Extracellular vesicles (EVs) hold great promise as potential therapeutic carriers. EVs are biologically active, intrinsically transporting cargo between cells. Moreover, they can be loaded with specific cargo for distribution and/or engineered to achieve enhanced uptake. Although studies have already demonstrated therapeutic delivery using EVs, various challenges must be overcome before EV technology is ready for the clinic. Since the properties of EVs are dependent upon their cell of origin and the conditions of their formation, establishing clear characterization practices is essential to ensuring reproducibility and safety. Identifying methods for mass production of EVs is crucial for achieving high EV yields necessary for clinical trials. This review introduces current theory behind EV formation and function, describes the latest methods for characterization and mass production, and discusses future opportunities for extracellular vesicles in therapeutic delivery.

Single modal cancer therapy that targets one pathological pathway often turns out to be inefficient. For example, relapse of chronic myelogenous leukemia (CML) after inhibiting BCR-ABL fusion protein using tyrosine kinase inhibitors (TKI) (e.g., Imatinib) is of significant clinical concern. This study developed a dual modal gene therapy that simultaneously tackles two key BCR-ABL-linked pathways using viral/nonviral chimeric nanoparticles (ChNPs). Consisting of an adeno-associated virus (AAV) core and an acid-degradable polymeric shell, the ChNPs were designed to simultaneously induce pro-apoptotic BIM expression by the AAV core and silence pro-survival MCL-1 by the small interfering RNA (siRNA) encapsulated in the shell. The resulting BIM/MCL-1 ChNPs were able to efficiently suppress the proliferation of BCR-ABL+ K562 and FL5.12/p190 cells in vitro and in vivo via simultaneously expressing BIM and silencing MCL-1. Interestingly, the synergistic antileukemic effects generated by BIM/MCL-1 ChNPs were specific to BCR-ABL+ cells and independent of a proliferative cytokine, IL-3. The AAV core of ChNPs was efficiently shielded from inactivation by anti-AAV serum and avoided the generation of anti-AAV serum, without acute toxicity. This study demonstrates the development of a synergistically efficient, specific, and safe therapy for leukemia using gene carriers that simultaneously manipulate multiple and interlinked pathological pathways.Keywords: AAV transduction; BIM expression; core−shell nanoparticles; hybrid vector; MCL-1 silencing; RNA interference; synergistic leukemia gene therapy

Polyplexes, complexed nucleic acids by cationic polymers, are the most common forms of nonviral gene delivery vectors. In contrast to a great deal of efforts in synthesizing novel cationic polymers and exploring their extracellular and intracellular delivery pathways, polyplex preparation methods of mixing nucleic acids and cationic polymers are often overlooked. In this study, the mixing sequence, that is adding nucleic acids to polymers or vice versa, was found to greatly affect complexation of both plasmid DNA and siRNA, polyplexes’ size, and polyplexes’ surface charge, which all collaboratively affected the transfection efficiency and cytotoxicity. Adding polyethylenimine (PEI), the most conventionally used standard in nonviral gene delivery, to plasmid DNA and siRNA resulted in larger polyplexes, higher gene expression and silencing, but higher cytotoxicity than polyplexes prepared in the reverse order. Based on the experimental results, the authors developed a model that gradual addition of cationic polymers (e.g., PEI) to nucleic acids (e.g., plasmid DNA and siRNA) incorporates more copies of nucleic acids in larger polyplexes in a smaller number, results in higher gene expression and silencing levels in transfected cells, and generates higher cytotoxicity by leaving more free polymers upon complete mixing than the other mixing sequence. The proposed model can be explored using a broad range of cationic polymers and nucleic acids, and provide insightful information about how to prepare polyplexed nonviral vectors for efficient and safe gene delivery.

