Multifunctional dendritic drug delivery systems: design, synthesis, controlled and triggered release

This review describes the strategy for the development of multifunctional dendrimeric and hyperbranched polymers, collectively named dendritic polymers, aiming at their application as drug and gene delivery systems. Employing well-characterized and mainly commercially available dendritic polymers, the functionalization of these polymers is aimed at providing drug carriers of low toxicity, high encapsulating capacity, specificity to certain type of cells and transport ability through their membranes. Following a step-wise functionalization strategy of the starting dendritic polymers one has the option to prepare products that fulfill one or more of these requirements. In particular, in addition to polyvalency which is a common feature of the dendritic polymers, these carriers bearing a number of targeting ligands exhibit specificity to certain cells, another type of groups secures stability in biological milieu and prolonged circulation, while other moieties facilitate their transport through cell membranes. Furthermore, dendritic polymers applied for gene delivery should be or become cationic in the biological environment for the formation of complexes with the negatively charged genetic material.     

Drug delivery using multifunctional dendrimers and hyperbranched polymers

Importance of the field: The review presents the design strategy and synthesis of multifunctional dendrimers and hyperbranched polymers with the objective to develop effective drug delivery systems.

Areas covered in this review: Well-characterized, commercially available dendritic polymers were subjected to functionalization for preparing drug delivery systems of low toxicity, high loading capacity, ability to target specific cells and transport through their membranes. This has been achieved by surface targeting ligands, which render the carriers specific to certain cells and polyethylene glycol groups, securing water solubility, stability and prolonged circulation. Moreover, transport agents facilitate transport through cell membranes while fluorescent probes detect their intracellular localization. A common feature of surface groups is multivalency, which considerably enhances their binding strength with complementary cell receptors. To these properties, one should also add the property of attaining high loading of active ingredients coupled with controlled and/or triggered release.

What the reader will gain: Readers will be exposed to the strategy of synthesizing multifunctional polymers, aimed at the development of effective drug delivery systems.

Take home message: Multifunctional systems upgrade the therapeutic potential of drugs and, in certain cases, may even lead to the application of new bioactive compounds that would otherwise not be feasible.     

Molecular engineering of dendritic polymers and their application as drug and gene delivery systems

This review discusses the development of functional and multifunctional dendrimeric and hyperbranched polymers, collectively called dendritic polymers, with the objective of being applied as drug and gene delivery systems. In particular, using as starting materials known and well-characterized basic dendritic polymers, the review deals with the type of structural modifications to which these dendritic polymers were subjected for the development of drug carriers with low toxicity, high encapsulating capacity, a specificity for certain biological cells, and the ability to be transported through their membranes. Proceeding from functional to multifunctional dendritic polymers, one is able to prepare products that fulfill one or more of these requirements, which an effective drug carrier should exhibit. A common feature of the dendritic polymers is the exhibition of polyvalent interactions, while for multifunctional derivatives, a number of targeting ligands determine specificity, another type of group secures stability in biological milieu and prolonged circulation, while others facilitate their transport through cell membranes. Furthermore, dendritic polymers employed for gene delivery should be or become cationic in the biological environment for the formation of complexes with the negatively charged genetic material.     

Evaluation of the transfection property of a peptide ligand for the fibroblast growth factor receptor as part of PEGylated polyethylenimine polyplex

Purpose: Experiments were conducted to evaluate the utility of a peptide receptor ligand to improve transfection efficiency as part of a polyethylenimine-polyethylene glycol (PEI-PEG) polyplex. The 7-mer peptide (MQLPLAT), targeted toward the fibroblast growth factor 2 (FGF2) receptor, was recently identified using a phage-display library method as possessing a high degree of specificity for the FGF2 receptor without the mutagenicity associated with FGF itself. Two approaches (pre-modification or post-modification) to incorporate the peptide into the PEGylated polyplex were compared in terms of their effect on particle size, surface charge, DNA condensation ability, toxicity, cellular uptake and transfection efficiency.

Methods: The peptide was conjugated to branched PEI (25 kDa) via a PEG spacer either before (pre-modified) or after (post-modified) complexation of PEI with DNA. Polyethyleneimine was conjugated to the PEG spacer (N-hydroxy succinimide (NHS) -PEG-maleimide (Mal)) through the NHS group. The FGF2 peptide was synthesized to contain a cysteine at the carboxyl end (MQLPLATC) and conjugated to the PEG spacer via the Maleimide group. Conjugates were evaluated using ¹H NMR, amino acid analysis, and picrylsulfonic acid assay. DNA condensation was evaluated using agarose gel electrophoresis and cellular toxicity was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cellular uptake was measured using flow cytometry and transfection efficiency was determined using a luciferase reporter gene assay.

