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Novel amphiphilic block-copolymer forming stable micelles and interpolyelectrolyte complexes with DNA for efficient gene delivery
Abstract
Novel amphiphilic poly(DMAEMA)-block-poly(NVP-co-BA-co-AEM) (BP83-1) forms stable micelles and BP83-1/pDNA complexes possessing controlled size, charge and enhanced aggregation degree. It was found that the formation of the micelles by BP83-1 is necessary for successful DNA binding and compaction. The polyamphiphile micelle aggregation degree defined their crucial effect on the compaction and morphology of polyplexes. Strong compaction of the DNA upon interaction with polymer at CMC value, positive charge, and high stability of the polyplex are key factors promoting the penetration of DNA through bio-surfaces that define the efficiency of gene delivery in mammalian cells.
(2-dimethyl amino)ethyl methacrylate (DMA) was co-polymerized with (2-dibutyl amino)ethyl methacrylate (DBA) to form random copolymer P(DMA-co-DBA) with DMA/DBA molar ratios in the range of 9:1 to 1:9 by Atom Transfer Radical Polymerization (ATRP). The (co)polymers were characterized with techniques such as GPC, ¹HNMR and acid-base titration. The cytotoxicity of the polymers was assessed by MTT. Size and zeta potential of P(DMA-co-DBA)/DNA polyplex were analyzed by dynamic light scattering. In vitro gene transfection was performed for P(DMA-co-DBA)/DNA polyplex in the medium with and without serum. In comparison with PDMA/DNA and PEI/DNA, P(DMA-co-DBA) 8:2/DNA showed much higher transfection efficiency than those of PDMA/DNA and PEI/DNA within the range of 0.125 − 1.25 μg·mL⁻¹ of DNA concentrations in the medium with 10% FBS. Confocal images showed that P(DMA-co-DBA) 8:2/DNA was superior to PDMA/DNA and PEI/DNA in the speed and capacity for endosomal release, which might be the main cause of its higher transfection efficiency.
The discovery of the genetic roots of various human diseases has motivated the exploration of different exogenous nucleic acids as therapeutic agents to treat these genetic disorders (inherited or acquired). However, the physicochemical properties of nucleic acids render them liable to degradation and also restrict their cellular entrance and gene translation/inhibition at the correct cellular location. Therefore, gene condensation/protection and guided intracellular trafficking are necessary for exogenous nucleic acids to function inside cells. Diversified cationic formulation materials, including natural and synthetic lipids, polymers, and proteins/peptides, have been developed to facilitate the intracellular transportation of exogenous nucleic acids. The chemical properties of different formulation materials determine their special features for nucleic acid delivery, so understanding the property–function correlation of the formulation materials will inspire the development of next-generation gene delivery carriers. Therefore, in this review, we focus on the chemical properties of different types of formulation materials and discuss how these formulation materials function as protectors and cellular pathfinders for nucleic acids, bringing them to their destination by overcoming different cellular barriers.
The field of polymeric nanoparticles is quickly expanding and playing a pivotal role in a wide spectrum of areas ranging from electronics, photonics, conducting materials, and sensors to medicine, pollution control, and environmental technology. Among the applications of polymers in medicine, gene therapy has emerged as one of the most advanced, with the capability to tackle disorders from the modern era. However, there are several barriers associated with the delivery of genes in the living system that need to be mitigated by polymer engineering. One of the most crucial challenges is the effectiveness of the delivery vehicle or vector. In last few decades, non-viral delivery systems have gained attention because of their low toxicity, potential for targeted delivery, long-term stability, lack of immunogenicity, and relatively low production cost. In 1987, Felgner et al. used the cationic lipid based non-viral gene delivery system for the very first time. This breakthrough opened the opportunity for other non-viral vectors, such as polymers. Cationic polymers have emerged as promising candidates for non-viral gene delivery systems because of their facile synthesis and flexible properties. These polymers can be conjugated with genetic material via electrostatic attraction at physiological pH, thereby facilitating gene delivery. Many factors influence the gene transfection efficiency of cationic polymers, including their structure, molecular weight, and surface charge. Outstanding representatives of polymers that have emerged over the last decade to be used in gene therapy are synthetic polymers such as poly(l-lysine), poly(l-ornithine), linear and branched polyethyleneimine, diethylaminoethyl-dextran, poly(amidoamine) dendrimers, and poly(dimethylaminoethyl methacrylate). Natural polymers, such as chitosan, dextran, gelatin, pullulan, and synthetic analogs, with sophisticated features like guanidinylated bio-reducible polymers were also explored. This review outlines the introduction of polymers in medicine, discusses the methods of polymer synthesis, addressing top down and bottom up techniques. Evaluation of functionalization strategies for therapeutic and formulation stability are also highlighted. The overview of the properties, challenges, and functionalization approaches and, finally, the applications of the polymeric delivery systems in gene therapy marks this review as a unique one-stop summary of developments in this field.
Background
Gene delivery systems are essentially necessary for the gene therapy of human genetic diseases. Gene therapy is the unique way that is able to use the adjustable gene to cure any disease. The gene therapy is one of promising therapies for a number of diseases such as inherited disorders, viral infection and cancers. The useful results of gene delivery systems depend open the adjustable targeting gene delivery systems. Some of successful gene delivery systems have recently reported for the practical application of gene therapy.
Main body
The recent developments of viral gene delivery systems and non-viral gene delivery systems for gene therapy have briefly reviewed. The viral gene delivery systems have discussed for the viral vectors based on DNA, RNA and oncolytic viral vectors. The non-viral gene delivery systems have also treated for the physicochemical approaches such as physical methods and chemical methods. Several kinds of successful gene delivery systems have briefly discussed on the bases of the gene delivery systems such as cationic polymers, poly(L-lysine), polysaccharides, and poly(ethylenimine)s.
Conclusion
The goal of the research for gene delivery system is to develop the clinically relevant vectors such as viral and non-viral vectors that use to combat elusive diseases such as AIDS, cancer, Alzheimer, etc. Next step research will focus on advancing DNA and RNA molecular technologies to become the standard treatment options in the clinical area of biomedical application.
