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Lv, H, Zhang, S, Wang, B, Cui, S and Yan, J. Toxicity of cationic lipids and cationic polymers in gene delivery. J Control Release 114: 100-109
Abstract
Gene therapy, as a promising therapeutics to treat genetic or acquired diseases, has achieved exciting development in the past two decades. Appropriate gene vectors can be crucial for gene transfer. Cationic lipids and polymers, the most important non-viral vectors, have many advantages over viral ones as non-immunogenic, easy to produce and not oncogenic. They hold the promise to replace viral vectors to be used in clinic. However, the toxicity is still an obstacle to the application of non-viral vectors to gene therapy. For overcoming the problem, many new cationic compounds have been developed. This article provides a review with respect to toxicity of cationic lipids and polymers in gene delivery. We evaluate the structural features of cationic compounds and summarize the relationship of toxicity and structure and hope to provide available suggestions on the development of these cationic compounds.
... Chemical transfection agents may have cytotoxicity depending on the nature of their constituent components [50,51]. This study shows that siRNA and PEI molecules separately do not exert cytotoxic effect on the MSC cultures but have moderate cytotoxicity as a part of polyplexes. ...
... siRNA molecules, acting by the RNA interference mechanism, are a highly accurate tool for genetic silencing of target mRNA transcripts without directly acting on the genome. However, the chemical transfection agents used for their delivery can have cytotoxicity [50,51]. In the course of this work, we determined the most effective agent for transfection of MSCs -PEI and the optimal concentration of siRNA molecules in polyplexes -50 pmol/mL. ...
... 21 O N L I N E F I R S T и в нецелевых органах, а продолжительность обнаружения долгосрочного сигнала является предметом научного рассмотрения. Если персистенция вектора, экспрессия трансгена и/или его продукта сохраняются в течение большего периода времени, чем указано в руководстве по доклиническим исследованиям безопасности в целях проведения клинических исследований и регистрации лекарственных препаратов 27 и рекомендациях ICH 28 , продолжительность периода наблюдения в токсикологическом исследовании при однократном или многократном введении должна как минимум отражать продолжительность экспрессии (может быть больше, чем для стандартных токсикологических исследований других биофармацевтических продуктов) и зависит также от степени и места экспрессии и/или ожидаемых дополнительных рисков 29 . ...
... Иммуногенность также является одной из возможных проблем при использовании невирусных векторов. Липосомальные составы широко используются в качестве невирусных систем доставки, однако из-за своей катионной природы липосомы неспецифически связываются с белками сыворотки, а также вызывают повреждения клеток наряду с активацией иммунной системы [29,30]. Сообщалось, что липидные наночастицы вызывали сильный иммунный ответ при достижении паренхимы головного мозга у мышей после внутривенной инъекции [31]. ...
Currently, gene therapy medicinal products (GTMPs) are actively developed in many countries, including the Russian Federation. However, the use of GTMPs raises class-specific safety concerns.
The aim of the study was to determine the main requirements for non-clinical safety testing of GTMPs, to identify risks associated with these medicinal products, to establish criteria for expert assessments, and to find optimisation opportunities for GTMP non-clinical safety programmes, using Russian and international experience in the assessment of submissions and the registration of medicinal products of this class.
The Russian Federation, the Eurasian Economic Union, the European Union, and the United States have created regulatory frameworks governing the lifecycle of GTMPs and continue improving these frameworks. The properties of GTMPs may create unique safety issues, such as insertional mutagenesis, unregulated transgene expression, long-term persistence and off-target spread, vertical germline transmission, and environmental risks. To account for these issues, a comprehensive non-clinical safety programme for GTMPs may require additional special studies along with the standard ones. This review focuses on the main approaches to designing non-cellular GTMP safety studies and evaluating the obtained results. The authors identified improvement opportunities for and problematic aspects of study design, as well as conditions for and limitations of nonclinical data extrapolation and clinical safety profile prediction. The continuous improvement and updating of the regulatory frameworks governing non-clinical studies of GTMPs mean that developers of non-clinical safety programmes for GTMPs should use all their experience, as well as relevant national and international guidelines and recommendations.
... 146 Unfortunately, toxicity and immunogenicity may be the main obstacles to its clinical application. 147 The cationic lipids, combined with other components forming nanoparticles, are named LNPs. 148 LNPs are the most extensively investigated and clinically advanced delivery system for mRNA-based therapy. ...
... 177 However, PEI has the disadvantages of poor biodegradability and high toxicity, limiting its broad clinical application. 147 Fortunately, with the development of chemical modification, the toxicity of PEI is reduced while maintaining its protonatable properties and high delivery efficiency. 178 Interestingly, the delivery of therapeutic FAH mRNA based on dendritic molecules can restore liver function and prolong the survival time of HT-1 mice. ...
Messenger RNA (mRNA) holds great potential in developing immunotherapy, protein replacement, and genome editing. In general, mRNA does not have the risk of being incorporated into the host genome and does not need to enter the nucleus for transfection, and it can be expressed even in nondividing cells. Therefore, mRNA‐based therapeutics provide a promising strategy for clinical treatment. However, the efficient and safe delivery of mRNA remains a crucial constraint for the clinical application of mRNA therapeutics. Although the stability and tolerability of mRNA can be enhanced by directly retouching the mRNA structure, there is still an urgent need to improve the delivery of mRNA. Recently, significant progress has been made in nanobiotechnology, providing tools for developing mRNA nanocarriers. Nano‐drug delivery system is directly used for loading, protecting, and releasing mRNA in the biological microenvironment and can be used to stimulate the translation of mRNA to develop effective intervention strategies. In the present review, we summarized the concept of emerging nanomaterials for mRNA delivery and the latest progress in enhancing the function of mRNA, primarily focusing on the role of exosomes in mRNA delivery. Moreover, we outlined its clinical applications so far. Finally, the key obstacles of mRNA nanocarriers are emphasized, and promising strategies to overcome these obstacles are proposed. Collectively, nano‐design materials exert functions for specific mRNA applications, provide new perception for next‐generation nanomaterials, and thus revolution of mRNA technology.
... Here we present a covalently layered Janus nanoparticle, jNP, with a 10 nm USPION core, tiered PEG brush, antibody targeting face and an opposing cationic carrier face. We selected linear-PEI (25 kDa) for the tiered-PEG cationic face based on studies reporting absence of acute adverse events for PEI-[DNA/siRNA] nano-polyplexes [26,27] during a 2-week observation period. We find -without cytokine elevation or adverse events -jNPs exhibit modular theranostic capabilities with high efficiency: i) jNP-microbubble(MB)-targeted delivery of functional DNA/miRNA to xenograft tumors in an endocytosis-independent manner via operatorcontrolled MB sonoporation, and ii) modular imaging: enhanced ultrasound molecular imaging of intratumoral vasculature (jNP-MBs) and enhanced T2* MR imaging of tumor-selective enhanced permeation retention (EPR) effects (jNPs). ...
... Absence of acute adverse events and non-activation of inflammatory pathways provide proof-ofconcept for a promising safety profile of jNPs and jNP-payload-MBs. Our results using L-PEI 25kDa for the payload-face in jNPs is consistent with studies reporting the absence of acute adverse events for PEI-[DNA/siRNA] nano-polyplexes [26,27]. This contrasts with 30-fold larger linear-PEI 750kDa and branched-PEI 25kDa , which account for reported PEI-based polyplex toxicities [64]. ...
Rationale: High mortality in pancreatic cancer (PDAC) and triple negative breast cancer (TNBC) highlight the need to capitalize on nanoscale-design advantages for multifunctional diagnostics and therapies. DNA/RNA-therapies can provide potential breakthroughs, however, to date, there is no FDA-approved systemic delivery system to solid tumors. Methods: Here, we report a Janus-nanoparticle (jNP)-system with modular targeting, payload-delivery, and targeted-imaging capabilities. Our jNP-system consists of 10 nm ultrasmall superparamagnetic iron oxide nanoparticles (USPION) with opposing antibody-targeting and DNA/RNA payload-protecting faces, directionally self-assembled with commercially available zwitterionic microbubbles (MBs) and DNA/RNA payloads. Results: Sonoporation of targeted jNP-payload-MBs delivers functional reporter-DNA imparting tumor-fluorescence, and micro-RNA126 reducing non-druggable KRAS in PDAC-Panc1 and TNBC-MB231 xenografted tumors. The targeting jNP-system enhances ultrasound-imaging of intra-tumoral microvasculature using less MBs/body weight (BW). The jNP-design enhances USPION's T2*-magnetic resonance (MR) and MR-imaging of PDAC-peritoneal metastases using less Fe/BW. Conclusion: Altogether, data advance the asymmetric jNP-design as a potential theranostic Janus-USPION Modular Platform - a JUMP forward.
... Furthermore, Galeano et al [390] found that viral transfection could release VEGF in diabetic wounds for 4 mo (even after wound healing). Furthermore, de Felipe proposed that a single viral vector capable of transfecting multiple genes could be used for treatment to address the issue that the transfection of a single GF or GF isoform gene only activated a single corresponding signaling pathway rather than promoting multiple stages of wound healing [395]. Jazwa et al [391] demonstrated the simultaneous delivery of VEGF-A and FGF4 genes via bicistronic AAV, and the results showed that the therapeutic effect of multiple gene delivery was better than that of single GF. ...
Wounds in diabetic patients, especially diabetic foot ulcers, are more difficult to heal compared with normal wounds and can easily deteriorate, leading to amputation. Common treatments cannot heal diabetic wounds or control their many complications. Growth factors are found to play important roles in regulating complex diabetic wound healing. Different growth factors such as transforming growth factor beta 1, insulin-like growth factor, and vascular endothelial growth factor play different roles in diabetic wound healing. This implies that a therapeutic modality modulating different growth factors to suit wound healing can significantly improve the treatment of diabetic wounds. Further, some current treatments have been shown to promote the healing of diabetic wounds by modulating specific growth factors. The purpose of this study was to discuss the role played by each growth factor in therapeutic approaches so as to stimulate further therapeutic thinking. Zheng SY et al. Therapeutic growth factors in diabetic wound WJD https://www.wjgnet.com 1 March 18, 2023 Volume 0 Issue 0 Core Tip: This review summarizes the main causes of poor wound healing in diabetes and the role of various therapeutically available growth factors in wound healing. In terms of treatment, it summarizes the treatment methods and drug delivery systems of various growth factors, and discusses the therapeutic effects of different methods and the special properties of drug delivery systems. We hope these discussions will provide the basis for more effective treatments, advance growth factor research, and help more people with diabetes heal their wounds. Therapeutic role of growth factors in treating diabetic wound. World J Diabetes 2023; 0(0): 0-0
... In a study, the macrophages effectively uptake multilamellar SA liposomes compared with negative or neutral liposomes [51]. In liposomes, the structure of the hydrophilic group determines the toxic effect of the cationic lipids [52]. Amphotericin B in lipid nano-emulsions was studied against L. amazonensis in murine macrophages (J774). ...
The uncontrolled increasing clinical resistance to the current anti-parasitic drugs towards important protozoan parasites (Plasmodium, Leishmania, Trypanosoma and Toxoplasma) has stimulated the search for novel and safe therapeutic agents at affordable prices for countries in which these parasites are endemic. For the past few decades, the criticality of the cationic lipid stearylamine (SA) in liposomes has been explored in these human parasites. Previously, SA was incorporated in the liposomal formulation to impart a net positive charge for enhanced cellular uptake. However, the discovery of SA in liposomes alone elicits a strong anti-parasitic activity with immunomodulatory potential. Additionally, the SA liposome possesses a significant inhibitory potential on multiple life stages of the parasite cycle and delivers an equal effect on both drug-sensitive and resistant parasites. Moreover, the delivery of standard anti-parasitic drugs using SA liposome vesicles has enhanced the efficacy of drugs due to the synergistic impacts without causing any apparent toxicity on the host cells. In addition, the delivery of antigens as vaccine candidates using SA liposomes elicits a pronounced immune response in clearing the infection compared to other cationic lipids and SA-free liposomes. Nonetheless, SA liposome mediates its anti-parasitic activity by targeting the negatively charged phosphatidylserine-exposed infected host cell surface or by interaction with negatively charged sialic acid of free-living parasites. Overall, SA liposome confers its protection by acting as a chemotherapeutic agent with immunomodulatory activity. Therefore, a broadly acting anti-parasitic agent (SA liposome) is promising in tackling the deadly parasitic infections in endemic regions and warrants further clinical investigations.