Results: Both pre- and post-modification approaches led to a decrease in the zeta potential of the resulting polyplexes but did not alter their size. The pre-modification of PEI did not affect its ability to condense DNA. However, polyplexes formed with the pre-conjugated PEI did not improve cell uptake or transfection efficiency. In contrast, polyplexes that were post-modified with the FGF2 peptide resulted in a 3-fold increase in cell uptake and a 6-fold increase in transfection efficiency. Both pre- and post-modified polyplexes resulted in lower toxicity compared with unmodified PEI.

Conclusions: The results indicate that the FGF2 peptide improves transfection efficiency when used as part of post-modified PEI/PEG polyplex. When used with pre-modified PEI/PEG, the beneficial effect of the peptide on transfection is not evident, probably because, in this case, the peptide ligand is not readily accessible to the FGF receptor.     

Identification of Novel Superior Polycationic Vectors for Gene Delivery by High-throughput Synthesis and Screening of a Combinatorial Library

Purpose Low efficiency and toxicity are two major drawbacks of current non-viral gene delivery vectors. Since DNA delivery to mammalian cells is a multi-step process, generating and searching combinatorial libraries of vectors employing high-throughput synthesis and screening methods is an attractive strategy for the development of new improved vectors because it increases the chance of identifying the most overall optimized vectors. Materials and Methods Based on the rationale that increasing the effective molecular weight of small PEIs, which are poor vectors compared to the higher molecular weight homologues but less toxic, raises their transfection efficiency due to better DNA binding, we synthesized a library of 144 biodegradable derivatives from two small PEIs and 24 bi- and oligo-acrylate esters. A 423-Da linear PEI and its 1:1 (w/w) mixture with a 1.8-kDa branched PEI were cross-linked with the acrylates at three molar ratios in DMSO. The resulting polymers were screened for their efficiency in delivering a β-galactosidase expressing plasmid to COS-7 monkey kidney cells. Selected most potent polymers from the initial screen were tested for toxicity in A549 human lung cancer cells, and in vivo in a systemic gene delivery model in mice employing a firefly luciferase expressing plasmid. Results Several polycations that exhibited high potency and low toxicity in vitro were identified from the library. The most potent derivative of the linear 423-Da PEI was that cross-linked with tricycle-[5.2.1.0]-decane-dimethanol diacrylate (diacrylate 14), which exhibited an over 3,600-fold enhancement in efficiency over the parent. The most potent mixed PEI was that cross-linked with ethylene glycol diacrylate (diacrylate 4) which was over 850-fold more efficient than the physically mixed parent PEIs. The relative efficiencies of these polymers were even up to over twice as high as that of the linear 22-kDa PEI, considered the “gold standard” for in vitro and systemic gene delivery. The potent cross-linked polycations identified were also less toxic than the 22-kDa PEI. The optimal vector in vivo was the mixed PEI cross-linked with propylene glycol glycerolate diacrylate (diacrylate 7); it mediated the highest gene expression in the lungs, followed by the spleen, with the expression in the former being 53-fold higher compared to the latter. In contrast, the parent PEIs mediated no gene expression at all under similar conditions, and injection of the polyplexes of the 22-kDa PEI at its optimal N/P of 10 prepared under identical conditions killed half of the mice injected. Conclusions High-throughput synthesis and transfection assay of a cross-linked library of biodegradable PEIs was proven effective in identifying highly transfecting vectors. The identified vectors exhibited dramatically superior efficiency compared to their parents both in vitro and in an in vivo systemic gene delivery model. The majority of these vectors mediated preferential gene delivery to the lung, and their in vivo toxicity paralleled that in vitro.     

Degradable poly(amido amine)s as gene delivery carriers

Introduction: In recent years, there has been a great deal of interest in the development of vectors which are being developed based on the capacity of polymers to mediate appropriate interactions with the cellular environment, or to interface with specific cellular processes. Several such vectors have been synthesized, resulting in biomacromolecules with low cytotoxicity and higher gene delivery ability.

Areas covered: This review briefly describes the recent success of poly(amido amine)s (PAAs) as non-viral vectors, and highlights their promising future in the development of nucleic acid-based therapy. It also provides an overview on the synthesis, characterization and application of PAAs as gene carriers, which will be useful for various biological motifs. This review helps the readers to better understand the emergence of non-viral vectors for gene therapy, especially PAAs, their properties, their advantages and disadvantages and the gene therapy based on them.