Cell-penetrating-peptides (CPPs) are small amino-acid sequences characterized by their ability to cross cellular membranes. They can transport various bioactive cargos inside cells including nucleic acids, large proteins, and other chemical compounds. Since 1988, natural and synthetic CPPs have been developed for applications ranging from fundamental to applied biology (cell imaging, gene editing, therapeutics delivery). In recent years, a great number of studies reported the potential of CPPs as carriers for the treatment of various diseases. Apart from a good efficacy due to a rapid and potent delivery, a crucial advantage of CPP-based therapies is the peptides low toxicity compared to most drug carriers. On the other hand, they are quite unstable and lack specificity. Higher specificity can be obtained using a cell-specific CPP to transport the therapeutic agent or using a non-specific CPP to transport a cargo with a targeted activity. CPP-cargo complexes can also be conjugated to another moiety that brings cell- or tissue-specificity. Studies based on all these approaches are showing promising results. Here, we focus on recent advances in the potential usage of CPPs in the context of cancer therapy, with a particular interest in CPP-mediated delivery of anti-tumoral proteins.
With its nearly unrestricted possibilities, gene therapy attracts more and more significance in modern-day research. The only issue still seeming to hold back its clinical success is the actual effective delivery of genetic material. Nucleic acids are in general challenging to administer to their intracellular targets because of their unfavorable pharmaceutical characteristics. Polymeric nanogels present a promising delivery platform for oligonucleotide-based therapies, as the growing number of reports deliberated in this review represents. Within the scope of this article, recent progress in the use of nanogels as gene delivery vectors is summarized and different examples of modified, stimuli-responsive, targeted, and codelivering nanogels are discussed in detail. Furthermore, major aspects of successful gene delivery are addressed and critically debated in regard to nanogels, giving insights into what progress has been made and which key issues still need to be further approached.
Novel, nontoxic star copolymers of N,N-dimethylaminoethyl methacrylate (DMAEMA) and hydroxyl-bearing oligo(ethylene glycol) methacrylate (OEGMA-OH) were synthesized via atom transfer radical polymerization (ATRP) using hyperbranched poly(arylene oxindole) as the macroinitiator. Stars with molar masses from 100,000 g/mol to 257,000 g/mol and with various amounts of OEGMA-OH in the arms were prepared. As these polymers can find applications, e.g., as carriers of nucleic acids, drugs or antibacterial or antifouling agents, in this work, much attention has been devoted to exploring their solution behavior and their stimuli-responsive properties. The behavior of the stars was studied in aqueous solutions under various pH and temperature conditions, as well as in PBS buffer, in Dulbecco’s modified Eagle’s medium (DMEM) and in organic solvents for comparison. The results indicated that increasing the content of hydrophilic OEGMA-OH units in the arms up to 10 mol% increased the cloud point temperature. For the stars with an OEGMA-OH content of 10 mol%, the thermo- and pH-responsivity was switched off. Since cytotoxicity experiments have shown that the obtained stars are less toxic than homopolymer DMAEMA stars, the presented studies confirmed that the prepared polymers are great candidates for the design of various nanosystems for biomedical applications.
The clinical applications of cationic polymer-based non-viral gene carriers are usually hindered by the instability of polyplex or complex between these cationic polymers and anionic genes. In order to increase the stability, an amphiphilic cationic block copolymer PHB-b-PDMAEMA was designed to form cationic micelle, which comprised hydrophobic poly[(R)-3-hydroxybutyrate] (PHB) block and cationic poly(2-(dimethylamino)ethyl methacrylate) (DMAEMA) block. Interestingly, this cationic micelle after complex formation with plasmid deoxyribonucleic acid (pDNA) exhibited enhanced stability within 24 h and showed improved gene transfection performance than PDMAEMA only or non-viral transfection gold standard poly(ethylene imine) with molecular weight of 25 kDa (PEI-25k). In short, the design of cationic amphiphilic polymer, with micelle formation ability to enhance stability of polyplex, might greatly benefit the designs of non-viral cationic polymers as potential gene vectors.
Reversible deactivation radical polymerizations (RDRPs) have proven to be the convenient tools for the preparation of polymeric architectures and nanostructured materials. When biodegradability is conferred to these materials, many biomedical applications can be envisioned. In this review, we discuss the synthesis and applications of biodegradable polymeric architectures using different RDRPs. These biodegradable polymeric structures can be designed as well-defined star-shaped, cross-linked or hyperbranched via smartly designing the chain transfer agents and/or post-polymerization modifications. These polymers can also be exploited to fabricate micelles, vesicles and capsules via either self-assembly or cross-linking methodologies. Nanogels and hydrogels can also be prepared via RDRPs and their applications in biomedical science are also discussed. In addition to the synthetic polymers, varied natural precursors such as cellulose and biomolecules can also be employed to prepare biodegradable polymeric architectures.
Mesoporous silica nanoparticles (MSNs) are attracting increasing interest for potential biomedical applications. With tailored mesoporous structure, huge surface area and pore volume, selective surface functionality, as well as morphology control, MSNs exhibit high loading capacity for therapeutic agents and controlled release properties if modified with stimuli-responsive groups, polymers or proteins. In this review article, the applications of MSNs in pharmaceutics to improve drug bioavailability, reduce drug toxicity, and deliver with cellular targetability are summarized. Particularly, the exciting progress in the development of MSNs-based effective delivery systems for poorly soluble drugs, anticancer agents, and therapeutic genes are highlighted.
Different generations (G3-G4) of amine-terminated Jeffamine® T-403 core poly(amidoamine) PAMAM dendrimers (JCPDs) were used as new macromolecular heavy metal chelating agent templates in polymer assisted ultrafiltration (PAUF) for the investigation of their removal ability for some of the divalent metal ions: Cu, Co, Ni, Cd, and Zn from aqueous solutions under competitive conditions. The effects of pH and generation size of JCPDs were also investigated. Extent of binding (EOB) data can be appropriately expressed by a tetradentate coordination for JCPDs at pH 9 where the maximum removal of metal ions was observed. At pH 9.0, the affinity of both generations towards heavy metal ions was also observed in the decreasing order of Zn(II) > Co(II) > Ni(II) > Cu(II) > Cd(II). Results revealed that the highest total binding capacity was observed for G3-NH2 (262.79 ± 1.62 mg/g) as a little bit higher than that of G4-NH2 (257.27 ± 2.57 mg/g). EOB studies also proved the active contribution of amide groups to metal binding ability of PAMAMs. Both generations were selective towards Cu(II) ions at lower pH 5 and pH 7. From these results, it was concluded that studied
JCPDs have the desired technical properties to be used for the removal of toxic metals from wastewaters.