... Another study on C-SLNs reported that cationic lipids could reduce the clearance interference via immune cells by neutralizing the negative charge of the LNP membrane [148]. Despite the various advantages of these cationic lipids, there have been ongoing discussions about their cytotoxicity [176,177]. Therefore, to continuously lower the cytotoxicity of delivery systems using cationic lipids, alternative methods, such as developing two-tailed cationic lipids with a lower cytotoxicity than existing ones, have been proposed [178,179]. ...
Numerous drugs have emerged to treat various diseases, such as COVID-19, cancer, and protect human health. Approximately 40% of them are lipophilic and are used for treating diseases through various delivery routes, including skin absorption, oral administration, and injection. However, as lipophilic drugs have a low solubility in the human body, drug delivery systems (DDSs) are being actively developed to increase drug bioavailability. Liposomes, micro-sponges, and polymer-based nanoparticles have been proposed as DDS carriers for lipophilic drugs. However, their instability, cytotoxicity, and lack of targeting ability limit their commercialization. Lipid nanoparticles (LNPs) have fewer side effects, excellent biocompatibility, and high physical stability. LNPs are considered efficient vehicles of lipophilic drugs owing to their lipid-based internal structure. In addition, recent LNP studies suggest that the bioavailability of LNP can be increased through surface modifications, such as PEGylation, chitosan, and surfactant protein coating. Thus, their combinations have an abundant utilization potential in the fields of DDSs for carrying lipophilic drugs. In this review, the functions and efficiencies of various types of LNPs and surface modifications developed to optimize lipophilic drug delivery are discussed.
... However, PEI is still highly toxic because commercial high-molecular-weight PEI is not degradable [155]. Some researchers have tried to solve this problem by adding acid-labile imine linkers [155] or by reducing its molecular weight and introducing branching [156]. ...
Over the past two decades, significant technological innovations have led to messenger RNA (mRNA) becoming a promising option for developing prophylactic and therapeutic vaccines, protein replacement therapies, and genome engineering. The success of the two COVID-19 mRNA vaccines has sparked new enthusiasm for other medical applications, particularly in cancer treatment. In vitro-transcribed (IVT) mRNAs are structurally designed to resemble naturally occurring mature mRNA. Delivery of IVT mRNA via delivery platforms such as lipid nanoparticles allows host cells to produce many copies of encoded proteins, which can serve as antigens to stimulate immune responses or as additional beneficial proteins for supplements. mRNA-based cancer therapeutics include mRNA cancer vaccines, mRNA encoding cytokines, chimeric antigen receptors, tumor suppressors, and other combination therapies. To better understand the current development and research status of mRNA therapies for cancer treatment, this review focused on the molecular design, delivery systems, and clinical indications of mRNA therapies in cancer.
... The use of cationic lipids may present translational challenges due to their recognized toxicity [261]. This has motivated the development of ionizable lipids, which are conditionally cationic, as well as the use of antibodies and peptides that bind to endothelial-specific receptors through molecular recognition rather than non-specific electrostatic interactions. ...
Endothelial cells play critical roles in circulatory homeostasis and are also the gateway to the major organs of the body. Dysfunction, injury, and gene expression profiles of these cells can cause, or are caused by, prevalent chronic diseases such as diabetes, cardiovascular disease, and cancer. Modulation of gene expression within endothelial cells could therefore be therapeutically strategic in treating longstanding disease challenges. Lipid nanoparticles (LNP) have emerged as potent, scalable, and tunable carrier systems for delivering nucleic acids, making them attractive vehicles for gene delivery to endothelial cells. Here, we discuss the functions of endothelial cells and highlight some receptors that are upregulated during health and disease. Examples and applications of DNA, mRNA, circRNA, saRNA, siRNA, shRNA, miRNA, and ASO delivery to endothelial cells and their targets are reviewed, as well as LNP composition and morphology, formulation strategies, target proteins, and biomechanical factors that modulate endothelial cell targeting. Finally, we discuss FDA-approved LNPs as well as LNPs that have been tested in clinical trials and their challenges, and provide some perspectives as to how to surmount those challenges.
... Ester bond is less toxic because of their easy hydrolysis by esterase that results in the higher biodegradability. However, ether bond is too stable to be biodegraded in the physiological condition and thus cause toxicity, albeit their excellent transfection efficiency [136]. Lipids with carbamate bonds also have low cytotoxicity [137,138]. ...
... As a result, transfection agents, mainly cationic polymers such as endocytosis, doxorubicin, and lipofectamine are frequently used to compensate for the charge to ensure efficient cellular uptake of DNA nanostructures. It has been documented that such DNA nanostructures have better uptake efficiency when combined with cationic and hydrophobic molecules (Lv et al., 2006;Zhdanov et al., 2002). Nevertheless, the new findings demonstrate that DNA nanostructures that can easily pass across the membrane barrier are of high interest in examining the delivery potential of these biological vehicles. ...
... As a result, transfection agents, mainly cationic polymers such as endocytosis, doxorubicin, and lipofectamine are frequently used to compensate for the charge to ensure efficient cellular uptake of DNA nanostructures. It has been documented that such DNA nanostructures have better uptake efficiency when combined with cationic and hydrophobic molecules (Lv et al., 2006;Zhdanov et al., 2002). Nevertheless, the new findings demonstrate that DNA nanostructures that can easily pass across the membrane barrier are of high interest in examining the delivery potential of these biological vehicles. ...
... Therefore, the extensive attention has Lipid-based systems such as lipofectamine, oligofectamine, and siPORT, and DharmaFECT could be used as the major carriers of RNA due to effective delivery and substantial transfection ability [101]. However, the in vitro and in vivo studies reported that their usage is constrained by their toxicity, nonspecific uptake, and immune and inflammatory side effects [102,103]. PEGylation is another strategy used to mitigate immunogenicity and prolonged systemic delivery by adding the D-α-tocopheryl polyethylene glycol succinate. This strategy can enhance the cellular uptake [104,105]. ...
Several research reports delineated the significant role of miRNAs in cancer proliferation, and their modulatory role in cancer mitigation, and drug resistance. Melanoma cells have been acquiring stemness to several chemotherapeutic agents through drug efflux proteins, epigenetic modulation, and DNA repair. miRNAs could be applied as novel therapeutic modalities for treating several kinds of cancers to modulate these mechanisms involved in stemness.Nanocarriers to carry these tumor‐targeting miRNAs to modulate stemness are a prominent strategy to overcome their low penetrability, minimal stability, and nonspecificity. We have searched several public databases such as Pubmed, Medline, Google scholar, and NLM and obtained the information pertinent to the miRNA‐based nanocarriers systems to target stemness through epigenetic modulation in melanomas. This review delineates that various miRNAs can modulate the stemness in melanomas by specific intricate epigenetic signaling, and other cell based signaling mechanisms. Specific nanocarrier formulations with specific miRNAs are optimal methods to deliver these miRNAs in order to achieve a significant entrapment efficiency, loading efficiency, and stability. Furthermore, the combinatorial regimen of FDA approved chemotherapeutic molecules with tumor‐targeting miRNAs and chemotherapycombined with nanocarriers can efficiently deliver the utmost therapeutic window by targeting tumor matrix, invasion, metastasis, and angiogenesis in melanomas. Substantial research should focus on the clinical application of this gene therapy in melanomas using these low immunogenic, highly degradable, and biocompatible combinatorial nanotherapeutic regimens.
... Although liposomes are widely used as in vitro transfection reagents due to their low immunogenicity, their in vivo application is often restricted by toxicity linked to compound composition and structure. 287 Encouragingly, several cationic lipid-based vectors have been successfully utilized to improve the in vivo delivery of therapeutic miRNAs in murine models of RA. ...
MicroRNAs (miRNAs), a class of endogenous single-stranded short noncoding RNAs, have emerged as vital epigenetic regulators of both pathological and physiological processes in animals. They direct fundamental cellular pathways and processes by fine-tuning the expression of multiple genes at the posttranscriptional level. Growing evidence suggests that miRNAs are implicated in the onset and development of rheumatoid arthritis (RA). RA is a chronic inflammatory disease that mainly affects synovial joints. This common autoimmune disorder is characterized by a complex and multifaceted pathogenesis, and its morbidity, disability and mortality rates remain consistently high. More in-depth insights into the underlying mechanisms of RA are required to address unmet clinical needs and optimize treatment. Herein, we comprehensively review the deregulated miRNAs and impaired cellular functions in RA to shed light on several aspects of RA pathogenesis, with a focus on excessive inflammation, synovial hyperplasia and progressive joint damage. This review also provides promising targets for innovative therapies of RA. In addition, we discuss the regulatory roles and clinical potential of extracellular miRNAs in RA, highlighting their prospective applications as diagnostic and predictive biomarkers.
... Various lipid materials, especially cationic and ionizable lipids, are the basis for efficient nucleic acid loading, cellular internalization, and endosomal escape. Compared with polymeric materials with unexpected cytotoxicity, the risk of adverse immune responses to lipids is lower, endowing LNPs with prospects for translation into clinical applications [44][45][46][47]. ...
In recent decades, advances in chemical synthesis and delivery systems have accelerated the development of therapeutic nucleic acids, several of which have been approved by the Us Food and Drug Administration (FDA). Oral nucleic acid delivery is preferred because of its simplicity and patient compliance, but it still presents distinct challenges. The negative charge, hydrophilicity, and large molecular weight of nucleic acids combined with in vivo gastrointestinal (GI) barriers (e.g., acidic pH, enzymes, mucus, and intestinal epithelial cells) severely hinder their delivery efficacy. Recently, various nanoparticles (NPs), ranging from polymeric to lipid-based (L)NPs and extracellular vesicles (EVs), have been extensively explored to address these obstacles. In this review, we describe the physiological barriers in the GI tract and summarize recent advances in NP-based oral nucleic acid therapeutics. Teaser: This review provides a concise summary of recent advances in the multiple physiological barriers in gastrointestinal tract, and the nanoparticle-based strategies explored to assist nucleic acids in overcoming barriers and treating diseases.
... reduction in GAPDH protein expression where its non-targeted PAMP-CP-rGO-Dox-siRNA derivatives only reduces the expression by 23.18%. In spite of equal protein content used for the assay to eliminate the toxic effect of Dox carrying positively charged carriers that were prepared by scrambled siRNA loading decreased the GAPDH protein expression by ~ 20% and ~ 30%, this decrease in protein levels observed upon treatment is indicative of their effects on cell viability [59][60][61]. The efficacy of the particles formed by combining therapeutic nucleic acids with PAMP-CP-rGO derivatives as gene and drug carriers was further supported by these results. ...
... One purpose of using a model with an intact immune system is to assure the therapeutic agent is not immunogenic and is not inactivated in an animal with the ability to generate neutralizing antibodies. Although cationic nanoliposomes provide an efficient way to complex with negatively charged siRNAs, these positively charged nanoparticles are highly toxic [49] eliciting dose-dependent toxicity and pulmonary inflammation, hepatotoxicity and a systemic interferon type I response [50]. Safety and off-target toxicity was examined in our study with immune competent mice over a short (4 weeks) or long (4 months) duration of NP administration. ...
Survival from pancreatic cancer is poor because most cancers are diagnosed in the late stages and there are no therapies to prevent the progression of precancerous pancreatic intraepithelial neoplasms (PanINs). Inhibiting mutant KRASG12D, the primary driver mutation in most human pancreatic cancers, has been challenging. The cholecystokinin-B receptor (CCK-BR) is absent in the normal pancreas but becomes expressed in high grade PanIN lesions and is over-expressed in pancreatic cancer making it a prime target for therapy. We developed a biodegradable nanoparticle polyplex (NP) that binds selectively to the CCK-BR on PanINs and pancreatic cancer to deliver gene therapy. PanIN progression was halted and the pancreas extracellular matrix rendered less carcinogenic in P48-Cre/LSL-KrasG12D/+ mice treated with the CCK-BR targeted NP loaded with siRNA to mutant Kras. The targeted NP also slowed proliferation, decreased metastases and improved survival in mice bearing large orthotopic pancreatic tumors. Safety and toxicity studies were performed in immune competent mice after short or long-term exposure and showed no off-target toxicity by histological or biochemical evaluation. Precision therapy with target-specific NPs provides a novel approach to slow progression of advanced pancreatic cancer and also prevents the development of pancreatic cancer in high-risk subjects without toxicity to other tissues.
