitate the future design of potent nanocarriers.
Nonviral nanoparticles have emerged as an attractive alternative to viral vectors for gene therapy applications, utilizing a range of lipid‐based, polymeric, and inorganic materials. These materials ...can either encapsulate or be functionalized to bind nucleic acids and protect them from degradation. To effectively elicit changes to gene expression, the nanoparticle carrier needs to undergo a series of steps intracellularly, from interacting with the cellular membrane to facilitate cellular uptake to endosomal escape and nucleic acid release. Adjusting physiochemical properties of the nanoparticles, such as size, charge, and targeting ligands, can improve cellular uptake and ultimately gene delivery. Applications in the central nervous system (CNS; i.e., neurological diseases, brain cancers) face further extracellular barriers for a gene‐carrying nanoparticle to surpass, with the most significant being the blood–brain barrier (BBB). Approaches to overcome these extracellular challenges to deliver nanoparticles into the CNS include systemic, intracerebroventricular, intrathecal, and intranasal administration. This review describes and compares different biomaterials for nonviral nanoparticle‐mediated gene therapy to the CNS and explores challenges and recent preclinical and clinical developments in overcoming barriers to nanoparticle‐mediated delivery to the brain.
This article is categorized under:
Therapeutic Approaches and Drug Discovery > Nanomedicine for Neurological Disease
Therapeutic Approaches and Drug Discovery > Emerging Technologies
Nanotechnology Approaches to Biology > Nanoscale Systems in Biology
Synthetic polymer‐based, lipid‐based, and inorganic nanoparticles can be employed to deliver nucleic acid cargo for gene therapy applications. For neurological diseases and brain cancers, systemic, intracranial, intrathecal, and intranasal administration are commonly utilized routes of administration.
Clinical translation of polymer‐based nanocarriers for systemic delivery of RNA has been limited due to poor colloidal stability in the blood stream and intracellular delivery of the RNA to the ...cytosol. To address these limitations, this study reports a new strategy incorporating photocrosslinking of bioreducible nanoparticles for improved stability extracellularly and rapid release of RNA intracellularly. In this design, the polymeric nanocarriers contain ester bonds for hydrolytic degradation and disulfide bonds for environmentally triggered small interfering RNA (siRNA) release in the cytosol. These photocrosslinked bioreducible nanoparticles (XbNPs) have a shielded surface charge, reduced adsorption of serum proteins, and enable superior siRNA‐mediated knockdown in both glioma and melanoma cells in high‐serum conditions compared to non‐crosslinked formulations. Mechanistically, XbNPs promote cellular uptake and the presence of secondary and tertiary amines enables efficient endosomal escape. Following systemic administration, XbNPs facilitate targeting of cancer cells and tissue‐mediated siRNA delivery beyond the liver, unlike conventional nanoparticle‐based delivery. These attributes of XbNPs facilitate robust siRNA‐mediated knockdown in vivo in melanoma tumors colonized in the lungs following systemic administration. Thus, biodegradable polymeric nanoparticles, via photocrosslinking, demonstrate extended colloidal stability and efficient delivery of RNA therapeutics under physiological conditions, and thereby potentially advance systemic delivery technologies for nucleic acid‐based therapeutics.
Nanocarriers are engineered to realize the potential of RNA therapeutics. This work reports the design of photocrosslinked bioreducible nanoparticles (XbNPs) for stable small interfering RNA (siRNA) encapsulation in high‐serum conditions, shielded surface charge, efficient intracellular trafficking, and triggered cytosolic RNA release. These attributes of XbNPs lead to robust siRNA‐mediated knockdown in cancer cells and potent systemic siRNA delivery to tumors in the lungs.
Current standard of care for many CNS tumors involves surgical resection followed by chemotherapy and/or radiation. Some pediatric brain tumor types are infiltrative and diffuse in nature, which ...reduces the role for surgery. Furthermore, children are extremely vulnerable to neurological sequelae from surgery and radiation therapy, thus alternative approaches are in critical need. As molecular targets underlying various cancers become more clearly defined, there is an increasing push for targeted gene therapies. Viral vectors and nonviral nanoparticles have been thoroughly investigated for gene delivery and show promise as vectors for gene therapy for pediatric brain cancer. Here, we review inorganic and organic materials in development for nanoparticle gene delivery to the brain with a particular focus on safety.
Purpose: Transient transfection is an essential tool for recombinant protein production, as it allows rapid screening for expression without stable integration of genetic material into a target cell ...genome. Poly(ethylenimine) (PEI) is the current gold standard for transient gene transfer, but transfection efficiency and the resulting protein yield are limited by the polymer’s toxicity. This study investigated the use of a class of cationic polymers, poly(beta-amino ester)s (PBAEs), as reagents for transient transfection in comparison to linear 25 kDa PEI, a commonly used transfection reagent. Methods: Transfection efficiency and protein production were assessed in human embryonic kidney 293F (HEK) and Chinese hamster ovary-S (CHO) cell suspensions using PBAE-based nanoparticles in comparison to linear 25 kDa PEI. Production of both a cytosolic reporter and secreted antibodies was investigated. Results: In both HEK and CHO cells, several PBAEs demonstrated superior transfection efficiency and enhanced production of a cytosolic reporter compared to linear 25 kDa PEI. This result extended to secreted proteins, as a model PBAE increased the production of 3 different secreted antibodies compared to linear 25 kDa PEI at culture scales ranging from 20 to 2000 mL. In particular, non-viral gene transfer using the lead PBAE/plasmid DNA nanoparticle formulation led to robust transfection of mammalian cells across different constructs, doses, volumes, and cell types. Conclusion: These results show that PBAEs enhance transfection efficiency and increase protein yield compared to a widespread commercially available reagent, making them attractive candidates as reagents for use in recombinant protein production.