Peptide-based biomaterials have been extensively investigated for biomedical applications due to their high structural and functional versatility. Particularly, peptides that can respond to different stimuli, such as pH, redox potential, temperature, light, and enzymes can offer controllable actions in target body locations. Therefore, smart, multifunctional peptide-based nanoparticle (NP) systems that respond to pathological stimuli with the ability to facilitate cellular internalization and enhance cytosolic release via disrupting membranes are of great interest in biomaterials research. This review predominantly focuses on synthetically designed stimuli-responsive peptides that serve different biomedical applications, with emphasis on drug and gene delivery, and touches upon recent advances in membrane-active peptide NPs for enhanced therapeutic activity. The review also provides an outlook on future directions on the maturing field of peptide-based therapeutics.

Multidrug-resistant microorganisms (MDRMOs) are progressively becoming an unavoidable challenge to worldwide health. Conventional antibiotics pressure resulted in increased bacterial efflux pumps lessening drug concentrations, up-regulated enzymes modifying/inactivating antibiotic compounds, or elevated mutations in the drug target site reducing antibiotic potency. Therefore, effective therapy for combating the emerging rate of MDRMOs requires innovative, combinatory strategies of generating conventional antimicrobial effects and simultaneously silencing drug-resistance processes in microbes. RNA interference (RNAi) is a revolutionary technology with high potential for obtaining synergistic therapies by knocking down antagonistic pathways with genomic specificity at a translational level. However, employing RNAi in antimicrobial therapy, particularly treating drug-resistant infections, has not received a great deal of attention. This paper briefly reviews key drug-resistance mechanisms in microbes, discusses the possibility of sensitizing MDRMOs to conventional antimicrobial therapy by combining it with RNAi, and introduces novel nano-scale formulation for efficient administration of such therapy (nanoantibiotics). The combined, synergistic antimicrobial therapy using antibiotics and RNAi may shed light when the current pipeline for new antibiotics is outrun by emergence of MDRMOs.

Development of efficient carriers for small interfering RNA (siRNA) delivery and validation tools for assessing in vivo RNA interference (RNAi) efficiency is crucial to advance RNAi-based therapeutics to the clinic. Here, acid-degradable ketalized linear polyethylenimine (KL-PEI) designed for efficient, stimuli-responsive, and biocompatible siRNA delivery was used to complex with GFP-silencing siRNA (GFP siRNA) for in vivo RNAi. The in vivo gene silencing efficiency of GFP siRNA/KL-PEI polyplexes was evaluated at mRNA, protein, and histological levels using a mouse bearing a GFP-expressing tumor. Intravenously injected GFP siRNA/KL-PEI polyplexes significantly reduced GFP expression in tumors and whole blood of mice, depending on the dosage of GFP siRNA and the time course. Average GFP mRNA levels in the tumors of siRNA/KL-PEI polyplex-injected mice were also reduced. The described siRNA carriers and RNAi validation methodologies in this study may provide insightful clues for the development of RNAi-based therapeutics and preclinical trials.

Silencing the expression of a target gene by RNA interference (RNAi) shows promise as a potentially revolutionizing strategy for manipulating biological (pathological) pathways at the translational level. However, the lack of reliable, efficient, versatile, and safe means for the delivery of small interfering RNA (siRNA) molecules, which are large in molecular weight, negatively charged, and subject to degradation, has impeded their use in basic research and therapy. Polyplexes of siRNA and polymers are the predominant mode of siRNA delivery, but innovative synthetic strategies are needed to further evolve them to generate the desired biological and therapeutic effects.This Account focuses on the design of polymeric vehicles for siRNA delivery based on an understanding of the molecular interactions between siRNA and cationic polymers. Ideal siRNA/polymer polyplexes should address an inherent design dilemma for successful gene silencing: (1) Cationic polymers must form tight complexes with siRNA via attractive electrostatic interactions during circulation and cellular internalization and (2) siRNA must dissociate from its cationic carrier in the cytoplasm before they are loaded into RNA-induced silencing complex (RISC) and initiate gene silencing. The physicochemical properties of polymers, which dictate their molecular affinity to siRNA, can be programmed to be altered by intracellular stimuli, such as acidic pH in the endosome and cytosolic reducers, subsequently inducing the siRNA/polymer polyplex to disassemble. Specific design goals include the reduction of the cationic density and the molecular weight, the loss of branched structure, and changes in the hydrophilicity/hydrophobicity of the polymeric siRNA carriers, via acid-responsive degradation and protonation processes within the endosome and glutathione (GSH)-mediated reduction in the cytoplasm, possibly in combination with gradual stimuli-independent hydrolysis.Acetals/ketals are acid-cleavable linkages that have been incorporated into polymeric materials for stimuli-responsive gene and drug delivery. Tailoring the ketalization ratio and the molecular weight of ketalized branched PEI (K-BPEI) offers molecular control of the intracellular trafficking of siRNA/polymer polyplexes and, therefore, the gene silencing efficiency. The ketalization of linear PEI (K-LPEI) enhances gene silencing in vitro and in vivo by improving siRNA complexation with the polymer during circulation and cellular internalization, supplementing proton buffering efficiency of the polymer in the endosome, and facilitating siRNA dissociation from the polymer in the cytoplasm, in a serum-resistant manner. Spermine polymerization via ketalization and esterification for multistep intracellular degradations provides an additional polymeric platform for improved siRNA delivery and highly biocompatible gene silencing. The chemistry presented in this Account will help lay the foundation for the development of innovative and strategic approaches that advance RNAi technology.