... Although immunogenicity of these lipids is not yet fully understood, system of complement and Toll-Like receptors may participate in innate immune activation. Cytotoxicity of lipid materials also poses safety concerns depending on dose, lipid properties and cell types [135,136]. In vivo application of lipid nanoparticles has been reported to induce liver and lung damage in rodents, which may relate to cytotoxicity of the materials and induction of pro-inflammatory factors. ...
Background:
The adventure of the mRNA vaccine began thirty years ago in the context of influenza. This consisted in encapsulating the mRNA coding for a viral protein in a lipid particle. We show how the mRNA encoding S protein has been modified for that purpose in the context of the anti-SARS-CoV-2 vaccination.
Results:
by using data coming from genetic and epidemiologic databases, we show the theoretical possibility of fragmentation of this mRNA into small RNA sequences capable of inhibiting important bio-syntheses such as the production of beta-globin.
Discussion:
we discuss two aspects related to mRNA vaccine: (i) the plausibility of mRNA fragmentation, and (ii) the role of liposomal nanoparticles (LNPs) used in the vaccine and their impact on mRNA biodistribution.
Conclusion:
we insist on the need to develop lipid nanoparticles allowing personalized administration of vaccines and avoiding adverse effects due to mRNA fragmentation and inefficient biodistribution. Hence, we recommend (i) adapting the mRNA of vaccines to the least mutated virus proteins and (ii) personalizing its administration to the categories of chronic patients at risk most likely to suffer from adverse effects.
... Liposomes are one of the most used transfection reagents in vitro, due to their biodegradability, biocompatibility and their high resemblance to the cell membrane [241,242]. Nevertheless, some studies have reported high toxicity rates in liposomes, alongside non-specific uptake and the triggering of unwanted immune responses [243,244]. It is worth mentioning that some of these drawbacks, such as low specificity, can be easily overcome by surface modification. ...
MicroRNAs (miRNAs) act as master regulators of gene expression in homeostasis and disease. Despite the rapidly growing body of evidence on the theranostic potential of restoring miRNA levels in pre-clinical models, the translation into clinics remains limited. Here, we review the current knowledge of miRNAs as T-cell targeting immunotherapeutic tools, and we offer an overview of the recent advances in miRNA delivery strategies, clinical trials and future perspectives in RNA interference technologies.
... Consequently, emphasizing on the methods that prioritize the pharmacological properties of the carrier would be cruicial. [10][11] . ...
This study was aimed at developing a novel platform for tetravalent conjugation of 4-arm polyethylene glycol (PEG) with an antisense oligonucleotide (ASO). The ASO technology has several limitations, such as low cellular uptake, poor nuclease stability, and short half-life. PEG-conjugated ASOs may result in an improvement in the pharmacokinetic behavior of the drug. Moreover, PEGylation can reduce enzymatic degradation and renal excretion of the conjugates, thereby, increasing its blood stability and retention time. In this study, we successfully synthesized PEG-ASO conjugate consisting of 4-arm-PEG and four molecules of ASO (4-arm-PEG-tetra ASO). Its hybridization ability with complementary RNA, enzymatic stability, and in-vitro gene silencing ability were evaluated. No significant difference in hybridization ability was observed between 4-arm-PEG-tetra ASO and the parent ASO. In addition, gene silencing activity of the 4-arm-PEG-tetra ASO was observed in vitro. However, the in vitro activity of the 4-arm-PEG-tetra ASO was slightly reduced as that of the parent ASO. Moreover, the 4-arm-PEG-tetra ASO showed appreciable stability in cellular extract, suggesting that it hybridizes with mRNA in its intact form, without being cleaved in the cell, and exhibits ASO activity.
... Commonly, liposomes are used to deliver mRNA as these particles most easily encapsulate mRNAs and can enter into APCs (Cullis & Hope, 2017). However, the cationic lipids used in these liposomes destabilize cell membranes (Lv et al., 2006) and induce inflammation (Ma et al., 2005). Therefore, ionizable lipid-like materials are explored, which are positively charged at low pH to allow complex formation with mRNA, and neutral at physiological pH to reduce the toxic effects (Kauffman et al., 2015). ...
Nanoparticles (NPs) are not only employed in many biomedical applications in an engineered form, but also occur in our environment, in a more hazardous form. NPs interact with the immune system through various pathways and can lead to a myriad of different scenarios, ranging from their quiet removal from circulation by macrophages without any impact for the body, to systemic inflammatory effects and immuno-toxicity. In the latter case, the function of the immune system is affected by the presence of NPs. This review describes, how both the innate and adaptive immune system are involved in interactions with NPs, together with the models used to analyse these interactions. These models vary between simple 2D in vitro models, to in vivo animal models, and also include complex all human organ on chip models which are able to recapitulate more accurately the interaction in the in vivo situation. Thereafter, commonly encountered NPs in both the environment and in biomedical applications and their possible effects on the immune system are discussed in more detail.
Not all effects of NPs on the immune system are detrimental; in the final section, we review several promising strategies in which the immune response towards NPs can be exploited to suit specific applications such as vaccination and cancer immunotherapy.
... This results in vector disintegration and release of genetic material. Although lipoplexes are less efficient than viral vectors when administered intravenously, lipid vectors still have greater safety for in vivo application [93,94]. ...
Several human pathogens can cause long-lasting neurological damage. Despite the increasing clinical knowledge about these conditions, most still lack efficient therapeutic interventions. Gene therapy (GT) approaches comprise strategies to modify or adjust the expression or function of a gene, thus providing therapy for human diseases. Since recombinant nucleic acids used in GT have physicochemical limitations and can fail to reach the desired tissue, viral and non-viral vectors are applied to mediate gene delivery. Although viral vectors are associated to high levels of transfection, non-viral vectors are safer and have been further explored. Different types of nanosystems consisting of lipids, polymeric and inorganic materials are applied as non-viral vectors. In this review, we discuss potential targets for GT intervention in order to prevent neurological damage associated to infectious diseases as well as the role of nanosized non-viral vectors as agents to help the selective delivery of these gene-modifying molecules. Application of non-viral vectors for delivery of GT effectors comprise a promising alternative to treat brain inflammation induced by viral infections.
... An excess of polymers with high positive charge content are used to ensure full and stable compaction of DNA to yield positively charged nanoparticles. However, the high charge content in carrier polymers can cause cytotoxic effects to cells by disrupting plasma and lysosomal membranes (Lv, Zhang, Wang, Cui, & Yan, 2006;Monnery et al., 2017;Nel et al., 2009) and/or compromising mitochondrial metabolic activity (Bhattacharjee et al., 2010). Thus, environmentally sensitive (e.g., biodegradable and bioreducible) polymers that break into small pieces in vivo have been developed and validated for enhanced safety profiles . ...
The advent of the first-ever retinal gene therapy product, involving subretinal administration of a virus-based gene delivery platform, has garnered hope that this state-of-the-art therapeutic modality may benefit a broad spectrum of patients with diverse retinal disorders. On the other hand, clinical studies have revealed limitations of the applied delivery strategy that may restrict its universal use. To this end, intravitreal administration of synthetic gene-delivery platforms, such as polymer-based nanoparticles (PNPs), has emerged as an attractive alternative to the current mainstay. To achieve success, however, it is imperative that synthetic platforms overcome key biological barriers in human eyes encountered following intravitreal administration, including the vitreous gel and inner limiting membrane (ILM). Here, we introduce a series of experiments, from the fabrication of PNPs to a comprehensive evaluation in relevant experimental models, to determine whether PNPs overcome these barriers and efficiently deliver therapeutic gene payloads to retinal cells. We conclude the article by discussing a few important considerations for successful implementation of the strategy. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Preparation and characterization of PNPs Basic Protocol 2: Evaluation of in vitro transfection efficacy Basic Protocol 3: Evaluation of PNP diffusion in vitreous gel Basic Protocol 4: Ex vivo assessment of PNP penetration within vitreoretinal explant culture Basic Protocol 5: Assessment of in vivo transgene expression mediated by intravitreally administered PNPs.
... Considerable progress has been made over the last decade in polymer-mediated CRISPR-Cas9 plasmid delivery for genome editing applications [149,[168][169][170][171]. The most widely used cationic polymers for pharmaceutical applications include poly-L-lysine (PLL), polyethylenimine (PEI), polyamidoamine (PAMAM) dendrimers, polyethylene glycol (PEG), and chitosan. Despite some evidences revealing the toxicity of PLL [172], PEG [173], PEI [174], and PAMAM [175], remarkable efforts have been made to modify and optimize these cationic polymers and render them as ideal vectors for effective gene delivery with lower toxicity, especially for the delivery of CRISPR-Cas9 [171,[176][177][178]. To date, no study reported the delivery of CRISPR-Cas9 machinery to the retina using this cationic polymer-based delivery strategy. ...
Inherited Retinal Diseases (IRDs) are considered one of the leading causes of blindness worldwide. However, the majority of them still lack a safe and effective treatment due to their complexity and genetic heterogeneity. Recently, gene therapy is gaining importance as an efficient strategy to address IRDs which were previously considered incurable. The development of the clustered regularly-interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) system has strongly empowered the field of gene therapy. However, successful gene modifications rely on the efficient delivery of CRISPR-Cas9 components into the complex three-dimensional (3D) architecture of the human retinal tissue. Intriguing findings in the field of nanoparticles (NPs) meet all the criteria required for CRISPR-Cas9 delivery and have made a great contribution toward its therapeutic applications. In addition, exploiting induced pluripotent stem cell (iPSC) technology and in vitro 3D retinal organoids paved the way for prospective clinical trials of the CRISPR-Cas9 system in treating IRDs. This review highlights important advances in NP-based gene therapy, the CRISPR-Cas9 system, and iPSC-derived retinal organoids with a focus on IRDs. Collectively, these studies establish a multidisciplinary approach by integrating nanomedicine and stem cell technologies and demonstrate the utility of retina organoids in developing effective therapies for IRDs.
... Additionally, interactions with negatively charged membrane-based phospholipid molecules affect their packing and subsequently their morphology leading ultimately to hemolysis (Zachowski and Durand, 1988;Kondo and Tomizawa, Nov. 1969). The lower toxicity of Arg-based surfactants might derive from their guanidinium function, which enables them to spread the positive charge (Lv et al., 2006). Furthermore, it remains questionable whether the lipophilic tail ...
The aim of this study was to evaluate the safety and efficacy for hydrophobic ion-pairing of surfactants based on arginine (Arg). The prepared Arg-cholesteryl ester (ACE) and Arg-diosgenyl ester (ADE) were characterized regarding solubility, pKa, critical micellar concentration (CMC), biodegradability as well as membrane- and aquatic toxicity using DOTAP as reference. The ability for hydrophobic ion-pairing was evaluated and the lipophilicity of formed complexes was determined.
NMR, FT-IR and MS confirmed successful synthesis of Arg-surfactants. The slightly soluble single-charged Arg-surfactants (pH < pKa3 (ACE = 10.42 ±0.52; ADE = 10.38 ±0.27)) showed CMCs of 27.17 µM for ACE and 35.67 µM for ADE. CMCs of the sparingly soluble double-charged species (pH < pKa2 (ACE = 5.30 ±0.20; ADE = 5.55 ±0.06)) were determined at concentrations of ≥ 250 µM for ACE and ≥ 850 µM for ADE. The enzymatic- and environmental biodegradability was proven by an entire cleavage of Arg-surfactants within 24 h, whereas DOTAP remained stable. Arg-surfactants exhibited lower membrane- (> 2-fold) and aquatic toxicity (> 15-fold) than DOTAP. The complexes formed with Arg-surfactants and insulin showed higher lipophilicity than the DOTAP-complex. According to these results, Arg-surfactants might be a promising safe tool for the delivery of peptide drugs.
... The cationic polymers and lipids that can complex with nucleic acids and disrupt lysosomes are the most used in building novel gene carriers. It should be noted, however, that cationic polymers and lipids might cause cytotoxicity and immunotoxicity [79]. Inadequate systemic stability and organ selectivity (except liver) are the other factors limiting the application of cationic delivery systems. ...