Hepatocellular carcinoma (HCC) has limited treatment options, and modest survival after systemic chemotherapy or procedures such as transarterial chemoembolization (TACE). There is therefore a need ...to develop targeted therapies to address HCC. Gene therapies hold immense promise in treating a variety of diseases, including HCC, though delivery remains a critical hurdle. This study investigated a new approach of local delivery of polymeric nanoparticles (NPs) via intra-arterial injection for targeted local gene delivery to HCC tumors in an orthotopic rat liver tumor model.
Poly(beta-amino ester) (PBAE) nanoparticles were formulated and assessed for GFP transfection in N1-S1 rat HCC cells in vitro. Optimized PBAE NPs were next administered to rats via intra-arterial injection with and without orthotopic HCC tumors, and both biodistribution and transfection were assessed.
In vitro transfection of PBAE NPs led to >50% transfected cells in adherent and suspension culture at a variety of doses and weight ratios. Administration of NPs via intra-arterial or intravenous injection demonstrated no transfection of healthy liver, while intra-arterial NP injection led to transfection of tumors in an orthotopic rat HCC model.
Hepatic artery injection is a promising delivery approach for PBAE NPs and demonstrates increased targeted transfection of HCC tumors compared to intravenous administration, and offers a potential alternative to standard chemotherapies and TACE. This work demonstrates proof of concept for administration of polymeric PBAE nanoparticles via intra-arterial injection for gene delivery in rats.
Tumor immunotherapy is a promising anticancer strategy; however, tumor cells may employ resistance mechanisms, including downregulation of major histocompatibility complex (MHC) molecules to avoid ...immune recognition. Here, we investigate reprogramming nanoparticles (NPs) that deliver immunostimulatory genes to enhance immunotherapy and address defective antigen presentation in skin cancer in vitro and in vivo. We use a modular poly(beta-amino ester) (PBAE)-based NP to deliver DNA encoding 4-1BBL, IL-12, and IFNγ to reprogram human Merkel cell carcinoma (MCC) cells in vitro and mouse melanoma tumors in vivo to drive adaptive antitumor immune responses. Optimized NP formulations delivering 4-1BBL/IL-12 or 4-1BBL/IL-12/IFNγ DNA successfully transfect MCC and melanoma cells in vitro and in vivo, respectively, resulting in IFNγ-driven upregulation of MHC class I and II molecules on cancer cells. These NPs reprogram the tumor immune microenvironment (TIME) and elicit strong T-cell-driven immune responses, leading to cancer cell killing and T-cell proliferation in vitro and slowing tumor growth and improving survival rates in vivo. Based on expected changes to the tumor immune microenvironment, particularly the importance of IFNγ to the immune response and driving both T-cell function and exhaustion, next-generation NPs codelivering IFNγ were designed. These offered mixed benefits, exchanging improved polyfunctionality for increased T-cell exhaustion and demonstrating higher systemic toxicity in vivo. Further profiling of the immune response with these NPs provides insight into T-cell exhaustion and polyfunctionality induced by different formulations, providing a greater understanding of this immunotherapeutic strategy.
Delivery of self‐amplifying mRNA (SAM) has high potential for infectious disease vaccination due to its self‐adjuvanting and dose‐sparing properties. Yet a challenge is the susceptibility of SAM to ...degradation and the need for SAM to reach the cytosol fully intact to enable self‐amplification. Lipid nanoparticles are successfully deployed at incredible speed for mRNA vaccination, but aspects such as cold storage, manufacturing, efficiency of delivery, and the therapeutic window can benefit from further improvement. To investigate alternatives to lipid nanoparticles, a class of >200 biodegradable end‐capped lipophilic poly(beta‐amino ester)s (PBAEs) that enable efficient delivery of SAM in vitro and in vivo as assessed by measuring expression of SAM encoding reporter proteins is developed. The ability of these polymers to deliver SAM intramuscularly in mice is evaluated, and a polymer‐based formulation that yields up to 37‐fold higher intramuscular (IM) expression of SAM compared to injected naked SAM is identified. Using the same nanoparticle formulation to deliver a SAM encoding rabies virus glycoprotein, the vaccine elicits superior immunogenicity compared to naked SAM delivery, leading to seroconversion in mice at low RNA injection doses. These biodegradable nanomaterials may be useful in the development of next‐generation RNA vaccines for infectious diseases.
Biodegradable end‐capped lipophilic poly(beta‐amino ester)s (PBAEs) enable efficient delivery of self‐amplifying mRNA (SAM) in vitro and in vivo. These nanomaterials yield up to 37‐fold higher intramuscular expression of SAM compared to injected naked SAM. PBAE nanoparticle formulations deliver SAM encoding rabies virus glycoprotein as a vaccine and elicit seroconversion in mice at low RNA injection doses.
Suprachoroidal nonviral gene therapy with biodegradable poly(β-amino ester) nanoparticles (NPs) provides widespread expression in photoreceptors and retinal pigmented epithelial (RPE) cells and ...therapeutic benefits in rodents. Here, we show in a human-sized minipig eye that suprachoroidal injection of 50 μl of NPs containing 19.2 μg of GFP expression plasmid caused GFP expression in photoreceptors and RPE throughout the entire eye with no toxicity. Two weeks after injection of 50, 100, or 200 μl, there was considerable within-eye and between-eye variability in expression that was reduced 3 months after injection of 200 μl and markedly reduced after three suprachoroidal injections at different locations around the eye. Reduction of bacterial
sequences in the expression plasmid resulted in a trend toward higher expression. These data indicate that nonviral suprachoroidal gene therapy with optimized polymer, expression plasmid, and injection approach has potential for treating photoreceptors throughout the entire retina of a human-sized eye.
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