Accounting for 16 million new cases and 9 million deaths annually, cancer leaves a great number of patients helpless. It is a complex disease and still a major challenge for the scientific and medical communities. The efficacy of conventional chemotherapies is often poor and patients suffer from off-target effects. Each neoplasm exhibits molecular signatures - sometimes in a patient specific manner - that may completely differ from the organ of origin, may be expressed in markedly higher amounts and/or in different location compared to the normal tissue. Although adding layers of complexity in the understanding of cancer biology, this cancer-specific signature provides an opportunity to develop targeting agents for early detection, diagnosis, and therapeutics. Chimeric antibodies, recombinant proteins or synthetic polypeptides have emerged as excellent candidates for specific homing to peripheral and central nervous system cancers. Specifically, peptide ligands benefit from their small size, easy and affordable production, high specificity, and remarkable flexibility regarding their sequence and conjugation possibilities. Coupled to imaging agents, chemotherapies and/or nanocarriers they have shown to increase the on-site delivery, thus allowing better tumor mass contouring in imaging and increased efficacy of the chemotherapies associated with reduced adverse effects. Therefore, some of the peptides alone or in combination have been tested in clinical trials to treat patients. Peptides have been well-tolerated and shown absence of toxicity. This review aims to offer a view on tumor targeting peptides that are either derived from natural peptide ligands or identified using phage display screening. We also include examples of peptides targeting the high-grade malignant tumors of the central nervous system as an example of the complex therapeutic management due to the tumor’s location. Peptide vaccines are outside of the scope of this review.
Development of modern agriculture and biotechnology is closely connected with the use of novel and effective genetic engineering methods. Presently, non-viral nanoparticle-mediated plant transformation methods gain more attention because of their stability, safety, and convenience of performance. In this work, new polymeric dimethylaminoethyl metacrylate (DMAEM)-based polymers were synthesized and investigated for their properties in gene delivery. Formation of stable complexes between TN 83/6, TN 84/5, DLM-9-DM and LM-8-DM polymers and plasmid DNA, as well as the DNA protection by the PDMAEM polymers against nuclease degradation were confirmed by electrophoresis in agarose gel. In addition, model organisms Allium cepa and Nicotiana tabacum L. were studied to evaluate cytotoxic effect of the PDMAEM carriers. The created PDMAEM-based carriers were effective in delivery of plasmid DNA into moss and tabacco protoplasts (obtaining stable transformants of Ceratodon purpureus moss, as well as in transient expression of the reporter yfp gene product in N. tabacum protoplasts). Thus, novel PDMAEM-based polymers were shown to be promising carriers for delivery of DNA into plant cells, and carriers possess high potential for further applications in this field.
Cationic polymers provide versatile traits that fit for non-viral gene delivery applications. However, cationic polymers could exhibit significant cytotoxicity. We chemically modified poly(acrylic acid) by conjugating side chains with either primary amine or with different number of alpha mine of Alanine (denoted as PAla polymers), and investigated the impact of amine groups of PAA regarding biocompatibility and intracellular gene delivery efficiency. Our design of PAla polymers proved that the incorporation of Alanine in cationic polymers may significantly decrease cytotoxicity of the resulting polymers while maintaining the efficiency of cellular uptake and the intracellular gene delivery of the DNA/cationic polymer complex.
Uses of viral vectors have thus far eclipsed uses of non-viral vectors for gene therapy delivery in the clinic. Viral vectors, however, have certain issues involving genome integration, the inability to be delivered repeatedly, and possible host rejection. Fortunately, development of non-viral DNA vectors has progressed steadily, especially in plasmid vector length reduction, now allowing these tools to fill in specifically where viral or other non-viral vectors may not be the best options. In this review, we examine the improvements made to non-viral DNA gene therapy vectors, highlight opportunities for their further development, address therapeutic needs for which their use is the logical choice, and discuss their future expansion into the clinic
Cationic polymers with hydrophobic side chains have gained great interest as DNA carriers since they form a compact complex with negatively charged DNA phosphate groups and interact with the cell membrane. Amphiphilic polyoxanorbornenes with different quaternary alkyl pyridinium side chains with ethyl‐p(OPy2) and hexyl units‐p(OPy6) bearing 10 kDa MWT were synthesized by living Ring‐Opening Metathesis Polymerization method.
The physicochemical characteristics: critical micellar concentration, size distribution, surface charge, and condensation of polymer/DNA complex were investigated. Morphology of complexes was monitored by Atomic force microscopy. Cytotoxicity and interaction of these complexes with model lipid vesicles mimicking the cell membrane were examined. These polymers were enabled to form small sized complexes of DNA, which interact with model membrane vesicles. It was found that the nature of hydrophobicity of the homopolymers significantly impacts rates of DNA complexation and the surface charge of the resulting complexes. These results highlight the prospect of the further examinations of these polymers as gene carriers.
Polyamines are positively charged organic cations under physiologic ionic and pH conditions and hence they interact with negatively charged macromolecules such as DNA and RNA. Although electrostatic interaction is the predominant mode of polyamine–nucleic acid interactions, site- and structure-specific binding has also been recognized. A major consequence of polyamine–DNA interaction is the collapse of DNA to nanoparticles of approximately 100 nm diameter. Electron and atomic force microscopic studies have shown that these nanoparticles are spheroids, toroids and rods. DNA transport to cells for gene therapy applications requires the condensation of DNA to nanoparticles and hence the study of polyamines and related compounds with nucleic acids has received technological importance. In addition to natural and synthetic polyamines, several amine-terminated or polyamine-substituted agents are under intense investigation for non-viral gene delivery vehicles.