The development of carrier systems that are able to transport and release therapeutics to target cells is an emergent strategy to treat cancer; however, they following endocytosis are usually trapped in the endo/lysosomal compartments. The efficacy of drug conjugates and nanotherapeutics relies critically on their intracellular drug release ability, for which advanced systems responding to the unique lysosomal environment such as acidic pH and abundant enzymes (e.g. cathepsin B, sulfatase and β-glucuronidase) or equipped with photochemical internalization property have been energetically pursued. In this review, we highlight the recent designs of smart systems that promote efficient lysosomal release and/or escape of anticancer agents including chemotherapeutics (e.g. doxorubicin, platinum, chloroquine and hydrochloroquine) and biotherapeutics (e.g. proteins, siRNA, miRNA, mRNA and pDNA) to cancer cells or immunotherapeutic agents (e.g. antigens, mRNA and immunoadjuvants) to antigen-presenting cells (APCs), thereby boosting cancer therapy and immunotherapy. Lysosomal-mediated drug release presents an appealing approach to develop innovative cancer therapeutics and immunotherapeutics.
... However, most polysaccharides are generally modified by cationic moieties before being used as delivery vehicles [49]. Although cationic materials interact sufficiently with the cell membrane to facilitate the delivery of DNA or RNA, the positive charges can often damage the cell membrane and cause high toxicity [50]. Recently, the development of non-cationic vectors has garnered increasing interest, but the number of non-cationic polysaccharide vectors remains limited. ...
Biomimetics plays an important role in cancer treatment since it can prolong the circulation of nanoparticles, enhance their delivery and retention in target tissues, and reduce the systemic toxicity of drugs and their carriers. In this study, we developed a biomimetic nanosystem consisting of chemotherapeutic and immunotherapeutic agents wrapped in cell membranes. Specifically, the anti-tumor drug doxorubicin (DOX) was loaded into a bacterial-derived immunomodulatory agent (low molecular weight curdlan, lCUR), and the lCUR-DOX was further wrapped in the red blood cell membrane for camouflage and prolonged circulation. The successful preparation of the [email protected] nanosystem was supported by various optical and morphological characterizations. In vitro studies indicated that the nanosystem can escape uptake by macrophages, inhibit the invasion of tumor cells, and reprogram M2 macrophages with an immunosuppressive phenotype into M1 macrophages with an immunopromoting phenotype via the MAPK signaling pathway while promoting the phagocytosis of macrophages. In vivo studies showed that the nanosystem effectively inhibits tumor growth in the A-375 tumor-bearing mouse model. Taken together, the above results support further development of the [email protected] platform for cancer immunochemotherapy in clinical applications.
With the development of newer biomarkers in the diagnosis of gastric cancer (GC), therapeutic targets are emerging and molecular-targeted therapy is in progress RNA interference has emerged as a promising method of gene targeting therapy. However, bare small interfering RNA (siRNA) is unstable and susceptible to degradation, so developing vectors for siRNA delivery is the focus of our research. We developed LMWP modified PEG-SS-PEI to deliver siRNA targeting BRD4 (L-NPs) for GC therapy. L-NPs were prepared by electrostatic interaction and characterized by dynamic light scattering (DLS) and transmission electron microscopy (TEM). The release characteristics, cellular uptake and intracellular localization were also investigated. The in vitro anticancer activity of the prepared nanoparticles was analysed by MTT, Transwell invasion and wound healing assay. Western blot was used to detect the effect of gene silencing. The results showed that the optimal N/P was 30 and the prepared L-NPs uniformly distributed in the system with a spherical and regular shape. L-NPs exhibited an accelerated release in GSH-containing media from 12h to 24h. The uptake of L-NPs was enhanced and mainly co-localized in the lysosomes. After 6h incubation, LMWP modified PEG-SS-PEI helped siRNA escape from the lysosomes and diffused into the cytoplasm. L-NPs significantly in hibited the proliferation, migration and invasion of cells. This might be related with the silence of BRD4, then inhibition of PI3K/Akt and c-Myc. Our results demonstrate that L-NPs are a novel delivery system with anticancer, which may provide a more effective strategy for GC treatment.
In the past two decades, thermoresponsive polymers based on tertiary amine groups have been studied extensively as a class of dual-responsive polymers. In particular, their temperature-dependent phase transition can be...
Lipid nanoparticles (LNPs) have achieved clinical success in delivering small interfering RNAs (siRNAs) for targeted gene therapy. However, endosomal escape of siRNA into the cytosol remains a fundamental challenge for LNPs. Herein, we report a strategy termed light-activated siRNA endosomal release (LASER) to address this challenge. We established a porphyrin-LNP by incorporating porphyrin-lipids into the clinically approved Onpattro formulation. The porphyrin-LNP maintained the physical properties of an LNP and generated reactive oxygen species (ROS) when irradiated with near-infrared (NIR) light. Using confocal microscopy, we revealed that porphyrin-lipids within the LNP translocate to endosomal membranes during endocytosis. The translocated porphyrin-lipids generated ROS under light irradiation and enabled LASER through endosomal membranes disruption as observed through GAL-9 recruitment and transmission electron microscopy (TEM). By establishing a quantitative confocal imaging method, we confirmed that porphyrin-LNPs can increase siRNA endosomal escape efficiency by up to 2-fold via LASER and further enhance luciferase target knockdown by 4-fold more in luciferase-transfected prostate cancer cells. Finally, we formulated porphyrin-LNPs encapsulated with gold nanoparticles (GNP) and visualized the LASER effect within prostate tumors via TEM, confirming the light-activated endosomal membrane disruption and subsequent GNP release into cytosols in vivo. Overall, porphyrin-LNPs and the LASER approach enhanced siRNA endosomal escape and significantly improved knockdown efficacy. We believe the versatility of this technology could be applied to various LNP-based RNA therapeutics.
Currently, organic solvents are necessary for the preparation of anionic liposomes for siRNA delivery. The removal of organic solvent is time-consuming and the residual organic solvent is not only a hidden danger, but also affects the stability of anionic liposomes. Glycerol, which is physiologically compatible and does not need to be removed, is used to promote the dispersion of lipids and the formation of anionic liposomes. Additionally, the preparation process is simple and not time-consuming. The results showed that anionic liposomes, which were typically spherical with a particle size of 188.9 nm were successfully prepared with glycerol. And with the help of Ca2+ , siRNA was encapsulated in anionic liposomes. The highest encapsulation efficiency at 2.4 mM Ca2+ reached 91%. And the formation of calcium phosphate could promote the endosomal escape of siRNA effectively. The results from cell viability showed that the anionic liposomes had no obvious cytotoxicity. It was also verified that anionic liposomes could improve the resistance of siRNA against degradation. Additionally, siRNA delivered by anionic liposomes could play an effective role in knockout. Therefore, anionic liposomes prepared with glycerol will be a safe and effective delivery platform for siRNA and even other nucleic acid drugs.
Messenger RNA (mRNA) based therapies offer great promise for the treatment of a variety of diseases. In 2020, two FDA approvals of mRNA-based vaccines have elevated mRNA vaccines to global recognition. However, the therapeutic capabilities of mRNA extend far beyond vaccines against infectious diseases. They hold potential for cancer vaccines, protein replacement therapies, gene editing therapies, and immunotherapies. For realizing such advanced therapies, it is crucial to develop effective carrier systems. Recent advances in material science have led to the development of promising non-viral mRNA delivery systems. In comparison to other carriers like lipid nanoparticles, polymer-based delivery systems often receive less attention, despite their unique ability to carefully tune their chemical features to promote mRNA protection, their favorable pharmacokinetics, and their potential for targeting delivery. In this review, we discuss the central features of polymer-based systems for mRNA delivery highlighting the molecular design criteria, stability, and biodistribution. Finally, we analyze the role of targeting ligands for the future of RNA therapies. This article is protected by copyright. All rights reserved.
Inside the field of neural engineering, many researchers have dedicated their work to investigating drug delivery technologies as vital tools to modulate neuro-inflammation, prevent neurodegeneration, promote regeneration, alleviate symptoms, and restore neurological function. Unfortunately, drug delivery into the central nervous system (CNS) tissue is as challenging as it is promising. The environment inside of the CNS is highly controlled, and active barriers between the blood and nervous systems often make traditional drug delivery methods ineffective. The major impediment to neural drug delivery is the blood-brain barrier (BBB). The BBB is not a single physical structure, but a series of tight junctions, cells, and an array of transporting mechanism which ensures that only certain compounds can enter the brain from the blood. However, the BBB is not insurmountable, and neural engineers have adapted and created many localized drug delivery platforms to circumvent filtering by the BBB. These platforms have the potential to reduce the inflammatory tissue response to implanted neural electrodes, add pharmacological manipulation to the recording/stimulation devices as a research tool, or enable closed-loop drug delivery therapy. Some delivery platforms, such as microfluidic channels and polymer coatings, are incorporated as components of existing devices. Others are engineered drug carriers that can be systemically administered to ferry compounds through the brain endothelium. Further, each platform is unique in material composition, method/mechanism of release, and drawbacks. The specific properties of these platforms must be examined in the context of the application. Although the field of neural drug delivery is still developing, it may one day be possible to treat diseases of the CNS as easily and efficaciously as any other ailment.
Background and Objectives: Today, PEI is used for high-efficiency gene transfection. This polycation can have toxic effects on cells due to charged amine groups. This study evaluated the toxic effects of PEI in both single and oligonucleotide-binding modes on VSMC cells. Methods: First, VSMC cells were cultured and then transfected by nanoparticles in two PEI states alone and containing oligonucleotides. After 24 hours, cell viability and morphology were assessed using MTT and electron microscopy methods respectively. Data were analysed using the one-way ANOVA, followed by the Tukey Post-Hoc test. Results: The results showed that nanoparticles significantly reduced the cell viability by causing toxicity to cells (P<0.0001) and also its images showed cell death and apoptosis. But PEI containing oligonucleotides had no toxic effects on cell viability (P= 0.7414). Its electron microscope image also showed a normal cell morphology. Conclusion: Overall, this study showed that the effect of PEI toxicity on VSMC cells is inhibited by oligonucleotide sequencing and thus neutralizing the positive charges of the amine group.
Cationic pH-responsive polymers promise to overcome critical challenges in cellular delivery. Ideally, the polymers become selectively charged along the endosomal pathway disturbing only the local membrane and avoiding unintended interactions or cytotoxic side effects at physiological conditions. Polypiperazines represent a novel, hydrophilic class of pH-responsive polymers whose response can be tuned within the relevant pH range (5 - 7.4). We discovered that the polypiperazines are effectively binding plasmid DNA (pDNA) and demonstrate high efficiency in transfection. By design of experiments (DoE), a wide parameter space (pDNA and polymer concentration) was screened to identify the range of effective concentrations for transfection. An isopropyl modified polypiperazine was highly efficient over a wide range of concentrations outperforming linear polyethylenimine (l-PEI, 25 kDa) in regions of low N*/P ratios. A quantitative polymerase chain reaction (qPCR) surprisingly revealed that the pDNA within the piperazine-based polyplexes can be amplified in contrast to polyplexes based on l-PEI. The pDNA must therefore be more accessible and bound differently than for other known transfection polymers. Considering the various opportunities to further optimize their structure, polypiperazines represent a promising platform for designing effective soluble polymeric vectors, which are charge-neutral at physiological conditions. This article is protected by copyright. All rights reserved.
Lipid nanoparticles (LNPs) are the most clinically advanced delivery vehicles for RNA and have enabled the development of RNA-based drugs such as the mRNA COVID-19 vaccines. Functional delivery of mRNA by an LNP greatly depends on the inclusion of an ionizable lipid, and small changes to these lipid structures can significantly improve delivery. However, the structure-function relationships between ionizable lipids and mRNA delivery are poorly understood, especially for LNPs administered intramuscularly. Here, we show that the iterative design of a novel series of ionizable lipids generates key structure-activity relationships and enables the optimization of chemically distinct lipids with efficacy that is on-par with the current state of the art. We find that the combination of ionizable lipids comprising an ethanolamine core and LNPs with an apparent pKa between 6.6 and 6.9 maximizes intramuscular mRNA delivery. Furthermore, we report a nonlinear relationship between the lipid-to-mRNA mass ratio and protein expression, suggesting that a critical mass ratio exists for LNPs and may depend on ionizable lipid structure. Our findings add to the mechanistic understanding of ionizable lipids and demonstrate that hydrogen bonding, ionization behavior, and lipid-to-mRNA mass ratio are key design parameters affecting intramuscular mRNA delivery. We validate these insights by applying them to the rational design of new ionizable lipids. Overall, our iterative design strategy efficiently generates potent ionizable lipids. This hypothesis-driven method reveals structure-activity relationships that lay the foundation for the optimization of ionizable lipids in future LNP-RNA drugs. We foresee that this design strategy can be extended to other optimization parameters beyond intramuscular expression.