Expert opinion: The future of gene-based therapy needs to identify approaches to develop new carriers, depending on the properties of the biological membranes they face, and their physicochemical properties, in order to successfully deliver the genes to the target sites. With the emergence of a variety of non-viral vectors, such as biodegradable polymers, it may not take long before non-viral vectors are observed that are not just safe and tissue-specific, but even more efficient than viral vectors.     

A promising targeted gene delivery system: Folate-modified dexamethasone-conjugated solid lipid nanoparticles

Context: Non-viral gene delivery could deliver drugs/genes through cellular membranes and nuclear membranes by some modification of materials.

Objective: This study develops a kind of vector to target the cells through receptor-mediated pathways. Nuclear localization signal (NLS) was also used to increase the nuclear uptake of genetic materials.

Materials and methods: A lipid containing dexamethasone (Dexa) was synthesized as the material of the preparation of solid lipid nanoparticles (SLNs) and folate (Fa)-conjugated PEG-PE (Fa-PEG-PE) ligands were used to modify the SLNs. The in vitro cytotoxicity of the carriers at various concentrations (10, 20, 50, 100, and 200?μg/ml) were evaluated in KB human carcinoma cells (KB cells). In vivo transfection efficiency of the novel modified vectors was evaluated in disseminated peritoneal tumors on mice bearing KB cells.

Results: Fa-PEG-PE modified SLNs/enhanced green fluorescence protein plasmid (pEGFP) has a particle size of 258?nm, and the gene loading quantity of the vector was 90%. The in vitro cytotoxicity of Fa-PEG-PE-modified SLNs/pEGFP (Fa-SLNs/pEGFP) was low (cell viabilities were between 80% and 100% compared with controls). Fa-SLNs/pEGFP displayed remarkably higher transfection efficiency (40%) than non-modified SLNs/pEGFP (24%) and the vectors not containing Dexa (30%) in vivo.

Conclusion: The results demonstrate that Fa and Dexa could function as excellent active targeting ligands to improve the cell targeting and nuclear targeting ability of the carriers and the resulting vectors could be promising active targeting drug/gene delivery systems.     

Enhancement of polymethacrylate-mediated gene delivery by Penetratin

Polymethacrylates are vinyl-based polymers that are used for DNA transfection. Cationic polymethacrylates efficiently condense DNA by forming inter-polyelectrolyte complexes. Their use for DNA transfection is, however, limited due to their low ability to interact with membranes. In order to increase their transfection efficiency, we combined polymethacrylates with Penetratin, a 16-residue water-soluble peptide that internalises into cells through membrane translocation. DNA condensation was assessed using physicochemical methods, while transfection efficiency and cellular internalisation were studied using Cos-1 cells. Agarose gel electrophoresis retardation, ethidium bromide exclusion tests and dynamic light scattering measurements showed that the stability of the polymethacrylate–DNA complexes is not affected by addition of Penetratin. Transfection efficiency of polymethacrylate–DNA complexes into Cos-1 cells increased by addition of Penetratin and was higher than that of polyethylenimine (PEI)–DNA complexes and comparable to Lipofectamine™. Confocal microscopy and flow cytometry indicated that Penetratin mainly enhances endolysosomal escape polymethacrylate–DNA complexes and increases their cellular uptake. Since the cellular toxicity of polymethacrylate–DNA–Penetratin complexes remains low, especially compared to PEI, this transfection system opens new perspectives for gene therapy.     

Synthesis and evaluation of functional hyperbranched polyether polyols as prospected gene carriers

Hyperbranched polyether polyols have been partially functionalized with quaternary or tertiary ammonium groups. Five derivatives have been prepared bearing 4, 8 and 12 quaternary or 4 and 21 tertiary ammonium groups. The resulting dendritic polymers interact with plasmid DNA affording the corresponding polyplexes. The complexes were physicochemically characterized while their transfection ability was assessed by gel retardation assay, ethidium bromide exclusion assay and cell culture transfection. All the investigated polymers were shown to have marginal to low cytotoxicity in mammalian cells. Transfection efficiency comparable to that of polyethylenimine was exhibited by selected quaternized polymers. However, the introduction of tertiary amino groups on polyglycerol did not improve the transfection of the ineffective parent polymer, despite the fact that the derivatives obtained exhibited additional buffering capacity (sponge effect). The observed transfection efficiency for the quaternized polymers has been attributed to the destabilization of the lysosomal membrane originating from the interaction between the cationic polymers and the anionic moieties located at the membrane. These results are encouraging for the prospective application of these polyols as gene delivery vectors.     