Protein expression after delivery of plasmid DNA to the cell nucleus depends on the processes of transcription and translation. Cytotoxic gene-delivery systems may compromise these processes and limit protein expression. This situation is perhaps most prevalent in current nonviral polycationic gene-delivery systems in which the polycationic nature of the delivery system can lead to cytotoxicity, To approach the problem of creating nontoxic but effective gene-delivery systems, we hypothesized that by optimizing the balance between polymer cationic density with endosomal escape moieties, effective gene transfer with low cytotoxicity could be created, As a model system, we synthesized a series of polymers whose side-chain termini varied with respect to the balance of cationic centers and endosomal escape moieties. Specifically, by polymer-analogous amidation we conjugated imidazole groups to the epsilon -amines of polylysine in varying mole ratios (73.5 mol % imidazole, 82.5 mol % imidazole, and 86.5 mol % imidazole), The primary epsilon -amine terminus of polylysine served as a model for the cationic centers, whereas the imidazole groups served as a model for the endosomal escape moieties. These polymers condensed plasmid DNA into nanostructures <150 nm and possessed little cytotoxicity in vitro, Transfection efficiency, as measured by luciferase protein expression, increased with increasing imidazole content of the polymers in a nonlinear relationship. The polymer with the highest imidazole content (86.5 mol %) mediated the highest protein expression, with levels equal to those mediated by polyethylenimine, but with little to no cytotoxicity.
A series of amphiphilic conjugates of dihydroxy selenolane (DHS) and monoamine selenolane (MAS), which we had previously reported to inhibit lipid peroxidation and assist the oxidative protein folding reaction respectively in cell free systems, were evaluated for cytotoxicity, associated mechanisms and antioxidant effects in cells. Our results indicated that a fatty acid/alkyl group of variable chain lengths (C6–14) as a lipophilic moiety of the DHS/MAS conjugates not only improved their ability to incorporate within the plasma membrane of cells but also modulated their cytotoxicity. In the concentration range of 1–50 μM, C6 conjugates were non-toxic whereas the long chain (≥C8) conjugates showed significant cytotoxicity. The induction of toxicity investigated by the changes in membrane leakage, fluidity, mitochondrial membrane potential and annexin-V–propidium iodide (PI) staining by using flow cytometry revealed plasma membrane disintegration and subsequent induction of necrosis as the major mechanism. Further, the conjugates of DHS and MAS also showed differential as well as nonlinear tendency in cytotoxicity with respect to chain lengths and this effect was attributed to their self-aggregation properties. Compared with the parent compounds, C6 conjugates not only exhibited better antioxidant activity in terms of the induction of selenoproteins such as glutathione peroxidase 1 (GPx1), GPx4 and thioredoxin reductase 1 (TrxR1) but also protected cells from the AAPH induced oxidative stress. In conclusion, the present study suggests the importance of hydrophilic–lipophilic balance (HLB) in fine tuning the toxicity and activity of bioinspired amphiphilic antioxidants.
There is much progress in application of genetic engineering for improving the biological properties of different organisms. Viral and nonviral carriers are used for delivery of genetic material into target cells. Polymeric materials of natural and synthetic origin are the most promising gene delivery agents. These polymers demonstrated high efficiency of DNA delivery into animal cells, although they were not very effective in plant cells. Here, the procedure for genetic transformation of Ceratodon purpureus (Hedw.) Brid. moss protoplasts is described. The method is based on the application of surface-active polymeric carriers of the poly-DMAEM structure and controlled length and charge. This allows obtaining more transient and stable moss transformants per microgram of plasmid DNA when compared with known protocol based on using polyethyleneglycol. It is easier, more convenient, and cheaper than the “gene gun” method. Prospects for further improvement of structure and functional characteristics of new polymeric carriers are considered for delivery of genetic material into plant cells.
Surface modification and endothelialization of vascular biomaterials are common approaches that are used to both resist the nonspecific adhesion of proteins and improve the hemocompatibility and long-term patency of artificial vascular grafts. Surface modification of vascular grafts using hydrophilic poly(ethylene glycol), zwitterionic polymers, heparin or other bioactive molecules can efficiently enhance hemocompatibility, and consequently prevent thrombosis on artificial vascular grafts. However, these modified surfaces may be excessively hydrophilic, which limits initial vascular endothelial cell adhesion and formation of a confluent endothelial lining. Therefore, the improvement of endothelialization on these grafts by chemical modification with specific peptides and genes is now arousing more and more interest. Several active peptides, such as RGD, CAG, REDV and YIGSR, can be specifically recognized by endothelial cells. Consequently, graft surfaces that are modified by these peptides can exhibit targeting selectivity for the adhesion of endothelial cells, and genes can be delivered by targeting carriers to specific tissues to enhance the promotion and regeneration of blood vessels. These methods could effectively accelerate selective endothelial cell recruitment and functional endothelialization. In this review, recent developments in the surface modification and endothelialization of biomaterials in vascular tissue engineering are summarized. Both gene engineering and targeting ligand immobilization are promising methods to improve the clinical outcome of artificial vascular grafts.
Delivery of the macromolecules including DNA, miRNA, and antisense oligonucleotides is typically mediated by carriers due to the large size and negative charge. Different physical (e.g., gene gun or electroporation), and chemical (e.g., cationic polymer or lipid) vectors have been already used to improve the efficiency of gene transfer. Polymer-based DNA delivery systems have attracted special interest, in particular via intravenous injection with many intra- and extracellular barriers. The recent progress has shown that stimuli-responsive polymers entitled as multi-functional nucleic acid vehicles can act to target specific cells. These non-viral carriers are classified by the type of stimulus including reduction potential, pH, and temperature. Generally, the physicochemical characterization of DNA-polymer complexes is critical to enhance the transfection potency via protection of DNA from nuclease digestion, endosomal escape, and nuclear localization. The successful clinical applications will depend on an exact insight of barriers in gene delivery and development of carriers overcoming these barriers. Consequently, improvement of novel cationic polymers with low toxicity and effective for biomedical use has attracted a great attention in gene therapy. This article summarizes the main physicochemical and biological properties of polyplexes describing their gene transfection behavior, in vitro and in vivo. In this line, the relative efficiencies of various cationic polymers are compared. This article is protected by copyright. All rights reserved.