The prevalence of allergic disorders has increased worldwide in recent decades. Polyphenols, including resveratrol and curcumin, are posited to have potential as therapeutic agents for allergy; however, their use has been limited by poor water solubility. Accordingly, we developed a highly concentrated, water dispersible, supramolecular complexes of polyphenols with polypeptides (poly‐L‐lysine, poly‐γ‐glutamic acid) and gelatin using high‐speed vibration milling. The complex exhibited resistance to photo‐bleaching and thermal radiation. Treatment of a rat basophilic leukemia cell line (RBL‐2H3) with polypeptide complexes containing resveratrol suppressed allergic responses in vitro. Moreover, aerosolized administration of polypeptide complexes demonstrated excellent bioavailability and inhibition of immediate hypersensitivity reactions in ear tissue in vivo. Further, our method avoids the use of organic solvent and therefore reduces undesirable biological responses. This article is protected by copyright. All rights reserved
Diabetic retinopathy (DR) is a vision-impairing complication of diabetes, damaging the retinal microcirculatory system. Overexpression of VEGF (vascular endothelial growth factor) is implicated in the pathogenesis of DR. Human antigen R (HuR) is an RNA-binding protein that favorably regulates VEGF protein expression by binding to VEGF-encoding mRNA. Downregulating HuR via RNA interference strategies using small interfering RNAs (siRNAs) may constitute a novel therapeutic method for preventing VEGF protein overexpression in DR. Delivery of siRNAs to the cellular cytoplasm can be facilitated by cationic peptides or polymers and lipids. In this study, a cationic polymer (polyethylenimine (PEI)) and lipid nanoparticles (liposomes) were co-formulated with siRNA to form lipopolyplexes (LPPs) for the delivery of HuR siRNA. LPPs-siRNA were analyzed for size, zeta potential, serum stability, RNase stability, heparin stability, toxicity, and siRNA encapsulation efficiency. Cellular uptake, downregulation of the target HuR (mRNA and protein), and associated VEGF protein were used to demonstrate the biological efficacy of the LPPs-HuR siRNA, in vitro (human ARPE-19 cells), and in vivo (Wistar rats). In vivo efficacy study was performed by injecting LPPs-HuR siRNA formulations into the eye of streptozotocin (STZ)-induced diabetic rats after the development of retinopathy. Our findings demonstrated that high retinal HuR and VEGF levels observed in the eyes of untreated STZ rats were lowered after LPPs-HuR siRNA administration. Our observations indicate that intravitreal treatment with HuR siRNA is a promising option for DR using LPPs as delivery agents.
The intracellular delivery of exogenous substances is an essential technical means in the field of biomedical research, including cell therapy and gene editing. Although many delivery technologies and strategies are present, each technique has its own limitations. The delivery cost is usually a major limiting factor for general laboratories. In addition, simplifying the operation process and shortening the delivery time are key challenges. Here, we develop a filter paper-syringe (FPS) delivery method, a new type of cell permeation approach based on filter paper. The cells in a syringe are forced to pass through the filter paper quickly. During this process, external pressure forces the cells to collide and squeeze with the fiber matrix of the filter paper, causing the cells to deform rapidly, thereby enhancing the permeability of the cell membrane and realizing the delivery of exogenous substances. Moreover, the large gap between the fiber networks of filter paper can prevent the cells from bearing high pressure, thus maintaining high cell vitality. Results showed that the slow-speed filter paper used can realize efficient intracellular delivery of various exogenous substances, especially small molecular substances (e.g., 3-5 kDa dextran and siRNA). Meanwhile, we found that the FPS method not only does not require a lengthy operating step compared with the widely used liposomal delivery of siRNA but also that the delivery efficiency is similar. In conclusion, the FPS approach is a simple, easy-to-operate, and fast (about 2 s) delivery method and may be an attractive alternative to membrane destruction-based transfection.
Cationic and ionizable cationic lipids are broadly applied as auxiliary agents, but their use is associated with adverse effects. If these excipients are rapidly degraded to endogenously occurring metabolites such as amino acids and fatty acids, their toxic potential can be minimized. So far, synthesized and evaluated biodegradable cationic and ionizable cationic lipids already showed promising results in terms of functionality and safety. Within this review, an overview about the different types of such biodegradable lipids, the available building blocks, their synthesis and cleavage by endogenous enzymes is provided. Moreover, the relationship between the structure of the lipids and their toxicity is described. Their application in drug delivery systems is critically discussed and placed in context with the lead compounds used in mRNA vaccines. Moreover, their use as preservatives is reviewed, guidance for their design is provided, and an outlook on future developments is given. Cationic and ionizable cationic lipids are widely used for pharmaceutical formulations. However, their toxicity is seemingly limiting their applicability. Formation of biodegradable cationic lipids overcomes adverse effects while maintaining functionality. The use of natural building blocks provides degradation into safe metabolites. Moreover, endogenous building blocks may improve their efficacy as complexing agents and preservatives.
Centrifugation is unsuitable for isolating liposomes from human plasma, and should not be the method of choice for researchers wanting to investigate the protein corona of liposomes for drug delivery.
This chapter will provide the drug delivery systems with nucleic acid–based nanocarrier. Nano‐DNA/RNA is an emerging field for self‐assembly of nanostructures with several advantages and potential therapeutic benefits. We will summarize state‐of‐the art strategies for drug delivery systems with DNA/RNA nanostructure‐based nanoarchitectures.
Joint diseases are one of the most common causes of morbidity and disability worldwide. The main diseases that affect joint cartilage are osteoarthritis and rheumatoid arthritis, which require chronic treatment focused on symptomatic relief. Conventional drugs administered through systemic or intra-articular routes have low accumulation and/or retention in articular cartilage, causing dose-limiting toxicities and reduced efficacy. Therefore, there is an urgent need to develop improved strategies for drug delivery, in particular, the use of micro- and nanotechnology-based methods. Encapsulation of therapeutic agents in delivery systems reduces drug efflux from the joint and protects against rapid cellular and enzymatic clearance following intra-articular injection. Consequently, the use of drug delivery systems decreases side effects and increases therapeutic efficacy due to enhanced drug retention in the intra-articular space. Additionally, the frequency of intra-articular administration is reduced, as delivery systems enable sustained drug release. This review summarizes various advanced drug delivery systems, such as nano- and microcarriers, developed for articular cartilage diseases.
Delivering functional substances across the cell membrane barriers has been a great challenge in biomedicine. Among various strategies to improve cellular uptake, DNA nanostructures, with different shapes and sizes, supply as suitable platforms for delivering a wide range of functional substances to cells. With great programmability, biocompatibility, and biostability, DNA nanostructure‐based delivery systems have shown and have succeeded in anticancer therapy, antibacterial treatment, gene editing, vaccine development, and stem cell bioengineering. Herein, the development and construction of various cellular ingestible DNA nanostructures are described, the loading strategies of functional substances are summarized, current biomedical applications are overviewed, and finally the remaining challenges and opportunities are discussed. DNA nanostructure‐based delivery systems have shown and have succeeded in anticancer therapy, antibacterial treatment, gene editing, vaccine development, and stem cell bioengineering. Herein, the development and construction of various cellular ingestible DNA nanostructures are described, the loading strategies of functional substances are summarized, current biomedical applications are overviewed, and finally the remaining challenges and opportunities are discussed.
By acting as effective biomimetics of the lipid bilayers, membrane-intercalating conjugated oligoelectrolytes (MICOEs) can spontaneously insert themselves into both synthetic lipid bilayers and biological membranes. The modular and intentional molecular design of MICOEs enable a range of applications, such as bioproduction, biocatalysis, biosensing, and therapeutics. This tutorial review provides a structural evolution of MICOEs, which originated from the broader class of conjugated molecules, and analyses the drivers behind this evolutionary process. Various representative applications of MICOEs, accompanied by insights into their molecular design principles, will be reviewed separately. Perspectives on the current challenges and opportunities in research on MICOEs will be discussed at the end of the review to highlight their potential as unconventional and value-added materials for biological systems.
Une exposition massive des populations aux actinides et au plutonium en particulier pourrait intervenir suite à l'inhalation, ou à l'entrée dans l'organisme, de petites particules dispersées par le vent après un accident (explosion dans l'industrie nucléaire, acte de guerre…). La rétention et le devenir des particules inhalées dépendent de leur taille et de leur forme physico-chimique. En règle générale, plus elles sont petites et moins solubles, plus elles sont dangereuses, car elles vont atteindre les alvéoles pulmonaires et seront principalement retenues pendant de longues périodes (décennies) dans les macrophages alvéolaires qui vont constituer de véritables sanctuaires vis-à-vis de ces radioéléments. Quant à la fraction soluble, capable de passer dans la circulation, ou en cas de blessures, le Pu(IV) (degré d'oxydation en milieu biologique) sera rapidement transféré et stocké dans le foie et les os où la dose délivrée pendant de longues périodes contribuera à divers effets délétères (cancers notamment). A l'heure actuelle, le seul traitement approuvé par la pharmacopée (en France notamment) et la FDA (Food and Drug Administration, aux Etats Unis) pour traiter une contamination interne par des actinides est l'administration, par voie intraveineuse (i.v), de sels de Diéthylène Triamine Pentaacétate (DTPA). L'efficacité de ce traitement reste surtout limitée aux formes solubles circulantes du Pu. De plus, la courte biodisponibilité du DTPA empêche cette thérapie par chélation d'exercer un effet réel sur les principaux compartiments de séquestration biologique (poumons, os, foie). Pour surmonter cette limitation et fournir une approche complémentaire à la thérapie de décorporation par le DTPA, nous développons des analogues polymériques du DTPA, basés sur un polyéthylèneimine (PEI) commercial, pour cibler indirectement ces sites de rétention. Ce travail de thèse fait suite à une thèse dans laquelle nous avons démontré que le PEI-MC (analogue polymérique structurel du DTPA) et le PEI-MP (analogue phosphonate) étaient capables, in vitro, de complexer le Th(IV), le Pu(IV) et l'U(VI) avec des efficacités comparables à celle du DTPA. Cette thèse constitue une première étude concernant les capacités de décontamination du PEI-MC et du PEI-MP vis-à-vis du Th(IV), utilisé comme analogue du Pu(IV), et incorporé sous forme soluble dans une matrice d'hydroxyapatite ou sous forme colloïdale (nanoparticules de Th) dans des macrophages afin de mimer au mieux des contaminations osseuses ou pulmonaires. La forte affinité du PEI-MP pour un des constituants majoritaires de la matrice osseuse, l'hydroxyapatite (HAp), son absence de cytotoxicité sur les cellules constitutives de l'os (ostéocytes et ostéoblastes), ainsi que des études thermodynamiques et cinétiques sur la décontamination de HAp, permettent d'envisager le développement du PEI-MP comme un candidat sérieux pour la décontamination du Pu(IV) séquestré au sein du tissus osseux. D'autre part, en combinant deux techniques (MET et ICP-MS) nous avons réalisé un suivi in cellulo des étapes de la phagocytose liées à la contamination de macrophages par des nanoparticules de thorium. Nous avons ensuite évalué l'efficacité de chélation de nos molécules vis-à-vis de ces formes insolubles à l'aide d'expériences in vitro (mise en contact avec les nanoparticules) et in cellulo (sur des macrophages contaminés). Ces résultats préliminaires mettent en évidence des paramètres importants liés à la contamination des macrophages par les actinides tels que la vitesse d'internalisation des nanoparticules de Th et l'effet des chélates en fonction de la dose et du temps. Globalement, cette thèse, a permis de mettre en place une méthodologie rigoureuse qui permet maintenant de tester ces polymères chélatants en conditions physiologiques sur leur(s) cible(s) biologique(s) réelle(s) : Os, poumons voire foie qui agissent tous les trois comme des sites de rétention vis-à-vis des actinides en cas de contamination.
Cardiovascular diseases are the leading cause of death in the world. This is partly due to the low regenerative capacity of adult hearts. mRNA therapy is a promising approach under development for cardiac diseases. In mRNA therapy, expression of the target protein is modulated by delivering synthetic mRNA. mRNA therapy benefits cardiac regeneration by increasing cardiomyocyte proliferation, reducing fibrosis, and promoting angiogenesis. Because mRNA is translated in the cytoplasm, the delivery efficiency of mRNA into the cytoplasm and nucleus significantly affects its therapeutic efficacy. To improve delivery efficiency, non-viral vehicles such as lipid nanoparticles have been developed. Non-viral vehicles can protect mRNA from enzymatic degradation and facilitate the cellular internalization of mRNA. In addition to non-viral vehicles, viral vectors have been designed to deliver mRNA templates into cardiac cells. This article reviews lipid nanoparticles, polymer nanoparticles, and viral vectors that have been utilized to deliver mRNA into the heart. Because of the growing interest in lipid nanoparticles, recent advances in lipid nanoparticles designed for cardiac mRNA delivery are discussed. Besides, potential targets of mRNA therapy for myocardial infarction are discussed. Gene therapies that have been investigated in patients with cardiac diseases are analyzed. Reviewing mRNA therapy from a clinically relevant perspective can reveal needs for future investigations.