Poly(amidoamine) dendrimer complexes as a platform for gene delivery

Introduction: Gene therapy is one of the most effective ways to treat major infectious diseases, cancer and genetic disorders. It is based on several viral and non-viral systems for nucleic acid delivery. The number of clinical trials based on application of non-viral drug and gene delivery systems is rapidly increasing.

Areas covered: This review discusses and summarizes recent advances in poly(amidoamine) dendrimers as effective gene carriers in vitro and in vivo, and their advantages and disadvantages relative to viral vectors and other non-viral systems (liposomes, linear polymers) are considered.

Expert opinion: In this regard, dendrimers are non-immunogenic and have the highest efficiency of transfection among other non-viral systems, and none of the drawbacks characteristic for viral systems. The toxicity of dendrimers both in vitro and in vivo is an important question that has been addressed on many occasions. Several non-toxic and efficient multifunctional dendrimer-based conjugates for gene delivery, along with modifications to improve transfection efficiency while decreasing cytotoxicity, are discussed. Twelve paradigms that affected the development of dendrimer-based gene delivery are described. The conclusion is that dendrimers are promising candidates for gene delivery, but this is just the beginning and further studies are required before using them in human gene therapy.     

End functionalized polymeric system derived from pyrrolidine provide high transfection efficiency

Chemical architecture and functionality play an important role in the physico-chemical properties of cationic polymers with applications as gene vectors. In this study, linear homopolymers of N-ethyl pyrrolidine methacrylamide (EPA), copolymers of EPA with N,N-dimethylacrylamide (DMA) and oligomers of EPA were synthesized, and the resulting structures were evaluated for their transfection efficiency as non-viral gene vectors. Specifically, polymer species with high and low molecular weights (120-2.6 kDa) and different functionalities (tertiary amines as side chains and primary amine as chain end) were prepared as non-crosslinked, linear homopolymers, copolymers and oligomers, respectively. Polymer/DNA complexes (polyplexes) formation was evaluated by agarose gel electrophoresis, showing that all systems complexed with DNA in all P/N ratios with the exception of the EPA homopolymer. Furthermore, light scattering measurements and transmission electronic microscopy (TEM) showed different size (50-450 nm) and morphology depending on the composition and concentration of the polyplex systems. Cell viability and proliferation after contact with polymer and polyplexes were studied using 3T3 fibroblasts, and the systems showed an excellent biocompatibility at 2 and 4 days. Transfection studies were performed with plasmid Gaussian luciferase kit and were found that the highest transfection efficiency in serum free was obtained with oligomers from the P/N ratio of 1/6 to 1/10. Transfection values of the functionalized oligomers with respect to the control linear poly (dimethylaminoethyl methacrylate) [poly (DMAEMA)] are very interesting in the presence of serum. Haemolysis for these polymers values below 1%, which provide attractive potential applications in gene therapy with these non-toxic readsorbable polymers.     

Decationized polyplexes for gene delivery

Gene therapy has received much attention in the field of drug delivery. Synthetic, nonviral gene delivery systems have gained increasing attention as vectors for gene therapy mainly due to a favorable immunogenicity profile and ease of manufacturing as compared to viral vectors. The great majority of these formulations are based on polycationic structures, due to their ability to interact with negatively charged nucleic acids to spontaneously form nanoparticles. In recent years, several polycationic systems have demonstrated high transfection in vitro. However, progress toward clinical applications has been slow, mainly because the cationic nature of these systems leads to intolerable toxicity levels, inappropriate biodistribution and unsatisfactory efficiency in vivo, particularly after systemic administration. Decationized polyplexes are a new class of gene delivery systems that have been developed as an alternative for conventional polycation-based systems. The major innovation introduced by decationized polyplexes is that these systems are based on neutral polymers, without any detrimental effect on the physicochemical stability or encapsulation ability, due to the transient presence of cationic charge and disulfide cross-links between the polymer chains by which the nucleic acids are physically entrapped in the particles. This editorial summarizes the most important features of decationized polyplexes and discusses potential implications for the development of new safe and efficient gene delivery systems.     