Stimuli-triggered degradation and facilitated intracellular release of nucleic acids are key design factors in developing efficient and biocompatible nonviral gene carriers. In this study, a cationic polymer was developed with aims to achieve enhanced gene transfection and low cytotoxicity via rapid biodegradation and subsequent intracellular de-complexation in response to dual stimuli in a cell. Diaminoethane was polymerized to synthesize cationic and hydrolytically degradable poly(amino ester), and then it was further conjugated with acid-degradable amino ketal branches. The resulting polymer, ketalized poly(amino ester) (K-PAE), differentially degrades via rapid hydrolysis of the ketal branches in the mildly acidic endosome and subsequent gradual degradation of the polyester backbone in the cytoplasm. DNA/K-PAE polyplexes underwent acid-triggered de-complexation at an endosomal pH, leading to efficient intracellular release of DNA and significantly enhanced transfection efficiency. In addition, DNA/K-PAE polyplexes demonstrated low cytotoxicity and serum-resistant gene transfection, in comparison with commercial counterpart polymers, promising for efficient and safe gene delivery in vivo. This study presents insightful design clues for developing efficient, versatile, stimuli-responsive, and biocompatible polymers for nonviral gene delivery.

Gene therapy is a promising tool to tackle challenging diseases at a molecular level. However, delivery of therapeutic nucleic acids to desired tissues and cells with high efficiency, versatility, and safety has been a fundamental technological gap in gene therapy. Viral and nonviral vectors offer advantages and disadvantages that can complement each other. Viral vectors exhibit high transduction efficiency with immunogenicity, mutagenesis, and limited versatility for structural and functional tenability. On the other hand, low transfection efficiency of nonviral vectors undermines their high flexibility for modification, low immunogenicity, and easy preparation. A number of attempts have been made to hybridize viral and nonviral vectors using genetic, physical, and chemical approaches. Synthetic engineering of viral vectors is reviewed here with (1) challenges in viral nucleic acid delivery pathways, in contrast to those of nonviral vectors, (2) design goals of incorporating synthetic molecules of broad types into viral vectors, and (3) methodology to modify and re-formulate viral vectors. Recent advances in synthetically engineered viral vectors for various biomedical applications are also discussed. This review clearly emphasizes the crucial roles of interdisciplinary approaches to developing ideal vectors in order to obtain desired properties for clinical success.Highlights► Overcoming the challenges in viral gene delivery by incorporating polymeric materials. ► Strategies to develop synthetically engineered viral particles for new, altered, and enhanced functions. ► Design of polymeric materials to be incorporated into viral vectors. ► Potentials and prospective of synthetically engineered viral particles in biomedical applications.