Recently, therapeutics based on mRNA (mRNA) have attracted significant interest for vaccines, cancer immunotherapy, and gene editing. However, the lack of biocompatible vehicles capable of delivering mRNA to the target tissue and efficiently expressing the encoded proteins impedes the development of mRNA-based therapies for a variety of diseases. Herein, we report mRNA-loaded polymeric nanoparticles based on diethylenetriamine-substituted poly(aspartic acid) that induce protein expression in the lungs and muscles following intravenous and intramuscular injections, respectively. Animal studies revealed that the amount of polyethylene glycol (PEG) on the nanoparticle surface affects the translation of the delivered mRNA into the encoded protein in the target tissue. After systemic administration, only mRNA-loaded nanoparticles modified with PEG at a molar ratio of 1:1 (PEG/polymer) induce protein expression in the lungs. In contrast, protein expression was detected only following intramuscular injection of mRNA-loaded nanoparticles with a PEG/polymer ratio of 10:1. These findings suggest that the PEG density on the surface of poly(aspartic acid)-based nanoparticles should be optimized for different delivery routes depending on the purpose of the mRNA treatment.
Pyridinium amphiphiles have found practical use for the delivery of DNA into cells. Starting from 4-methylpyridine, a general synthesis has been devised for the production of pyridinium amphiphiles which allows variation in both the hydrophobic part and in the headgroup area of the compounds. By means of differential scanning microcalorimetry, zeta potential, particle size measurements and cryo electron microscopy, some characteristics of the pyridinium amphiphile/DNA complexes have been determined.
A DNA-transfection protocol has been developed that makes use of a synthetic cationic lipid, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA). Small unilamellar liposomes containing DOTMA interact spontaneously with DNA to form lipid-DNA complexes with 100% entrapment of the DNA, DOTMA facilitates fusion of the complex with the plasma membrane of tissue culture cells, resulting in both uptake and expression of the DNA. The technique is simple, highly reproducible, and effective for both transient and stable expression of transfected DNA. Depending upon the cell line, lipofection is from 5- to greater than 100-fold more effective than either the calcium phosphate or the DEAE-dextran transfection technique.
We present, here, evidence that foreign DNA can be specifically delivered to cells by a soluble carrier system that takes advantage of receptor-mediated endocytosis. Our experiments were based on the following concepts: hepatocytes possess a unique receptor that binds and internalizes galactose-terminal (asialo-)glycoproteins; DNA can bind to polycations in a strong but noncovalent manner forming soluble complexes; and the gene for chloramphenicol acetyltransferase, a bacterial enzyme that acetylates chloramphenicol, is not present in mammalian cells. We coupled asialoorosomucoid (ASOR) to poly-L-lysine to form an asialoorosomucoid-poly-L-lysine conjugate. The plasmid, pSV2 CAT, was complexed to the conjugate in a molar ratio of 1:2. To test this complex, a model system was used consisting of hepatoma cell lines, Hep G2, asialoglycoprotein receptor (+), and SK-Hep 1, receptor (-). Each cell line was incubated with filtered ASOR X poly-L-lysine X DNA complex, or controls consisting of DNA plus ASOR, DNA plus poly-L-lysine, or DNA alone. Cells were assayed for the presence of chloramphenicol acetyltransferase activity as a measure of gene transformation. SK-Hep 1, receptor (-) cells, produced no detectable acetylated chloramphenicol derivatives under any condition. However, Hep G2, receptor (+) cells, incubated with the ASOR X poly-L-lysine X DNA complex were transformed as indicated by the presence of chloramphenicol acetyltransferase activity (0.028 chloramphenicol acetyltransferase units/10(6) cells). Mixtures of individual components of the complex failed to transform these cells. Competition by a 10-fold excess of ASOR prevented gene transformation by the ASOR X poly-L-lysine X DNA complex.
Several polycations possessing substantial buffering capacity below physiological pH, such as lipopolyamines and polyamidoamine polymers, are efficient transfection agents per se--i.e., without the addition of cell targeting or membrane-disruption agents. This observation led us to test the cationic polymer polyethylenimine (PEI) for its gene-delivery potential. Indeed, every third atom of PEI is a protonable amino nitrogen atom, which makes the polymeric network an effective "proton sponge" at virtually any pH. Luciferase reporter gene transfer with this polycation into a variety of cell lines and primary cells gave results comparable to, or even better than, lipopolyamines. Cytotoxicity was low and seen only at concentrations well above those required for optimal transfection. Delivery of oligonucleotides into embryonic neurons was followed by using a fluorescent probe. Virtually all neurons showed nuclear labeling, with no toxic effects. The optimal PEI cation/anion balance for in vitro transfection is only slightly on the cationic side, which is advantageous for in vivo delivery. Indeed, intracerebral luciferase gene transfer into newborn mice gave results comparable (for a given amount of DNA) to the in vitro transfection of primary rat brain endothelial cells or chicken embryonic neurons. Together, these properties make PEI a promising vector for gene therapy and an outstanding core for the design of more sophisticated devices. Our hypothesis is that its efficiency relies on extensive lysosome buffering that protects DNA from nuclease degradation, and consequent lysosomal swelling and rupture that provide an escape mechanism for the PEI/DNA particles.
The application of cationic liposome reagents has advanced DNA and mRNA transfection research in vitro, and data are accumulating which show their utility for in vivo gene transfer. However, chemical structure-activity data leading to a better mechanistic understanding of their biological activity is still limited. Most of the cationic lipid reagents in use today for this application are formulated as liposomes containing two lipid species, a cationic amphiphile and a neutral phospholipid, typically dioleoylphosphatidylethanolamine (DOPE). The studies reported here examine the effects of some systematic chemical structural changes in both of these lipid components. Cationic and neutral phospholipids were formulated together as large multilamellar vesicles (MLV) or small sonicated unilamellar vesicles (SUV) in water, and each formulation was assayed quantitatively in 96-well microtiter plates under 64 different assay conditions using COS.7 cells and an RSV-beta-galactosidase expression plasmid. The cationic lipid molecules used for these studies were derived from a novel series of 2,3-dialkyloxypropyl quaternary ammonium compounds containing a hydroxyalkyl moiety on the quaternary amine. A homologous series of dioleylalkyl (C18:1) compounds containing increasing hydroxyalkyl chain lengths on the quaternary amine were synthesized, formulated with 50 mol % DOPE, and assayed for transfection activity. The order of efficacy was ethyl > propyl > butyl > pentyl > 2,3-dioleyloxypropyl-1-trimethyl ammonium bromide (DOTMA). DOTMA, which is commercially available under the trademark Lipofectin Reagent, lacks a hydroxyalkyl moiety on the quaternary amine. A homologous series of hydroxyethyl quaternary ammonium derivatives with different alkyl chain substitutions were synthesized, formulated with 50 mol % DOPE, and assayed in the transfection assay. The order of transfection efficacy was dimyristyl (di-C14:0) > dioleyl (di-C18:1) > dipalmityl (di-C16:0) > disteryl (di-C18:0). The addition of 100 microM chloroquine in the transfection experiment enhanced the activity of the dioleyl compound by 4-fold and decreased the activity of the dimyristyl compound by 70%. For each of the compounds and formulations examined in this report, large multilamellar vesicles (MLV; diameter 300-700 nm) were more active than small unilamellar vesicles (SUV; diameter 50-100 nm). The neutral phospholipid requirements for transfection activity in COS.7 cells with these cationic lipid molecules were examined.(ABSTRACT TRUNCATED AT 400 WORDS)
Novel, double-chained pyridinium compounds have been developed that display highly efficient DNA transfection properties. The transfection efficiency of several of these compounds is enhanced by an order of magnitude, when compared with the transfection efficiency accomplished with the widely used cationic lipid system, lipofectin. Most importantly, the pyridinium compounds were found to be essentially nontoxic toward cells. Using various reporter genes, such as beta-galactosidase and pNEO (a gene construct that renders cells resistent to antibiotic derivatives of neomycin like G418), we demonstrate that the enhanced efficiency relates to the fact that a relative higher number of cells in the population is transfected (approximately 50% in the case of COS cells) by the pyridinium derivatives, whereas the delivery of DNA per cell is also enhanced. Furthermore, application of the pyridinium derivatives shows little cellular preference in their ability to transfect cells. By systematically modifying the structure of the pyridinium amphiphile, i.e., by changing either the headgroup structure or the alkyl chains, some insight was obtained that may lead to unraveling the mechanism of amphiphile-mediated transfection, and thus to protocols that further optimize the carrier properties of the amphiphile. Our results reveal that unsaturated alkyl chains enhance the transfection properties of the pyridinium-based amphiphiles. Preliminary experiments suggest that the structure-dependent improvement of transfection efficiency, when comparing pyridinium derivatives with lipofectin, likely relates to the mechanism of delivery rather than the packaging of the amphiphile/DNA complex.
A previous study has shown an efficient, systemic transgene expression in mice via intravenous administration of a LPD formulation composed of DOTAP liposomes, protamine sulfate and plasmid DNA. In this study, factors affecting the in vivo performance of this formulation were further evaluated. A protocol in which liposomes were mixed with protamine before the addition of plasmid DNA was shown to produce small condensed particles with a diameter of about 135 nm. These particles were stable over time and gave a high level of gene expression in all tissues examined including lung, heart, spleen, liver and kidney with the highest level of expression in the lung. Inclusion of dioleoylphosphatidylethanolamine (DOPE) as a helper lipid significantly decreased the in vivo activity of LPD. In contrast, inclusion of cholesterol as a helper lipid increased the in vivo transfection efficiency of LPD and more importantly, decrease the amount of cationic lipid required for the maximal level of gene expression. Studies on the interaction between mouse serum and LPD showed that LPD became negatively charged after exposure to serum, and LPDs containing different helper lipids varied in the amount of associated serum proteins. LPD containing DOPE was more enriched in a protein corresponding to albumin in molecular weight. These results suggest that the mechanism of LPD-mediated intravenous gene delivery might be different from that of in vitro lipofection and that serum protein association might be a major factor limiting the in vivo transfection by LPD.
Since the first published examination of poly(ethylenimine) (PEI) as a gene delivery vehicle, there has been a flurry of research aimed at this polycation and its role in gene therapy. Here we will briefly review PEI chemistry and the characterization of PEI/DNA complexes used for gene delivery. Additionally, we will note various PEI transfection considerations and examine findings involving other polycationic gene delivery vehicles used with cellular targeting ligands. The current state of our knowledge regarding the mechanism of PEI/DNA transfection will also be discussed. Finally, we will survey toxicity issues related to PEI transfection.
We have developed a modified poly(ethylenimine) (PEI) transfection procedure that significantly increases PEI's transfection efficiency. While the basic transfection procedure had a transfection efficiency of 37%, our modified procedure yielded a 53% transfection efficiency. The altered procedure gives improved results because of two simultaneous actions: free polycations are removed from the transfecting solutions, and the composition of the PEI complexes that are administered to cells has been modified. The reduction in the amount of free polycations in transfecting solutions reduced the toxicity sometimes associated with the administration of polycations to cellular environments. The structural modification of PEI/DNA transfecting complexes involves improved PEI packing around the delivered plasmid to yield a greater buffering capacity without a change in the complex's surface charge concentration. These structural properties were confirmed by titration and zeta potential analyses. Whether the modified PEI/DNA complexes are more effective because of increased cellular uptake or an enhanced ability to escape from endolysosomes has been addressed. The increase in transfection efficiency was obtained when the buffering capacity of the PEI/DNA was increased without a change in surface charge concentration, which implies that it is the property of enhanced lysosomal buffering that is responsible for successful PEI transfection.
