Nanotechnology as a Delivery Tool for Precision Cancer Therapies

Genomic analyses from patients with cancer have improved the understanding of the genetic elements that drive the disease, provided new targets for treating this relentless disease, and offered criteria for stratifying patient populations that will benefit most from treatments. In the last decade, several new targeted therapies have been approved by the FDA based on these omics findings, leading to significantly improved survival and quality of life for select patient populations. However, many of these precision medicines, e.g., nucleic acid-based therapies and antibodies, suffer from poor plasma stability, suboptimal pharmacokinetic properties, and immunological toxicities that prohibit their clinical translation. Nanotechnology is being explored as a delivery platform that can enable the successful delivery of these precision medicine treatments without these limitations. These precision nanomedicines are able to protect the cargo from degradation or premature/burst release prior to accumulation at the tumor site and improve the selectivity to cancer cells by incorporating ligands that can target receptors overexpressed on the cancer cell surface. Here, we review the development of several precision nanomedicines based on genomic analysis of clinical samples, actively targeted nanoparticle delivery systems in the clinic, and the pathophysiological barriers of the tumor microenvironment. Successful translation of these precision nanomedicine initiatives will require an effective collaboration between basic and clinical investigators to match the right patient with the right therapies and to deliver them at therapeutic concentrations which will improve overall treatment responses.     

Lipid and polymeric carrier-mediated nucleic acid delivery

Importance of the field: Nucleic acids such as plasmid DNA, antisense oligonucleotide, and RNA interference (RNAi) molecules, have a great potential to be used as therapeutics for the treatment of various genetic and acquired diseases. To design a successful nucleic acid delivery system, the pharmacological effect of nucleic acids, the physiological condition of the subjects or sites, and the physicochemical properties of nucleic acid and carriers have to be thoroughly examined.

Areas covered in this review: The commonly used lipids, polymers and corresponding delivery systems are reviewed in terms of their characteristics, applications, advantages and limitations.

What the reader will gain: This article aims to provide an overview of biological barriers and strategies to overcome these barriers by properly designing effective synthetic carriers for nucleic acid delivery.

Take home message: A thorough understanding of biological barriers and the structure–activity relationship of lipid and polymeric carriers is the key for effective nucleic acid therapy.     

Lipid-based Nanoparticles for Nucleic Acid Delivery

Abstract Lipid-based colloidal particles have been extensively studied as systemic gene delivery carriers. The topic that we would like to emphasize is the formulation/assembly of lipid-based nanoparticles (NP) with diameter under 100 nm for delivering nucleic acid in vivo. NP are different from cationic lipid–nucleic acid complexes (lipoplexes) and are vesicles composed of lipids and encapsulated nucleic acids with a diameter less than 100 nm. The diameter of the NP is an important attribute to enable NP to overcome the various in vivo barriers for systemic gene delivery such as: the blood components, reticuloendothelial system (RES) uptake, tumor access, extracellular matrix components, and intracellular barriers. The major formulation factors that impact the diameter and encapsulation efficiency of DNA-containing NP include the lipid composition, nucleic acid to lipid ratio and formulation method. The particle assembly step is a critical one to make NP suitable for in vivo gene delivery. NP are often prepared using a dialysis method either from an aqueous-detergent or aqueous-organic solvent mixture. The resulting particles have diameters about 100 nm and nucleic acid encapsulation ratios are >80%. Additional components can then be added to the particle after it is formed. This ordered assembly strategy enables one to optimize the particle physico-chemical attributes to devise a biocompatible particle with increased gene transfer efficacy in vivo. The components included in the sequentially assembled NP include: poly(ethylene glycol) (PEG)-shielding to improve the particle pharmacokinetic behavior, a targeting ligand to facilitate the particle–cell recognition and in some case a bioresponsive lipid or pH-triggered polymer to enhance nucleic acid release and intracellular trafficking. A number of groups have observed that a PEG-shielded NP is a robust and modestly effective system for systemic gene or small interfering RNA (siRNA) delivery.     