© 2015 Wiley Periodicals, Inc.
Cervical cancer is the fourth most common cancer among women worldwide and its development is mainly associated with human papillomavirus infection, a highly sexually transmissible virus. The expression of E6 and E7 viral oncoproteins deregulates cell repairing mechanisms through impairment of tumor suppressor protein functions, such as p53 or retinoblastoma protein. Although the implementation of new preventive vaccines has decreased the infection rate and cervical cancer progression, there are still many women who are affected by this pathology. Nowadays, the main treatment often requires the use of invasive techniques. From well-established strategies, like DNA vaccines and gene therapy, to innovative gene silencing technologies; different methodologies are currently under scrutiny that target the E6 and E7 oncoproteins and/or their modes of action.
Micelles have demonstrated excellent ability to deliver several different types of therapeutic agents, including chemotherapy drugs, proteins, siRNA, and DNA into tumor cells. Cationic micelles, self‐assemblies of amphiphilic cationic polymers, have exhibited tremendous promise in delivery of therapy genes and gene transfection. Present time, researches in the field have focused on achieving enhanced stability of the micellar assembly, prolonged circulation times and controlled release of the gene. This review focuses on the micelles as a nano‐sized carrier system for gene delivery, the system‐related modifications for the cytoplasm release, the stability and biocompatibility, as well as the clinic trials. As the development of synthetic chemistry and self‐assembly technology, the micelles’ structures and functionalities can be precisely controlled, and hence the synthetic micelles can not only efficiency condense DNA, but also facilitate DNA endocytosis, endosomal escape, DNA uptaking and nuclear transport, resulting in comparable gene transfection of virus.
For clinical application of therapeutic gene delivery, it is urgent to develop the safe and in vivo efficient delivery systems. Nowadays, gene delivery carriers based on functional peptides have attached...
Gene delivery systems with well biocompatibility and high transfection efficiency play a major role on the clinic application of gene therapy. It is of great interesting to develop a functional...
Gene therapy as a strategy for disease treatment requires safe and efficient gene delivery systems that encapsulated nucleic acids and deliver them to effective sites in the cell. Due to the unexpected security of viral vectors, non-viral vectors have gained great attention recently. Additional advantages of non-viral vectors include their safety, flexibility in packaging nucleic acids, and ease of production. To construct an ideal gene carrier, peptides can be incorporated into non-viral gene delivery systems as functional motifs to overcome the current barriers in gene delivery. In this review, we summarize recent developments peptide-based gene delivery vector research.
Colloidal-chemical characteristics of block/branched cationic and non-ionic polyamphiphiles containing poly(fluorine-alkyl methacrylate) (poly(FMA)) block and their intermolecular complexes with biopolymers were studied. The dependences of their surface activity and micelle size on the length of hydrophobic and hydrophilic blocks, as well as the length of side fluorine-alkyl branches were established. Poly(FMA)-block-poly(DMAEMA) was used for formation of interpolyelectrolyte complexes with plasmid DNA (pDNA) via their electrostatic interaction. Novel non-viral polyplexes were tested as gene delivery systems for mammalian cells. The results of DLS, TEM and MALDI-ToF studies demonstrated disaggregation of lysozyme (LYZ) aggregates in the presence of poly(FMA)-block-poly(NVP) and formation of the polyamphiphile…LYS complex possessing antibacterial action.
SA-SP-PEI were synthesized via acylation reaction between carboxyl-modified soybean protein (SA-SP) and branched polyethylenimine (PEI) with molecular weight of 600, 1200, and 1800 Da, and designed as SA-SP-PEI600, SA-SP-PEI1200, and SA-SP-PEI1800, respectively. SA-SP-PEI could effectively condense plasmid DNA into nanoscale polyplexes and protect them from enzymatic digestion. MTT assay revealed that SA-SP-PEI exhibited reduced cytotoxicity on 293 T and SH-SY5Y cells. SA-SP-PEI1800/DNA complexes hold highest transfection efficiency on 293 T and SH-SY5Y cells with or without 10% serum, which was owing to its better serum stable and improved biocompatibility. Such polycationic soybean proteins have great potentials as gene carriers by further optimization.
Effective endosomal escape is required for practical application of nucleic acid therapeutics. In this study, we prepared siRNA micelleplexes with photothermally triggered endosomal escape and improved gene silencing activity in vitro. The micelleplexes were prepared from cholesterol‐modified and CXCR4‐inhibiting poly(amido amine)s (PAMD‐Ch). Near‐infrared dye IR780 was encapsulated in cationic PMAD‐Ch micelles, which then were used to form IR780 micelleplexes with siRNA. The micelleplexes displayed improved gene silencing efficiency upon laser irradiation, which was attributed to enhanced endosomal escape because of the photothermal effects of the encapsulated IR780. The IR780 micelleplexes retained the CXCR4 antagonism and inhibition of cancer cell invasion of the parent PAMD. Overall, this study validates codelivery of IR780 in siRNA micelleplexes as promising photothermally controlled siRNA delivery approach.
The goal of this work was to understand the key factors determining the DNA compacting capacity of single-chained cationic surfactants. Fluorescence, zeta potential, circular dichroism, gel electrophoresis and AFM measurements were carried out in order to study the condensation of the nucleic acid resulting from the formation of the surfactant-DNA complexes. The apparent equilibrium binding constant of the surfactants to the nucleic acid, Kapp, estimated from the experimental results obtained in the ethidium bromide competitive binding experiments, can be considered directly related to the ability of a given surfactant as a DNA compacting agent. The plot of ln(Kapp) vs. ln(cmc), cmc being the critical micelle concentration, for all the bromide and chloride surfactants studied, was found to be a reasonably good linear correlation. This result shows that hydrophobic interactions mainly control the surfactant DNA compaction efficiency.