Conjugation of desired molecules onto retroviral surfaces through the ease of the bioorthogonal functionalization method was demonstrated. Oxidation of surface sialic acids using periodate and further p-anisidine-catalyzed conjugation with aminooxy-bearing molecules were used to directly label retroviral envelope with a fluorescent dye. The retroviral particles that were produced from a bioorthogonally functionalized virus producing cell surface and further tethered with magnetic nanoparticles were efficiently purified by simple magnetic column separation and capable of magnet-directed transduction.

Combined diagnosis and therapy for cancer has been of great interest in medicine. Small interference RNA (siRNA)-encapsulating polyplexes were covalently coated with small gold nanoparticles (Au NPs) via acid-cleavable linkages in order to explore the possibility of achieving combined stimuli-responsive multimodal optical imaging and stimuli-enhanced gene silencing. In a mildly acidic tumor environment, Au NPs are dissociated from the siRNA-carrying polyplexes, generating various optical signal changes such as diminished scattering intensity, increased variance of Doppler frequency, and blue-shifted UV absorbance (stimuli-responsive imaging). Simultaneously, Au NP dissociation exposes the siRNA-carrying polyplex with elevated surface charge and results in enhanced cellular uptake and transfection (stimuli-enhanced therapy). In this study, the feasibility of achieving combined diagnosis and therapy for cancer (theragnostics) is demonstrated by (1) microscopic and spectrophotometric confirmation of acid-transformation of the nanoparticles, (2) reduced scattering intensity and increased variance of Doppler frequency in an acidic pH upon the nanoparticle’s transformation, and (3) simultaneous optical signal changes and gene silencing in vitro under a tumor pH-mimicking condition. This novel type of stimuli-responsive nanotheragnostics will provide a new paradigm for pinpointed, multimodal, and combined imaging and therapy for cancer.

To maximize therapeutic effects, targeted delivery of nucleic acids (e.g., DNA and RNA) in their appropriate intracellular targets is highly desirable. In this study, primary amines of a model polymeric nonviral carrier, polyethylenimine (PEI), at two molecular weights (0.8 and 25 kDa) were differentially ketalized (i.e., 17–96%) in order to explore the possibility of precisely modulating intracellular localization of plasmid DNA- and siRNA-containing polyplexes. The size of the polyplexes revealed that the ketalization ratios of 35 to 70% were found to be the most efficient in condensing nucleic acids with the ketalized low molecular weight PEI (LMW PEI), while high molecular weight PEI (HMW PEI) ketalized at the ratio of 23% condensed nucleic acids most efficiently. Ketalization of LMW PEI (up to 70%) enhanced transfection; however, ketalization of HMW PEI reduced its transfection capability. On the contrary, HMW PEI ketalized at 23 and 37% ratios showed significant RNA interference, while LMW PEI could not successfully inhibit gene expression regardless of ketalization ratios. The results were explained by confocal microscopic studies demonstrating that ketalization ratios, molecular weights of ketalized PEI, and types of nucleic acids complexed in the polyplexes play crucial roles in intracellular localization of nucleic acids/ketalized PEI polyplexes and affect DNA transfection and RNA interference efficiencies. All ketalized PEI showed negligible cytotoxicity. This study implies a feasibility of selectively localizing nucleic acids in their intracellular targets by employing differentially tailored polymeric gene carriers.

Efficient intracellular processes including cytosolic release and unpackaging of siRNA from the carrier in the cytoplasm are efficiency-determining steps in achieving successful gene silencing. In this study, acid-degradable ketalized linear polyethylenimine (KL-PEI) was synthesized for efficient, intracellular target-specific, and biocompatible siRNA delivery. The siRNA/KL-PEI polyplexes resulted in much higher RNA interference efficiency than unmodified L-PEI via selective cytoplasmic localization of the polyplexes and efficient disassembly of siRNA from the polyplexes, which were promoted upon acid-hydrolysis of amino ketal linkages. Confocal laser scanning microscopy demonstrated that siRNA was efficiently disassembled from the siRNA/KL-PEI polyplexes that were selectively localized in the cytoplasm. On the contrary, siRNA and unmodified linear PEI were colocalized in both the cytoplasm and the nucleus, and limited unpackaging of siRNA from the polyplexes was observed. In addition, ketalization further reduced the cytotoxicity of linear PEI but did not alter its serum-independent gene delivery efficiency. Therefore, KL-PEI is a promising nonviral vector for efficient and biocompatible siRNA delivery.