Advances in polymeric and inorganic vectors for nonviral nucleic acid delivery

Nonviral systems for nucleic acid delivery offer a host of potential advantages compared with viruses, including reduced toxicity and immunogenicity, increased ease of production and less stringent vector size limitations, but remain far less efficient than their viral counterparts. In this article we review recent advances in the delivery of nucleic acids using polymeric and inorganic vectors. We discuss the wide range of materials being designed and evaluated for these purposes while considering the physical requirements and barriers to entry that these agents face and reviewing recent novel approaches towards improving delivery with respect to each of these barriers. Furthermore, we provide a brief overview of past and ongoing nonviral gene therapy clinical trials. We conclude with a discussion of multifunctional nucleic acid carriers and future directions.     

Proteomic analysis on cellular response induced by nanoparticles reveals the nano-trafficking pathway through epithelium

The application of nanomedicines in oral drug delivery effectively promotes the drug absorption and transportation through enterocytes. Nevertheless, the absence of mechanism studies on efficacy and safety limits their final translation in humans. Although the vesicular trafficking has been verified as the general character for transport of nanomedicines, the deeper mechanism in molecular mechanism is still unclear. Moreover, the cellular transport of nanomedicines is a dynamic process involved by different organelles and components. However, most of existing studies just pay attention to the static location of nanomedicines, but neglect the dynamic biological effects on cells caused by them. Here, we prepared gold nanoparticles (AuNPs) as the model and cultured epithelial cell monolayer to explore the nano-bio interactions at the molecular level. The traditional pharmacological inhibition strategy and subcellular imaging technology elucidated the macropinocytosis/endosome/MVB/lysosome pathway during the transportation of AuNPs. Proteomics strategy based on mass spectrometry (MS) was utilized to identify and quantify proteins involved in the cellular transport of nanomedicines. Multiple proteins related to subcellular structure, signal transduction, energy transformation and metabolism regulation were demonstrated to be regulated by nanoparticle transport. These alterations of protein expression clarified the effects of intracellular proteins and verified the conventional findings. More importantly, it revealed a feedback mechanism of cells to the nano-trafficking. We believed that these new regulatory mechanisms provided new insights into the efficient transport of nanomedicines through epithelial barriers.     

Nonviral Approaches for Neuronal Delivery of Nucleic Acids

The delivery of therapeutic nucleic acids to neurons has the potential to treat neurological disease and spinal cord injury. While select viral vectors have shown promise as gene carriers to neurons, their potential as therapeutic agents is limited by their toxicity and immunogenicity, their broad tropism, and the cost of large-scale formulation. Nonviral vectors are an attractive alternative in that they offer improved safety profiles compared to viruses, are less expensive to produce, and can be targeted to specific neuronal subpopulations. However, most nonviral vectors suffer from significantly lower transfection efficiencies than neurotropic viruses, severely limiting their utility in neuron-targeted delivery applications. To realize the potential of nonviral delivery technology in neurons, vectors must be designed to overcome a series of extra- and intracellular barriers. In this article, we describe the challenges preventing successful nonviral delivery of nucleic acids to neurons and review strategies aimed at overcoming these challenges.     

Modulation of gene expression by antisense and antigene oligodeoxynucleotides and small interfering RNA

Antisense oligodeoxynucleotides, triplex-forming oligodeoxynucleotides and double-stranded small interfering RNAs have great potential for the treatment of many severe and debilitating diseases. Concerted efforts from both industry and academia have made significant progress in turning these nucleic acid drugs into therapeutics, and there is already one FDA-approved antisense drug in the clinic. Despite the success of one product and several other ongoing clinical trials, challenges still exist in their stability, cellular uptake, disposition, site-specific delivery and therapeutic efficacy. The principles, strategies and delivery consideration of these nucleic acids are reviewed. Furthermore, the ways to overcome the biological barriers are also discussed so that therapeutic concentrations at their target sites can be maintained for a desired period.     

Dendrimers in gene delivery

Dendrimers have unique molecular architectures and properties that make them attractive materials for the development of nanomedicines. Key properties such as defined architecture and a high ratio of multivalent surface moieties to molecular volume also make these nanoscaled materials highly interesting for the development of synthetic (non-viral) vectors for therapeutic nucleic acids. Rational development of such vectors requires the link to be made between dendrimer structure and the morphology and physicochemistry of the respective nucleic acid complexes and, furthermore, to the biological performance of these systems at the cellular and systemic level. The review focuses on the current understanding of the role of dendrimers in those aspects of synthetic vector development. Dendrimer-based transfection agents have become routine tools for many molecular and cell biologists but therapeutic delivery of nucleic acids remains a challenge.     