The clinical success of chimeric antigen receptor (CAR) T cell immunotherapy in treating multiple blood cancers has created a need for efficient methods of ex vivo gene delivery to primary human T cells for cell engineering. Here, we synthesize and evaluate a panel of cationic polymers for gene delivery to both cultured and primary human T cells. We show that a subset of comb- and sunflower-shaped pHEMA-g-pDMAEMA polymers can mediate transfection with efficiencies up to 50% in the Jurkat human T cell line with minimal concomitant toxicity (>90% viability). We then optimize primary human T cell transfection conditions including activation time, cell density, DNA dose, culture media, and cytokine treatment. We demonstrate transfection of both CD4+and CD8+primary human T cells with messenger RNA and plasmid DNA at efficiencies up to 25 and 18%, respectively, with similarly high viability.
Micelleplexes are promising gene delivery vehicles that form when DNA complexes with polycationic micelles. In this study, the influence of DNA length and topology on the structure and colloidal stability of micelleplexes was explored using a model system. The cationic micelles were composed of poly(2-(dimethylamino)ethyl methacrylate)-block-poly(n-butyl methacrylate) and were complexed with linear DNA and circular plasmids of 2442, 4708, and 7537 base pairs. The cationic micelles had a mean core radius of 8 ± 1 nm and a mean hydrodynamic radius of 34 ± 1 nm in buffer at pH 5 and 100 mM ionic strength. The formation of micelleplexes was monitored by turbidimetric titration as a function of N/P ratio (amine in micelle corona/phosphate on DNA) in acetate buffers of various ionic strengths. The structure and size evolution of micelleplexes were studied by dynamic light scattering and cryo-TEM, while the composition of micelleplexes was estimated using static light scattering. The combination of these techniques revealed that increasing DNA length resulted in increased micelleplex size at N/P > 1; this was attributed to an increased propensity for longer DNA to bridge between micelles. At N/P < 1, however, longer DNA enhances the stability of micelleplexes against aggregation by providing additional steric repulsions between micelleplexes. At high ionic strength, increasing DNA length also shifts titration curves to higher N/P ratios as the structure of micelleplexes changes with DNA length. On the other hand, DNA topology showed minimal influence on the titration curves, structure, and long-term stability of micelleplexes. Overall, this work illustrates how polycationic micelles may serve as compaction agents for long chain DNA and how factors such as DNA length can be used to tune the structure and colloidal stability of micelleplexes.
Over the past few years, the scientific community has attributed the association between social, environmental and genetic factors as the major contribution to the increase of the number of different diseases, like cancer and neurologic disorders. For the majority of diseases, once the current options of treatment available are reduced and limited, there arises a need to overcome these barriers and to develop new and efficient therapeutic strategies. In this sense, polymer-based nanotechnology emerged as a promising strategy. Through the development of smart micelleplexes that are capable of transporting genetic material/gene-based drugs and delivery them efficiently into the target cell, tissue or organ, as well as surpassed the biochemical and physiological barriers, leading to a higher success rate of treatment with low side effects.
This third edition of Electron Microscopy: Methods and Protocols expands upon the previous editions with current, detailed protocols on biological and molecular research techniques based on TEM and SEM as well as other closely related imaging and analytical methods. With new chapters on conventional and microwave assisted specimen, cryo-specimen preparation, negative staining and immunogold labelling techniques, DNA and RNA tracking using hybrization in TEM or Atomic Force Microscopy, TEM crystallography and cryoTEM 3D tomography, 3D tomography of resin embedded tissues using FIB-SEM , Correlative microscopy using fluorescence microscopy, confocal microscopy or immune labelling techniques for both TEM and FIB-SEM, and Elemental and isotopic identification and their distribution in cells and tissues using TEM, SEM, Scanning Transmission Electron Microscopy (STEM), Secondary Ion Mass Spectrometry (SIMS) and NanoSIMS. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.
Authoritative and practical, Electron Microscopy: Methods and Protocols, Third Edition provides the most up-to-date and essential information in electron microscopy techniques and methods provided in this edition will assist in advancing future molecular and biological research.
Cationic polymers are characterized as the macromolecules that possess positive charges, which can be either inherently in the polymer side chains and/or its backbone. Based on their origins, cationic polymers are divided in two category including natural and synthetic, in which the possessed positive charges are as result of primary, secondary or tertiary amine functional groups that could be protonated in particular situations. Cationic polymers have been employed commonly as drug delivery agents due to their superior encapsulation efficacy, enhanced bioavailability, low toxicity and improved release profile. In this paper, we focus on the most prominent examples of cationic polymers which have been revealed to be applicable in drug delivery systems and we also discuss their general synthesis and surface modification methods as well as their controlled release profile in drug delivery.
Statement of significance:
Polymers are widely studied as non-viral gene vectors, but suffer from high cytotoxicity and low transfection efficiency. Incorporating short, bioactive peptides into polymer-based gene delivery systems can address this issue. Peptides demonstrate various functions in polymer-based gene delivery systems, such as targeting to specific cells, breaching membrane barriers, facilitating DNA condensation and release, and lowering cytotoxicity. In this review, we highlight the peptides' roles in polymer-based gene delivery, and elaborate how to utilize various functional peptides to enhance the transfection efficiency of polymers. The optimized peptide-polymer vectors should be able to alter their structures and functions according to biological microenvironments and utilize inherent intracellular pathways of cells, and consequently overcome the barriers during gene delivery to enhance transfection efficiency.
Cancer has been the most deleterious disease since few decades and its prevalence is increasing day by day. Scientists have developed many novel carrier systems to modify the delivery of anti-cancer drugs specifically towards the cancer sites. Liposomes seemed to be the particles of choice to carry anti-cancer drugs owing to their bio-membrane friendly structure. However, poor stability and storage problems remain a drawback. Polymeric nanoparticles (PNPs) offer their rigid structure making controlled release of drugs possible. PNPs also maintain their integrity for a longer period of time. Recently, the benefits of the two carrier systems mentioned above have been combined into a single hybrid carrier system i.e., Lipid polymer hybrid nanoparticles (LPNP). Such a setting makes a useful carrier system taking advantages of both the counter parts and minimizing their limitations at the same time. In this review, special types of lipid and polymer hybrid structures are discussed in detail. Also the methods of preparation along with controlling formulation parameters have been discussed. Various targeting moieties have been enlisted along with their mechanisms of active targeting. These moieties can be functionalized onto the surfaces of these hybrid particles. Special focus has been given to the colorectal cancer (CRC), throwing light upon its prevalence and already available treatment options. Lipid polymer hybrid nanoparticles enable the researchers to formulate a carrier system which will be able to provide targeted anti-cancer drug delivery.