Transgene expression and tumor regression after direct injection of plasmid DNA encoding cytokine genes, such as mIL-12 and mIFN-gamma, remain very low. The objective of this study is to develop nontoxic biodegradable polymer-based cytokine gene delivery systems, which should enhance mIL-12 expression, increasing the likelihood of complete tumor elimination. We synthesized poly[alpha-(4-aminobutyl)-l-glycolic acid] (PAGA), a biodegradable nontoxic polymer, by melting condensation. Plasmids used in this study encoded luciferase (pLuc) and murine interleukin-12 (pmIL-12) genes. PAGA/plasmid complexes were prepared at different (+/-) charge ratios and characterized in terms of particle size, zeta potential, osmolality, surface morphology, and cytotoxicity. Polyplexes prepared by complexing PAGA with pmIL-12 as well as pLuc were used for transfection into cultured CT-26 colon adenocarcinoma cells as well as into CT-26 tumor-bearing BALB/c mice. The in vitro and in vivo transfection efficiency was determined by luciferase assay (for pLuc), enzyme-linked immunosorbent assay (for mIL-12, p70, and p40), and reverse transcriptase-polymerase chain reaction (RT-PCR) (for Luc and mIL-12 p35). PAGA condensed and protected plasmids from nuclease degradation. The mean particle size and zeta potential of the polyplexes prepared in 5% (w/v) glucose at 3:1 (+/-) charge ratio were approximately 100 nm and 20 mV, respectively. The surface characterization of polyplexes as determined by atomic force microscopy showed complete condensation of DNA with an ellipsoidal structure in Z direction. The levels of mIL-12 p40, mIL-12 p70, and mIFN-gamma were significantly higher for PAGA/pmIL-12 complexes compared to that of naked pmIL-12. This is in good agreement with RT-PCR data, which showed significant levels of mIL-12 p35 expression. The PAGA/pmIL-12 complexes did not induce any cytotoxicity in CT-26 cells as evidenced by 3-¿4, 5-dimethylthiazol-2-yl¿-2,5-diphenyltetrazolium bromide assay and showed enhanced antitumor activity in vivo compared to naked pmIL-12. PAGA/pmIL-12 complexes are nontoxic and significantly enhance mIL-12 expression at mRNA and protein levels both in vitro and in vivo.
Pyridinium amphiphiles have found practical application for the delivery of DNA into eukaryotic cells. A general synthetic method starting from (iso)nicotinoyl chloride has been devised for the preparation of pyridinium amphiphiles based on (bio)degradable esters, allowing structural variation both in the hydrophobic part and in the headgroup area. By means of differential scanning calorimetry, transmission electron microscopy and UV measurements, some characteristics, including hydrolytic behaviour, have been determined. In vitro transfection ability and toxicity have been determined using the eukaryotic COS-7 cell line.
Bifunctional cationic compound carrying trivalent galactosides as the cell targeting ligand and DAB-dendr-(NH2)8 (generation 2.0) as the DNA binding domain was synthesized for gene delivery to hepatocytes. DAB-dendr-(NH2)4 (generation 1.0) conjugated with a hydrocarbon chain was used as a scaffold for the attachment of three galactosides, while the other hydrocarbon end was linked with DAB-dendr-(NH2)8 (generation 2.0) through a carbamate bond. This design provided an effective entry for the synthesis of a polyamine compound having the cell targeting galactosyl ligand. Preliminary in vitro transfection results demonstrated that the bifunctional cationic compound could effectively deliver the gene into HepG2 cells.
To achieve effective plasmid-based gene therapy, the control of cellular access and uptake, intracellular trafficking and nuclear retention of plasmids must be achieved. Inefficient endosomal release, cytoplasmic transport and nuclear entry of plasmids are amongst some of the key limiting factors in the use of plasmids for effective gene therapy. A number of non-viral gene delivery systems have been designed to overcome these limiting factors. The most common approach to protect and control plasmid distribution is to complex plasmids with cationic lipids or polymers through electrostatic interactions. Endosomal release of plasmids can be achieved, for instance, by using pH-sensitive lipids, inactivated viral particles, endosomolytic peptides and polymers. Among the least explored gene delivery systems are those that consist mainly of synthetic, short peptides. Peptides can be incorporated into multicomponent gene delivery complexes for specific purposes, such as for DNA condensation, cell-specific targeting, endosomolysis or nuclear transport. The aims of this review are to: (i) explore the conceptual and experimental aspects of peptide-DNA interactions; (ii) critically assess the possible use of peptides for efficient gene transfer; and (iii) present an overview on the use of peptides to enhance the effectiveness of other gene delivery systems. On balance, peptide-based gene delivery systems appear to have a significant potential as commercially viable gene delivery products.
Cationic polysaccharides based on spermine−dextran conjugates were synthesized and tested as vectors for gene transfection. Dextrans of 10−380 kDa were oxidized under mild conditions by potassium periodate to obtain the respective polyaldehydes in 90% overall yield. The oxidized dextrans were reacted by reductive amination with increasing amounts of spermine, and the efficacy of conjugation between the oligoamine and polysaccharides was studied as a function of spermine/aldehyde mole ratio, pH, and temperature of medium. The optimal conjugation yields were obtained at 1.25 mole ratio (spermine/aldehyde groups) and pH 11 at room temperature. Under these conditions, 2 μmol/mg (spermine/polysaccharide) conjugation was achieved with 25−30% of the spermine moieties were conjugated in both sides to form branched polymers. The water-soluble polymers obtained were interacted with pCMV-GFP plasmid to form nanoparticles that were introduced to HEK293 and NIH3T3 cells in vitro for transfection efficacy assessment. Out of about 50 different polymer structures, only spermine−dextran of 6000−8000 Da, spermine content of 2 μmol/mg, and degree of branching of 25−30% was active in transfecting about 50% of the cells while all other polymers were significantly less active.
Cationic lipids are a promising alternative to viral vectors for gene therapy, allowing the delivery of larger plasmids without immunogenicity, despite their lower transfection efficiency. Among them, heterocyclic systems with imidazolium or pyridinium polar head groups have definite advantages such as the excellent transfection profiles and low cytotoxicity. Our approach for synthesizing heterocyclic cationic lipids differs from those previously described because we synthesize a pyridinium ring from simple starting materials. First a pyrylium salt is formed via diacylation of alkenes. The pyrylium salt is then converted by primary amines into pyridinium salts. Appropriate choice of the primary amine allows the attachment of two hydrophobic chains yielding compounds 21A and 25A (with various chain lengths derived from palmitic, stearic and oleic acids). The same strategy allowed the preparation of lipophilic derivatives 21B, 25B useful as strongly fluorescent markers for the study of the properties of biological membranes. Preliminary tests with some of the compounds 21A and 25A, on several cell lines, showed comparable transfection efficiencies and lower cytotoxicity than those obtained with standard commercial transfection agents. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2003)
Several novel cationic amphiphiles, based on a hydrophibic cholesteryl or dioleoylglyceryl moiety, have been prepared whose hydrophobic and cationic portions are linked by ester bonds to facilitate degradation in animal cells. Dispersons combining such cationic species with phosphatidylethanolamine (PE), certain structural analogues of PE or diacylglycerol can mediate efficient transfer of both nonexchangeable lipid probes and the DNA plasmid pSV2cat into cultured mammalian (CV-2 and 3T3) cells. The abilities of different types of cationic lipid dispersions to mediate transfection of mammalian cells with pSV2cat could not be directly correlated with their abilities to coalesce with other membranes, as assessed by their ability to intermix lipids efficiently with large unilamellar phospatidylcholine/phospatidylserine vesicles in the presence or absence of DNA. The cytotoxicities toward CV-1 cells of dispersions combining PE with most of the degradable cationic amphiphiles studied here compare favorably with those reported previously for similar dispersions containing other types of cationic amphiphiles. Fluorescent analogues of two of the diacylglycerol-based cationic amphiphiles examined in this study are shown to be readily degraded after incorporation into CV-1 cells from PE/cationic lipid dispersions.
The preparation of cationic amphiphiles that induce minor cytotoxic response during polynucleotide delivery into mammalian cells has been limited by the conventional use of ester, amide, or carbamate linkages to tether either the polar or the hydrophobic domains. The deleterious effects of ammonium-based lipidic salts on cellular processes have been well-established. The present report is the first example of a linchpin tetraester construct that utilizes ester linkages to tether both the polar and hydrophobic domains. Dimyristoyl and dioleoyl analogues were prepared from pentaerythritol, N,N-dimethylglycine, and their corresponding fatty acyl groups via successive diesterifications followed by amine quaternization. The resultant cationic tetraesters were examined in transfection (luciferase) and cell proliferation (MTS) assays using NIH 3T3 and 16HBE14o- cells. The tetraesters exhibited transfection activity comparable to the well-studied lipids DOTAP and DC-cholesterol (DC-chol) in both cell lines. The tetraester construct afforded no cytotoxicity in NIH3T3 cells and provided a significant lowering of cytotoxicity relative to DC-chol in the 16HBE14o- cells. The expression of green fluorescent protein (GFP) in both cell lines also was examined using the lipid panel. Comparison of fluorescent and corresponding phase-contrast images confirmed the chemical cytotoxicity results and revealed that the cytotoxic response was not dependent on transgene expression. Phase-contrast micrographs of cells treated with the cationic lipid panel in the absence of GFP plasmid showed identical morphology to the GFP-transfected cells, suggesting that the onset of a lipid-mediated cytotoxic response might occur at a stage prior to endosomal encapsulation.
Hox genes, which play key roles in the development of body plans, have
been described from a variety of metazoans. Here we report the presence
of Hox class genes that are typical of triploblasts in Myxozoa, formerly
a protozoan taxon. This finding confirms Myxozoa's phylogenetic affinity
with the Bilateria and reveals an extreme example of parasitic
degeneracy.
The aim of the present study was to compare different regularly used transfection agents, i.e.poly(L-lysine) (PLL), branched poly(ethyleneimine) (BPEI), linear PEI (LPEI), poly(2-(dimethylamino)ethyl methacrylate)(pDMAEMA) and 1,2-dioleyl-3-trimethylammonium-propane (DOTAP), regarding their in vivo transfection behavior after intravenous administration. An attempt was made to study whether the in vitro behavior of these transfectants is relevant for the in vivo situation in terms of their transfection efficiency and serum aggregation properties. In vivo, at an N/P ratio of 5, transfection mediated by the various cationic transfectants mainly occurred in the lungs. The order of decreasing lung transfection was: DOTAP > LPEI > pDMAEMA > BPEI > PLL. Similar rankings were found in in vitro experiments regarding the extent of serum-induced aggregation, suggesting that in vitro studies are certainly relevant for the in vivo situation. As a whole, the in vitro data suggest that the induction of aggregates in the circulation is a major mechanistic factor underlying the phenomenon of dominant lung transfection.
A large number of PKC inhibitors are positively charged. We evaluated the structural features of cationic amphiphiles which are necessary for inhibiting PKC. Many of these compounds were derivatives of cholesterol, which possesses a hydrophobic backbone which does not perturb hydrocarbon packing in membrane bilayers. In addition, they contain a tertiary or quaternary nitrogen functionality in the head group. All designed cholesterol-based amphiphiles inhibit PKC activity; the potency of the amphiphile correlates with the presence of positive charge. Quaternary ammonium amphiphiles are 10-fold more potent than their tertiary amine counterparts, generally inhibiting in the 10-60 microM range using the Triton mixed micelle assay. Aside from charge, factors such as the structure of the amine-containing head group, its length from the hydrocarbon moiety, or the number of amine groups on the amphiphile did not markedly influence inhibitor potency. In contrast, the hydrocarbon backbone did influence potency: cationic amphiphiles containing a steroid backbone were more potent inhibitors of PKC than their straight-chain analogues. Changing the nature of the hydrocarbon from a sterol to an alkyl group lowers the pK of the amine head group so that the straight-chain analogues are no longer cationic in the conditions in the PKC assay. The results of these studies suggest that a combination of positive charge and a bilayer-stabilizing structural characteristic provides a basis for the rational design of PKC inhibitors.
Four different cationic derivatives of cholesterol were synthesized which contain either a tertiary or a quaternary amino head group, with and without a succinyl spacer-arm. Their ability to inhibit protein kinase C (PKC) activity was measured in a detergent mixed micellar solution. Derivatives containing a quaternary amino head group were effective inhibitors (Ki approx. 12 and 59 microM) of PKC and derivatives containing a tertiary amino head group were approx. 4-20-fold less inhibitory. Liposomes containing an equimolar mixture of dioleoylphosphatidylethanolamine (DOPE) and a cationic cholesterol derivative were tested for the DNA-mediated transfection activity in mouse L929 cells. Highest activity was found with the derivative with low PKC inhibitory activity and with a succinyl spacer-arm. The transfection activity of this tertiary amine derivative, N,N-dimethylethylenediaminyl succinyl cholesterol was dependent on DOPE as a helper lipid; liposomes containing dioleoylphosphatidylcholine and this derivative had little activity. The transfection protocol of this new cationic liposome reagent was optimized with respect to the ratio of liposome/DNA, dose of the complex and time of incubation with cells. Several adherent cell lines could be efficiently transfected with this liposome reagent without any apparent cytotoxicity. However, the transfection activity was strongly inhibited by the presence of serum components.