A new polyethylenimine (PEI)-derived biodegradable polymer was synthesized as a nonviral gene carrier. Branches of PEI were ketalized, and capabilities of nucleic acid condensation and delivery efficiency of the modified polymers were compared with ones of unketalized PEI. Ketalized PEI was able to efficiently compact both plasmid DNA and siRNA into nucleic acids/ketalized PEI polyplexes with a range of 80–200 nm in diameter. Nucleic acids were efficiently dissociated from the polyplexes made of ketalized PEI upon hydrolysis. In vitro study also demonstrated that ketalization enhanced transfection efficiency of the polyplexes while reducing cytotoxicity, even at high N/P ratios. Interestingly, transfection efficiency was found to be inversely proportional to molecular weights of ketalized PEI, while RNA interference was observed in the opposite way. This study implies that selective delivery of plasmid DNA and siRNA to the nucleus and the cytoplasm can be achieved by tailoring the structures of polymeric gene carriers.

RNA interference (RNAi) has shown immense potential for treating diseases by selectively silencing an abnormal/pathological pathway through the degradation of target messenger RNA (mRNA). However, exploitation of RNAi as a prevalent drug in the clinic has been hampered by the inability to successfully deliver siRNA to cells with adequate pharmacokinetics, while avoiding side effects. This review examines the current technologies to circumvent multiple challenges for siRNA-based therapeutics to be established as a conventional and effective class of medicine.

Multiple extra- and intracellular obstacles, including low stability in blood, poor cellular uptake, and inefficient endosomal escape and disassembly in the cytoplasm, have to be overcome in order to deliver nucleic acids for gene therapy. This review introduces the recent advances in tackling the key challenges in achieving efficient, targeted, and safe nonviral gene delivery using various nucleic acid-containing nanomaterials that are designed to respond to various extra- and intracellular biological stimuli (e.g., pH, redox potential, and enzyme) as well as external artificial triggers (e.g., light and ultrasound). Gene delivery in combination with molecular imaging and targeting enables diagnostic assessment, treatment monitoring and quantification of efficiency, and confirmation of cure, thus fulfilling the great promise of efficient and personalized medicine. Nanomaterials platform for combined imaging and gene therapy, nanotheragnostics, using stimuli-responsive materials is also highlighted in this review. It is clear that developing novel multifunctional nonviral vectors, which transform their physico-chemical properties in response to various stimuli in a timely and spatially controlled manner, is highly desired to translate the promise of gene therapy for the clinical success.Download high-res image (301KB)Download full-size image

•Suboptimal SELEX methodologies have hindered aptamer application usage.•Innovations in SELEX methods made aptamer selection more favorable.•Modified and unnatural nucleobases expanded the aptamer repertoire.•Automation and single-round selection improved on the rates of aptamer discovery.•Advantages and disadvantages of the modified SELEX methods are reviewed.It has been more than two decades since the first aptamer molecule was discovered. Since then, aptamer molecules have gain much attention in the scientific field. This increasing traction can be attributed to their many desirable traits, such as 1) their potentials to bind a wide range of molecules, 2) their malleability, and 3) their low cost of production. These traits have made aptamer molecules an ideal platform to pursue in the realm of pharmaceuticals and bio-sensors. Despite the broad applications of aptamers, tedious procedure, high resource consumption, and limited nucleobase repertoire have hindered aptamer in application usage. To address these issues, new innovative methodologies, such as automation and single round SELEX, are being developed to improve the outcomes and rates in which aptamers are discovered.Download high-res image (152KB)Download full-size image