Recently, interdisciplinary research in cancer diagnosis and therapy has evolved to the point where nanotechnology particularly polymeric nano delivery systems are utilized for theragnostic applications. Nanoscale are being trialed for specific targeting-delivery of drugs, micelles, antibody, DNA, protein etc. to cancer sites in order to improve the therapeutic efficacy due to improved distribution specificity, increased internalization and intracellular drug delivery, that minimizing side effects. Polymeric micelles have been subjected to extensive studies in the field of drug delivery, functioning as drug solibilizers and carriers. More recently, a micelle constructed as a hybrid from hydrophilic oligonucleotide and hydrophobic polymer has drawn close attention. Mostly used micelles are synthesized with polymer and have several physical properties, including molecular weight and copolymer block composition, which can be tailored to alter the vesicle structure. In this review, we focused on the different polymeric nanodelivery systems is association with different type of cancer therapeutics such as micelles, drug, aptamer, DNA, recombinant protein, miRNA, siRNA, small inhibitors, gene, antibody, proteins etc are some conjugating molecules that involved in cancer therapy have been discussed.
Gene therapy is a promising strategy for angiogenesis, but developing gene carriers with low cytotoxicity and high gene delivery efficiency in vivo is a key issue. In the present study, we synthesized the CAGW peptide- and poly(ethylene glycol) (PEG)-modified amphiphilic copolymers. CAGW peptide serves as a targeting ligand for endothelial cells (ECs). Different amounts of CAGW peptide were effectively conjugated to the amphiphilic copolymer via heterofunctional poly(ethylene glycol). These CAG- and PEG-modified copolymers could form nanoparticles (NPs) by self-assembly method and were used as gene carriers for the pEGFP-ZNF580 (pZNF580) plasmid. CAGW and PEG modification coordinately improved the hemocompatibility and cytocompatibility of NPs. The results of cellular uptake showed significantly enhanced internalization efficiency of pZNF580 after CAGW modification. Gene expression at mRNA and protein levels demonstrated that EC-targeted NPs possessed high gene delivery efficiency, especially the NPs with higher content of CAGW peptide (1.16 wt %). Furthermore, in vitro and in vivo vascularization assays also showed outstanding vascularization ability of human umbilical vein endothelial cells treated by the NP/pZNF580 complexes. This study demonstrates that the CAGW peptide-modified NP is a promising candidate for gene therapy in angiogenesis.
Gene therapy demonstrates promising prospects on cardiovascular diseases. However, nonviral gene delivery system has relatively low transfection efficiency, especially for endothelial cells (ECs). Herein, typical cell-penetrating peptide (TAT), nuclear localization signals (NLSs), and REDV functional peptide have been used to prepare multitargeting complexes. These complexes exhibit higher transfection efficiency owing to the targeting sequences of REDV and NLSs as well as the cell-penetrating function of TAT. The multifunction of the complexes provides high cell uptake, endo/lysosomal escape, and nucleus accumulation of the encapsulated DNA. Thus these multitargeting complexes can provide a potential platform for gene delivery, especially for EC transfection.
Generally, the major obstacles for efficient gene delivery are cellular internalization and endosomal escape of nucleic acid such as plasmid DNA (pDNA) or small interfering RNA (siRNA). We previously developed Pluronic P123 modified polypropyleneimine (PPI)/pDNA (P123-PPI/pDNA) polyplexes as a gene delivery system. The results showed that P123-PPI/pDNA polyplexes revealed higher transfection efficiency than PPI/pDNA polyplexes in multidrug resistant breast cancer cells. As a continued effort, the present investigation on the factors influencing the transfection efficiency, cellular uptake mechanisms, and intracellular fate of P123-PPI/pDNA polyplexes is reported. The presence of P123 was the main factor influencing the transfection efficiency of P123-PPI/pDNA polyplexes in MCF-7/ADR cells, but other parameters, such as N/P ratio, FBS concentration, incubation time and temperature were important as well. The endocytic inhibitors against clathrin-mediated endocytosis (CME), caveolae-mediated endocytosis (CvME), and macropinocytosis were involved in the internalization to investigate their effects on the cellular uptake and transfection efficiency of P123-PPI/pDNA polyplexes in vitro. The data showed that the internalization of P123-PPI/pDNA polyplexes was obtained from both CME and CvME. Colocalization experiments with TRITC-transferrin (CME indicator), Alexa Fluor 555-CTB (CvME indicator), monoclonal anti-α-tubulin (microtubule indicator), and LysoTracker Green (Endosome/lysosome indicator) were carried out to confirm the internalization routes. The results showed that both CME and CvME played vital roles in the effective transfection of P123-PPI/pDNA polyplexes. Endosome/lysosome system and skeleton, including actin filament and microtubule, were necessary for the transportation after internalization.
The basic concept of this new edition remains unchanged: Emphasis is again put on a broader description of the general methods and processes for the synthesis, modification and characterization of macromolecules. These more fundamental chapters will be supplemented by 115 selected and detailed experiments. In addition to the preparative aspects the book also gives the reader an impression on the relation of chemical constitution and morphology of Polymers to their properties, as well as on their application areas. Thus, an additional textbook will not be needed in order to understand the experiments. The 4th edition nevertheless contains numerous changes: Chapter 1 and 2 are enlarged, 15 older examples have been replaced by new ones and background text is added. Suitable for students in organic and polymer chemistry as well as for chemists in industry who want to acquaint themselves with the theoretical and practical aspects of macromolecular chemistry. From the reviews:"This is an excellent book for all polymer chemsits engaged in synthesis research studies and education. It is educationally sound and has excellent laboratory synthetic examples. The fundamentals are well done for the teaching of students and references are resonably up-to-date. As in previous issues, there are sections dealing with an introduction; structure and nomenclature; methods and techniques for synthesis, characterization, processing and modification of polymers.....The authors have noted the following changes from previous editions- a new section on correlayions of structure, morphology and properties; revision and enlargement of other property and characterization procedures; additional new experiments such as controlled radical polymerization; enzymatix polymerizations; microelmulsions; and electrical conducting polymers.This is a high quality textbook at a reasonable price and should be considered as a suitable references for all engaged in synthetic areas of polymer research."