Recent advances in polymeric materials for the delivery of RNA therapeutics

ABSTRACT

Introduction: The delivery of nucleic acid therapeutics through non-viral carriers face multiple biological barriers that reduce their therapeutic efficiency. Despite great progress, there remains a significant technological gap that continues to limit clinical translation of these nanocarriers. A number of polymeric materials are being exploited to efficiently deliver nucleic acids and achieve therapeutic effects.

Areas covered: We discuss the recent advances in the polymeric materials for the delivery of nucleic acid therapeutics. We examine the use of common polymer architectures and highlight the challenges that exist for their development from bench side to clinic. We also provide an overview of the most notable improvements made to circumvent such challenges, including structural modification and stimuli-responsive approaches, for safe and effective nucleic acid delivery.

Expert opinion: It has become apparent that a universal carrier that follows ‘one-size’ fits all model cannot be expected for delivery of all nucleic acid therapeutics. Carriers need to be designed to exhibit sensitivity and specificity toward individual targets diseases/indications, and relevant subcellular compartments, each of which possess their own unique challenges. The ability to devise synthetic methods that control the molecular architecture enables the future development that allow for the construction of ‘intelligent’ designs.     

Novel non-endocytic delivery of antisense oligonucleotides

Antisense oligonucleotides (ONs) have several properties that make them attractive as therapeutic agents. Hybridization of antisense ONs to their complementary nucleic acid sequences by Watson-Crick base pairing is a highly selective and efficient process. Design of therapeutic antisense agents can be made more rationally as compared to most traditional drugs, i.e., they can be designed on the basis of target RNA sequences and their secondary structures. Despite these advantages, the design and use of antisense ONs as therapeutic agents are still faced with several obstacles. One major obstacle is their inefficient cellular uptake and poor accessibility to target sites. In this article, we will discuss key barriers affecting ON delivery and approaches to overcome these barriers. Current methods of ON delivery will be reviewed with an emphasis on novel non-endocytic methods of delivery. ONs are taken up by cells via an endocytic process. The process of ON release from endosomes is a very inefficient process and, hence, ONs end up being degraded in the endosomes. Thus, ONs do not reach their intended site of action in the cytoplasm or nucleus. Delivery systems ensuring a cytoplasmic delivery of ONs have the potential to increase the amount of ON reaching the target. Here, we shall examine various ON delivery methods that bypass the endosomal pathway. The advantages and disadvantages of these methods compared to other existing methods of ON delivery will be discussed.     

Innate and adaptive immune responses toward nanomedicines

Since the commercialization of the first liposomes used for drug delivery, Doxil/Caelyx® and Myocet®, tremendous progress has been made in understanding interactions between nanomedicines and biological systems. Fundamental work at the interface of engineering and medicine has allowed nanomedicines to deliver therapeutic small molecules and nucleic acids more efficiently. While nanomedicines are used in oncology for immunotherapy or to deliver combinations of cytotoxics, the clinical successes of gene silencing approaches like patisiran lipid complexes (Onpattro®) have paved the way for a variety of therapies beyond cancer. In parallel, the global severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has highlighted the potential of mRNA vaccines to develop immunization strategies at unprecedented speed. To rationally design therapeutic and vaccines, chemists, materials scientists, and drug delivery experts need to better understand how nanotechnologies interact with the immune system. This review presents a comprehensive overview of the innate and adaptative immune systems and emphasizes the intricate mechanisms through which nanomedicines interact with these biological functions.KEY WORDS: Cancer immunotherapy, mRNA vaccine, Complement activation, Macrophage, In vivo clearance, Anti-PEG antibody, Nanoparticle, mRNA-1273, BNT162b2, Immunology     

Nuclear delivery of macromolecules: barriers and carriers

Recent evidence for efficient delivery of macromolecules, such as peptides and nucleic acids, from the cell exterior to the nucleus offers the interesting possibility of developing novel treatments directed at intranuclear targets. The findings should also stimulate the search for physiological ligands that utilize similar transport mechanisms to regulate pathobiological processes. Cytokines, growth factors and their receptors, as well as morphogens have all been shown to enter the nucleus to evoke biological responses in target cells. The rational design of intracellular drug delivery vehicles requires an increased understanding of the elaborate systems that mediate cellular communication and coordination with the extracellular environment without inflicting on the integrity of the cell. This review discusses some aspects of the carriers and barriers in macromolecular transport.     