The dynamic and versatile nature of diseases such as cancer has been a pivotal challenge for developing efficient and safe therapies. Cancer treatments using a single therapeutic agent often result in limited clinical outcomes due to tumor heterogeneity and drug resistance. Combination therapies using multiple therapeutic modalities can synergistically elevate anti-cancer activity while lowering doses of each agent, hence, reducing side effects. Co-administration of multiple therapeutic agents requires a delivery platform that can normalize pharmacokinetics and pharmacodynamics of the agents, prolong circulation, selectively accumulate, specifically bind to the target, and enable controlled release in target site. Nanomaterials, such as polymeric nanoparticles, gold nanoparticles/cages/shells, and carbon nanomaterials, have the desired properties, and they can mediate therapeutic effects different from those generated by small molecule drugs (e.g., gene therapy, photothermal therapy, photodynamic therapy, and radiotherapy). This review aims to provide an overview of developing multi-modal therapies using nanomaterials (“combo” nanomedicine) along with the rationale, up-to-date progress, further considerations, and the crucial roles of interdisciplinary approaches.Download high-res image (246KB)Download full-size image

Peptide-based biomaterials have been extensively investigated for biomedical applications due to their high structural and functional versatility. Particularly, peptides that can respond to different stimuli, such as pH, redox potential, temperature, light, and enzymes can offer controllable actions in target body locations. Therefore, smart, multifunctional peptide-based nanoparticle (NP) systems that respond to pathological stimuli with the ability to facilitate cellular internalization and enhance cytosolic release via disrupting membranes are of great interest in biomaterials research. This review predominantly focuses on synthetically designed stimuli-responsive peptides that serve different biomedical applications, with emphasis on drug and gene delivery, and touches upon recent advances in membrane-active peptide NPs for enhanced therapeutic activity. The review also provides an outlook on future directions on the maturing field of peptide-based therapeutics.

Polyplexes, complexed nucleic acids by cationic polymers, are the most common forms of nonviral gene delivery vectors. In contrast to a great deal of efforts in synthesizing novel cationic polymers and exploring their extracellular and intracellular delivery pathways, polyplex preparation methods of mixing nucleic acids and cationic polymers are often overlooked. In this study, the mixing sequence, that is adding nucleic acids to polymers or vice versa, was found to greatly affect complexation of both plasmid DNA and siRNA, polyplexes’ size, and polyplexes’ surface charge, which all collaboratively affected the transfection efficiency and cytotoxicity. Adding polyethylenimine (PEI), the most conventionally used standard in nonviral gene delivery, to plasmid DNA and siRNA resulted in larger polyplexes, higher gene expression and silencing, but higher cytotoxicity than polyplexes prepared in the reverse order. Based on the experimental results, the authors developed a model that gradual addition of cationic polymers (e.g., PEI) to nucleic acids (e.g., plasmid DNA and siRNA) incorporates more copies of nucleic acids in larger polyplexes in a smaller number, results in higher gene expression and silencing levels in transfected cells, and generates higher cytotoxicity by leaving more free polymers upon complete mixing than the other mixing sequence. The proposed model can be explored using a broad range of cationic polymers and nucleic acids, and provide insightful information about how to prepare polyplexed nonviral vectors for efficient and safe gene delivery.

Development of efficient carriers for small interfering RNA (siRNA) delivery and validation tools for assessing in vivo RNA interference (RNAi) efficiency is crucial to advance RNAi-based therapeutics to the clinic. Here, acid-degradable ketalized linear polyethylenimine (KL-PEI) designed for efficient, stimuli-responsive, and biocompatible siRNA delivery was used to complex with GFP-silencing siRNA (GFP siRNA) for in vivo RNAi. The in vivo gene silencing efficiency of GFP siRNA/KL-PEI polyplexes was evaluated at mRNA, protein, and histological levels using a mouse bearing a GFP-expressing tumor. Intravenously injected GFP siRNA/KL-PEI polyplexes significantly reduced GFP expression in tumors and whole blood of mice, depending on the dosage of GFP siRNA and the time course. Average GFP mRNA levels in the tumors of siRNA/KL-PEI polyplex-injected mice were also reduced. The described siRNA carriers and RNAi validation methodologies in this study may provide insightful clues for the development of RNAi-based therapeutics and preclinical trials.