Artificial vascular scaffolds have been developed and used clinically for many years, but the lack of a living functional layer of human umbilical vein endothelial cells (HUVECs) remains a significant challenge, especially for small diameter artificial blood vessels. Endothelialization of artificial vascular scaffolds has been proved as one of the most potential approaches to improve their hemocompatibility and long-term patency. A variety of non-viral gene carriers have been investigated to mediate the transfection and proliferation of HUVECs. In this mini-review, we will summarize the recent development of the non-viral gene carriers for transfecting HUVECs. The gene transfection with targeting ligand immobilized carriers is a promising approach to enhance endothelialization of artificial vascular scaffolds. Copyright © 2015 John Wiley & Sons, Ltd.
We report that the dye nile red, 9-diethylamino-5H-benzo[alpha]phenoxazine-5-one, is an excellent vital stain for the detection of intracellular lipid droplets by fluorescence microscopy and flow cytofluorometry. The specificity of the dye for lipid droplets was assessed on cultured aortic smooth muscle cells and on cultured peritoneal macrophages that were incubated with acetylated low density lipoprotein to induce cytoplasmic lipid overloading. Better selectivity for cytoplasmic lipid droplets was obtained when the cells were viewed for yellow-gold fluorescence (excitation, 450-500 nm; emission, greater than 528 nm) rather than red fluorescence (excitation, 515-560 nm; emission, greater than 590 nm). Nile red-stained, lipid droplet-filled macrophages exhibited greater fluorescence intensity than did nile red-stained control macrophages, and the two cell populations could be differentiated and analyzed by flow cytofluorometry. Such analyses could be performed with either yellow-gold or red fluorescence, but when few lipid droplets per cell were present, the yellow-gold fluorescence was more discriminating. Nile red exhibits properties of a near-ideal lysochrome. It is strongly fluorescent, but only in the presence of a hydrophobic environment. The dye is very soluble in the lipids it is intended to show, and it does not interact with any tissue constituent except by solution. Nile red can be applied to cells in an aqueous medium, and it does not dissolve the lipids it is supposed to reveal.
Our original concept of the mono-ion complex (MIC) between plasmid DNA (pDNA) and a mono-cationic biocompatible polymer has been stabilized by hydrogen bond formation. To form the hydrogen bond with pDNA, omega-amide-pentylimidazolium end-modified poly(ethylene glycol), that is, APe-Im-PEG, has been synthesized. Agarose gel retardation assay and circular dichroism measurement have revealed that the MIC between pDNA and APe-Im-PEG has been stabilized by the hydrogen bond between pDNA and the omega-amide group, and that the stable MIC has surprisingly further migrated into gel, as compared with naked pDNA. The rise of melting temperature suggests that the specific hydrogen bond forms between an adenine-thymine base pair and the omega-amide group. The resulting pDNA MIC with APe-Im-PEG has enhanced gene expression by intramuscular administration in mice, as compared with a poly(ethyleneimine) polyion complex (PIC). These results suggest that the pDNA MIC is diffusive in vivo administration site, as compared with pDNA PICs. Our methodology for MIC stabilization by a omega-amide group is expected to offer superior suprarmolecular systems to those by ubiquitous PICs for in vivo diffusive gene delivery.
Cancer remains a major killer and a leading cause of death in the world; thus, a growing number of new treatments have been focused on cancer therapy over the past few decades. Chemotherapy, which is thought to be a powerful strategy for cancer treatment, has been widely used in clinical therapy in recent years. However, due to the complexity of cancer, a single therapeutic approach is insufficient for the suppression of cancer growth and migration. Therefore, increasing attention has been paid to the use of smart multifunctional carriers and combinatorially delivers chemotherapeutic drugs and functional genes in order to maximize therapeutic efficiency. Combination therapy using selected drugs and genes can not only overcome multidrug resistance and inhibit the cellular anti-apoptotic process but also achieve a synergistic therapeutic effect. Because multifunctional nanocarriers are important for achieving these goals, this review will illustrate and discuss some advanced biomaterial nanocarriers for co-delivering therapeutic genes and drugs, including multifunctional micelles, liposomes, polymeric conjugates and inorganic nanoparticles. In addition, the challenges and future perspectives for co-delivery systems, containing therapeutic drugs and genes to achieve better therapeutic effects for cancer treatment will be discussed.
ABTRACT Gene therapy represents a potential efficient approach of disease prevention and therapy. However, due to their poor in vivo stability, gene-molecules need to be associated with delivery systems to overcome extracellular and intracellular barriers and allow access to the site of action. Cationic polymeric nanoparticles are popular carriers for small interfering RNA (siRNA) and DNA-based therapeutics for which efficient and safe delivery are important factors that need to be optimized. Micelle-like nanoparticles (MNP) (half micelles, half polymeric nanoparticles) can overcome some of the disadvantages of such cationic carriers by unifying in one single carrier the best of both delivery systems. In this review, we will discuss how the unique properties of MNP including self-assembly, condensation and protection of nucleic acids, improved cell association and gene transfection, low toxicity may contribute to the successful application of siRNA and DNA-based therapeutics into the clinic. Recent developments of MNP involving the addition of stimuli-sensitive functions to respond specifically to pathological or externally applied 'triggers' (e.g. temperature, pH or enzymatic catalysis, light, or magnetic fields) will be discussed. Finally, we will overview the use of MNP as two-in-one carriers for the simultaneous delivery of different agents (small molecules, imaging agents) and nucleic acids combinations.