Pharmacokinetics and biodistribution of nanoparticles

Nanoparticles show their promise for improving the efficacy of drugs with a narrow therapeutic window or low bioavailability, such as anticancer drugs and nucleic acid-based drugs. The pharmacokinetics (PK) and tissue distribution of the nanoparticles largely define their therapeutic effect and toxicity. Chemical and physical properties of the nanoparticles, including size, surface charge, and surface chemistry, are important factors that determine their PK and biodistribution. The intracellular fate of the nanoparticles after cellular internalization that affects the drug bioavailability is also discussed. Strategies for overcoming barriers for intracellular delivery and drug release are presented. Finally, future directions for improving the PK of nanoparticles and perspectives in the field are discussed.     

European Commission-supported development of targeted nanomedicines: did MediTrans make a difference?

Nanotechnology-inspired approaches to particle design and formulation, an improved understanding of (patho) physiological processes and biological barriers to drug targeting, as well as the limited input of new chemical entities in the 'pipeline' of pharmaceutical companies, suggest a bright future for targeted nanomedicines as pharmaceuticals. There is an increased consensus to the view that a major limitation hampering the entry of targeted delivery systems into the clinic is that new concepts and innovative research ideas within academia are not being developed and exploited in close collaboration with the pharmaceutical industry. Thus, an integrated 'bench-to-clinic' approach realized within a structural collaboration between industry and academia, will facilitate and promote the progression of targeted nanomedicines towards clinical application. The MediTrans project performed under the EU Framework Program 6, was designed to contribute to this ambition. The objectives of this collaborative initiative were: to apply nanotechnology for development of innovative targeted drug-delivery systems; to optimize targeted nanomedicines by using imaging guidance; to promote structural collaboration between industry and academia; and to forward targeted nanomedicines towards the clinic and the market. In this article, we will briefly address the research content, outcome and impact of the MediTrans project.     

Endocytosis and intracellular trafficking as gateways for nanomedicine delivery: opportunities and challenges

More than 40 nanomedicines are already in routine clinical use with a growing number following in preclinical and clinical development. The therapeutic objectives are often enhanced disease-specific targeting (with simultaneously reduced access to sites of toxicity) and, especially in the case of macromolecular biotech drugs, improving access to intracellular pharmacological target receptors. Successful navigation of the endocytic pathways is usually a prerequisite to achieve these goals. Thus a comprehensive understanding of endocytosis and intracellular trafficking pathways in both the target and bystander normal cell type(s) is essential to enable optimal nanomedicine design. It is becoming evident that endocytic pathways can become disregulated in disease and this, together with the potential changes induced during exposure to the nanocarrier itself, has the potential to significantly impact nanomedicine performance in terms of safety and efficacy. Here we overview the endomembrane trafficking pathways, discuss the methods used to determine and quantitate the intracellular fate of nanomedicines, and review the current status of lysosomotropic and endosomotropic delivery. Based on the lessons learned during more than 3 decades of clinical development, the need to use endocytosis-relevant clinical biomarkers to better select those patients most likely to benefit from nanomedicine therapy is also discussed.     

Nanomedicine(s) under the microscope

Depending on the context, nanotechnologies developed as nanomedicines (nanosized therapeutics and imaging agents) are presented as either a remarkable technological revolution already capable of delivering new diagnostics, treatments for unmanageable diseases, and opportunities for tissue repair or highly dangerous nanoparticles, nanorobots, or nanoelectronic devices that will wreak havoc in the body. The truth lies firmly between these two extremes. Rational design of "nanomedicines" began almost half a century ago, and >40 products have completed the complex journey from lab to routine clinical use. Here we critically review both nanomedicines in clinical use and emerging nanosized drugs, drug delivery systems, imaging agents, and theranostics with unique properties that promise much for the future. Key factors relevant to the design of practical nanomedicines and the regulatory mechanisms designed to ensure safe and timely realization of healthcare benefits are discussed.     