Liposomes with positively-charged lipid components have previously demonstrated efficacy in animal models for human diseases, and are currently being evaluated in human clinical studies. Cationic lipids can improve entrapment efficiency of drugs and other substances which are negatively charged, and facilitate penetration of biological membranes in vitro, e.g. in transfection. However, toxic effects have also been reported for positively-charged liposomes containing stearylamine. In this report we have examined gross interactions between plasma components or erythrocytes with cholesterol-rich SUV composed of PC or DPPC and having 0-50 mol% of phospholipid replaced with positively-charged stearylamine, DOTMA, or BisHOP. Plasma interactions observed included increased turbidity of the usually clear stroma and/or formation of a clot-like mass. At plasma concentrations of 0.25 mumol/ml or more, the extent of plasma interactions depended upon the concentration of positive charge, the charge density of cationic lipid initially present in the liposomes, and to a lesser degree, the nature of the lipid providing the positive charge. At liposomal positive charge concentrations of greater than 0.5 mumol/ml plasma, stearylamine provoked a strong increase in plasma turbidity, whereas liposomes incorporating DOTMA or BisHOP provoked a strong clotting response. Some hemolysis of erythrocytes in vitro occurred on interaction with cationic liposomes where positive charge was contributed by DOTMA or stearylamine, but not BisHOP. Implications for the clinical use of liposomes containing cationic lipids, is discussed.
Sonicated liposomes composed of dioleoylphosphatidylethanolamine (DOPE) and a quaternary ammonium detergent (dodecyl-, tetradecyl-, or cetyl-trimethylammonium bromide) mediates functional transfer of pSV2 CAT plasmid DNA to mouse L929 fibroblasts. Successful transfection was determined by assaying for chloramphenicol acetyltransferase activity in cell lysates collected 40 h after exposure to the lipid-DNA complexes. Liposomes prepared with the quaternary ammonium detergents were less toxic than the free detergents at the same concentrations and were more efficient in their delivery of the plasmid DNA to the cells. Analysis of the three detergents in combination with the lipid showed that cetyltrimethylammonium bromide was least toxic to the cells. This detergent, at a minimal concentration of 20 mol% in DOPE, allowed for stable liposome preparations and efficient transfection. Optimal efficiency of transfection occurred with 30 micrograms of DNA. Further increases in the DNA concentration caused a decrease in the transfection efficiency, perhaps due to charge repulsions between the liposomes now saturated with negatively charged DNA and the negatively charged cell surface. The transfection activity of the liposome was limited by its cytotoxicity at high liposome concentrations. These results are compared with that of the Lipofectin, another positively charged liposome preparation which is commercially available. Although the overall transfection activity of the liposome containing the quaternary ammonium detergent is somewhat lower than that of the Lipofectin, it may serve as an inexpensive and convenient alternative.
We have developed three catioinic amphiphiles based on the structure 1-[2-(acyloxy)ethyl]-2-alkyl(alkenyl)-3-(2-hydroxyethyl)imidazolinium chloride. Although these three compounds differ only in the structure of the hydrophobic acyl chains, they differ greatly in their ability to mediate in vivo and in vitro gene delivery. Moreover, in vitro efficiency is not predictive of in vivo efficiency. The myristoyl form is the most effective compound in vitro, and the oleoyl form is the most effective compound in vivo. The compounds readily form suspensions in aqueous media, both in the pure form and as mixtures with either cholesterol or dioleoylphosphatidylethanolamine. These suspensions can be sonicated to produce smaller particles. Particle size, electron microscopy, and the ability to capture glucose suggest that these lipids form liposomes on suspension in aqueous media. When mixed with plasmid DNA, the lipid particles appear to fuse and form larger particles. Fusion is maximal at the critical DNA:lipid ratio where extensive aggregation and precipitation are observed. Therefore, these compounds behave similarly to other cationic liposome-forming lipids upon interaction with DNA.
A nasal solution formulation of the cationic material chitosan was shown to greatly enhance the absorption of insulin across the nasal mucosa of rat and sheep. The absorption promoting effect was concentration dependent with the optimal efficacy obtained for concentrations higher than 0.2% and 0.5% in rats and sheep, respectively. The absorption promoting effect was reversible with time in a "pulse-chase" study. Histological examination of the nasal mucosa of rats exposed to a chitosan solution for 60 minutes showed little change.
The present study compares different cytotoxicity and cell proliferation assays including cell morphology, mitochondrial activity, DNA synthesis, and cell viability and toxicity assays. CaSki cells were exposed to two cationic liposomal preparations containing dimethyldioctadecyl-ammonium bromide (DDAB), dioleoylphosphatidylethanolamine (DOPE) and a commercial transfection-reagent DOTAP (N[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium-methylsulfat e). The results provided by these assays were similar. However, the lactate dehydrogenase assay was more sensitive in measuring early damages of cell membranes than the Trypan blue assay. Also, cell morphology showed early toxic changes, such as cytoplasmic vacuolization and cell shrinking, and it should be included with such toxicity evaluations. DDAB:DOPE was more toxic than DOTAP. The cells treated with DOTAP at 10 microM were surviving as well as the control cells, while DOTAP at 40 uM and DDAB:DOPE at 10 microM had slight toxic effects on CaSki cells. The most toxic effects were seen in CaSki cells after treatment with DDAB:DOPE at 40 microM.
All types (cationic, anionic and non-ionic) amphiphilic detergents significantly inhibited the production of both IgG and IgM by human peripheral blood mononuclear cells after polyclonal activation in vitro. The most potent inhibitors were didodecyldimethylammonium bromide (DDAB) and (1-methyldodecyl)dimethylamine N-oxide (2-ATDNO). They were able to suppress effectively the immunoglobulin production in 10(-3)-10(-8) M concentrations. A medium inhibitory effect was observed with Slovapon, sodium dodecyl sulphate (SDS) and Triton X-100, while Slovanik showed an inhibition only in concentrations higher than 10(-2)%. These results suggest that amphiphilic detergents may be characterized as potential immunotoxic substances with very negative effects on the immunoglobulin production.
Complexes formed between cationic liposomes and nucleic acids represent a highly efficient vehicle for delivery of DNA and RNA molecules into a large variety of eukaryotic cells. By using fluorescence, gel electrophoresis, and metal-shadowing electron microscopy techniques, the factors that affect the, yet unclear, interactions between DNA and cationic liposomes as well as the structural features of the resulting complexes have been elucidated. A model is suggested according to which cationic liposomes bind initially to DNA molecules to form clusters of aggregated vesicles along the nucleic acids. At a critical liposome density, two processes occur, namely, DNA-induced membrane fusion, indicated by lipid mixing studies, and liposome-induced DNA collapse, pointed out by the marked cooperativity of the encapsulation processes, by their modulations by DNA-condensing agents, and also by their conspicuous independence upon DNA length. The DNA collapse leads to the formation of condensed structures which can be completely encapsulated within the fused lipid bilayers in a fast, highly cooperative process since their exposed surface is substantially smaller than that of extended DNA molecules. The formation of the transfecting DNA-liposome complexes in which the nucleic acids are fully encapsulated within a positively-charged lipid bilayer is proposed, consequently, to be dominated by mutual effects exerted by the DNA and the cationic liposomes, leading to interrelated lipid fusion and DNA collapse.
Cationic liposomes mediate efficient transfection of mammalian cells, but the manner in which cells internalize and process cationic liposome-DNA complexes has not been well characterized. We exposed several cell types, including human and murine erythroleukemia cells. African green monkey kidney cells (CV-1), isolated rat alveolar type II cells and alveolar macrophages to DNA-cationic liposome complexes containing N-(1-2,3-dioleyloxypropyl)-N,N,N-triethylammonium (DOTMA) and Dioleylphosphatidylethanolamine (DOPE). The morphology of liposome-cell interactions was assessed by electron microscopy. Liposome preparations were complexed to colloidal gold particles or to both plasmid DNA and gold particles. Cells treated with DOTMA liposome-DNA complexes demonstrated endocytosis of the liposome-DNA complexes in coated pits, which were seen in early endosomes, late endosomes, and lysosomes. In isolated alveolar type II cells, the gold-labelled DOTMA lipid apparently mixed with the contents of lamellar bodies. In most cells, gold particles were dispersed throughout the cytoplasmic matrix. In a small proportion of CV-1 and U937 cells, a membrane system resembling the endoplasmic reticulum developed within the nucleus. This novel structure was also present in nuclei after they were isolated from CV-1 cells and then mixed with DOTMA-containing liposomes. Membranes which form after exposure to DOTMA-containing liposomes were 10 nm in thickness as compared to the approx. 8 nm thickness of endogenous cellular membranes. Based on these morphologic observations, we propose that the main route of entry of cationic liposomes into cells is by endocytosis. In some instances, the endosomal compartment releases its cationic liposome-DNA contents into the cytoplasmic matrix. Occasionally, liposomes may enter the nucleus by fusion with the nuclear envelope, creating vesicular and reticular intranuclear membranes. It is not clear at present which, if any of these morphological observations correlates with transfection mediated by cationic liposomes.
Prostate tumor cell lines have been shown to both produce interleukin-6 (IL-6) and express the IL-6 receptor, suggesting a potential autocrine growth regulatory role for IL-6. We explored the role of IL-6 in the proliferation of the human prostatic carcinoma cell line, DU145, using ribozymes to inhibit IL-6 expression. Hammerhead-type ribozymes targeted against IL-6 mRNA sequences were prepared, and in vitro analyses were used to demonstrate that these molecules catalyzed the cleavage of IL-6 mRNA poly- nucleotide fragments. To test in situ activity, these ribozymes were transfected into DU145 cells using cationic transfection lipids, cytofectins. Treatment of cultured cells with ribozyme/cationic lipid complexes resulted in a reduction of IL-6 protein levels in the supernatant and reduced numbers of DU145 cells 48 h after treatment. However, similar results were also seen following treatment with control RNA/lipid complexes. This reduction in IL-6 levels and cell numbers was a function of the RNA/lipid complexes and was not seen with either lipid or RNA alone. Therefore, the reductions in IL-6 levels and cell numbers observed were not due to ribozyme-mediated cleavage of IL-6 mRNA, but rather reflected a dose-dependent, nonspecific toxic effect of the treatment with ribozyme/cytofectin complexes. This effect can resemble functional ribozyme activity, complicating analysis of the activity of synthetic ribozymes after transfection into cultured cells.
Stable complexes of cationic liposomes with plasmid DNA were prepared by (1) including a small amount of poly(ethylene glycol)-phospholipid conjugate or (2) condensing the DNA with polyamines prior to the formation of liposome-plasmid complexes. These preparations were stable for months at 4 degrees C and gave reproducible high transfection activity for in vivo gene delivery after intravenous injection in mice. Under these conditions, the expression of marker gene (luciferase) was primarily in the lungs (reaching values up to 3 ng expression per mg tissue protein), but also in other tissues to a lesser extent. Non-stabilized formulations lost all their transfection activity in 4 days. In these formulations cholesterol, not dioleoylphosphatidylethanolamine, was the helper lipid effective for sustaining high transfection activity in vivo. These new developments in formulation technology should enhance the potential for liposome-mediated gene therapy.
To increase cationic liposome-mediated intravenous DNA delivery extruded DOTAP:cholesterol liposomes were used to form complexes with DNA, resulting in enhanced expression of the chloramphenicol acetyltransferase gene in most tissues examined. The DNA:liposome ratio, and mild sonication, heating, and extrusion steps used for liposome preparation were crucial for improved systemic delivery. Size fractionation studies showed that maximal gene expression was produced by a homogeneous population of DNA:liposome complexes between 200 to 450 nm in size. Cryo-electron microscopy examination demonstrates that the DNA:liposome complexes have a novel morphology, and that the DNA is condensed on the interior of invaginated liposomes between two lipid bilayers. This structure could account for the high efficiency of gene delivery in vivo and for the broad tissue distribution of the DNA:liposome complexes. Ligands can be placed on the outside of this structure to provide for targeted gene delivery.