Structure and biological activity of pathogen-like synthetic nanomedicines

Here we characterize the structure, stability and intracellular mode of action of DermaVir nanomedicine that is under clinical development for the treatment of HIV/AIDS. This nanomedicine comprises pathogen-like pDNA/PEIm nanoparticles (NPs) having the structure and function resembling spherical viruses that naturally evolved to deliver nucleic acids to the cells. Atomic force microscopy demonstrated spherical 100 - 200 nm NPs with a smooth polymer surface protecting the pDNA in the core. Optical absorption determined both the NP structural stability and biological activity relevant to their ability to escape from the endosome and release the pDNA at the nucleus. Salt, pH and temperature influence nanomedicine shelf-life and intracellular stability. This approach facilitates the development of diverse polyplex nanomedicines where the delivered pDNA-expressed antigens induce immune responses to kill infected cells. FROM THE CLINICAL EDITOR: The authors investigated DermaVir nanomedicine comprised of pathogen-like pDNA/PEIm nanoparticles with structure and function resembling spherical viruses. DermaVir delivery of pDNA expresses antigens that induce immune responses to kill HIV infected cells.     

Non-viral nucleic acid containing nanoparticles as cancer therapeutics

ABSTRACT

Introduction: The delivery of nucleic acids such as DNA and short interfering RNA (siRNA) is promising for the treatment of many diseases, including cancer, by enabling novel biological mechanisms of action. Non-viral nanoparticles are a promising class of nucleic acid carriers that can be designed to be safer and more versatile than traditional viral vectors.

Areas covered: In this review, recent advances in the intracellular delivery of DNA and siRNA are described with a focus on non-viral nanoparticle-based delivery methods. Material properties that have enabled successful delivery are discussed as well as applications that have directly been applied to cancer therapy. Strategies to co-deliver different nucleic acids are highlighted, as are novel targets for nucleic acid co-delivery.

Expert opinion: The treatment of complex genetically-based diseases such as cancer can be enabled by safe and effective intracellular delivery of multiple nucleic acids. Non-viral nanoparticles can be fabricated to deliver multiple nucleic acids to the same cell simultaneously to prevent tumor cells from easily compensating for the knockdown or overexpression of one genetic target. The continued innovation of new therapeutic modalities and non-viral nanotechnologies to provide target-specific and personalized forms of gene therapy hold promise for genetic medicine to treat diseases like cancer in the clinic.     

Oligonucleotide delivery: a cellular prospective.

The use of oligonucleotides (ONs) for gene therapy of certain diseases has been discussed since the late 1970s. ONs are single stranded chains of nucleic acids that can hybridize with target nucleic acid sequences to inhibit specific proteins, and therefore allow selective treatment of various diseases. The use of ONs is limited due to their instability in biological tissues and difficulty in delivery to the intracellular compartments of the cell. Chemical analog approaches have been used to address the instability issue and delivery systems have been developed to increase cellular uptake of ONs. It is generally thought that ONs with or without a delivery system are transported into cells by endocytosis, and then accumulate within endosomes where they are significantly inactivated. The rate and extent of movement of ON from endosomes appears to be important in determining ON effects. Consequently, developing accessory compounds or delivery methods that enhance endosome to cytoplasm transfer may be vital to ON therapy. This review focuses on investigating mechanisms of various delivery approaches at the cellular/intracellular level that have demonstrated utility in increasing ON activity or cellular accumulation. The future prospects of ON delivery are also addressed.     

Lipid nanoparticles as vehicles for macromolecules: nucleic acids and peptides

Traditional drug delivery systems are not efficient for peptide, protein and nucleic acid (plasmid DNA, oligonucleotides or short interfering RNA) delivery, thereby LNP have been exploited as potential delivery and targeting systems of these molecules. Since their discovery in the early 90's several research groups have focused their efforts on the improvement of this kind of nanocarriers in terms of effectiveness and safety. This review features the recent and most relevant patents related to these topics, with particular attention to targeting and protection from environmental agents. Moreover, in the case of nucleic acids strategies to improve transfection mediated by lipid nanoparticles (entrance to the cells, intracellular distribution or going through nuclear envelope) will be assessed. Regarding peptides and proteins, enhancement of encapsulation efficiency and absorption through mucoses are the main studied drawbacks. Finally, this work also includes a summary of the existing patents about the use of LNP as immune response adjuvants by using either plasmid DNA or proteins.