The role of the disulfide group in disulfide-based polymeric gene carriers

Background: An essential prerequisite for successful gene therapy is the development of safe and efficient gene delivery carriers. For this purpose, cationic polymers have been widely studied as non-viral carriers, but they generally suffer from low transfection efficiency and/or high cytotoxicity. To address these problems, disulfide-based cationic polymers have been designed as intelligent gene carriers that are capable of inducing highly efficient gene transfection with low cytotoxicity. Objective: The present review discusses the effects of the disulfide linker on the gene delivery properties of cationic polymers in relation to various gene delivery barriers. Methods: The literature regarding the gene delivery barriers encountered by polymeric gene delivery is reviewed and discussed in relation to the presence of the disulfide moiety in these gene carriers. Conclusions: The presence of disulfide linkages in cationic polymers can in many aspects favorably influence the gene delivery properties, such as increasing DNA binding ability, enabling de-shielding of ‘stealth’ (PEG) groups, fine-tuning of the buffer capacity for enhanced endosomal escape, improving carrier-unpacking and decreasing cytotoxicity. Therefore, disulfide-based cationic polymers are promising candidates for the next generation of non-viral carriers.     

Dendrosome-dendriplex inside liposomes: as a gene delivery system

Dendrosomes are lipid vesicular entities containing entrapped dendrimer-DNA complexes and possessing low toxicity, acceptable transfection efficiency, and good in vivo tolerance. Herein, an attempt was made to explore the potential of dendrosomes as a gene delivery system combining the advantages of both polyamidoamine (PAMAM) dendrimer (nucleic acid condensation, facilitated endosomal release) and of non-cationic liposomes (increased cellular uptake, low cytotoxicity), and at the same time overcoming the drawbacks of these system (low encapsulation efficiency of non-cationic liposome and toxicity of dendrimers). Dendrosomes were assembled by loading optimized DNA-dendrimer complexes into liposomes prepared by solvating of dried lipid films made of DOPE/EggPC/Cholesterol (4.74:4.75:1.5 mole ratio). Dendrosomes were characterized in terms of size, zeta, encapsulation efficiency and the ability to protect the system from DNA degradation. The transfection efficiency and toxicity of the preparations were evaluated in HeLa cells using flow cytometry and CellTiter-Blue^® methods. The efficient transfection and low toxicity makes them an appealing alternative to be further explored for gene delivery in vivo.     

Current status of non-viral gene therapy for CNS disorders

ABSTRACT

Introduction: Viral and non-viral vectors have been used as methods of delivery in gene therapy for many CNS diseases. Currently, viral vectors such as adeno-associated viruses (AAV), retroviruses, lentiviruses, adenoviruses and herpes simplex viruses (HHV) are being used as successful vectors in gene therapy at clinical trial levels. However, many disadvantages have risen from their usage. Non-viral vectors like cationic polymers, cationic lipids, engineered polymers, nanoparticles, and naked DNA offer a much safer option and can therefore be explored for therapeutic purposes.

Areas covered: This review discusses different types of viral and non-viral vectors for gene therapy and explores clinical trials for CNS diseases that have used these types of vectors for gene delivery. Highlights include non-viral gene delivery and its challenges, possible strategies to improve transfection, regulatory issues concerning vector usage, and future prospects for clinical applications.

Expert opinion: Transfection efficiency of cationic lipids and polymers can be improved through manipulation of molecules used. Efficacy of cationic lipids is dependent on cationic charge, saturation levels, and stability of linkers. Factors determining efficacy of cationic polymers are total charge density, molecular weights, and complexity of molecule. All of the above mentioned parameters must be taken care for efficient gene delivery.     

SAHA (vorinostat) facilitates functional polymer-based gene transfection via upregulation of ROS and synergizes with TRAIL gene delivery for cancer therapy

Abstract

Non-viral gene delivery is an attractive approach for the treatment of many diseases including cancer, benefiting from its safety and large-scale production concerns. However, the relatively low transfection efficacy compared with viral vectors restricts the clinical applications of non-viral gene vectors. Reactive oxygen species (ROS) triggered charge reversal polymers (named B-PDEAEA) presented improved transfection efficacy, because of fast release of plasmid DNA responding to enhanced oxidative stress in cancer cells. But inadequate dissociation can still occur owing to the insufficient intracellular ROS generation. Here, we report SAHA (vorinostat), which is a clinical histone deacetylase inhibitor and anticancer drug, induces the ROS accumulation in cancer cells, and facilitates the charge reversal process of B-PDEAEA and the cellular dissociation of the delivered gene from the vectors. As a result, SAHA remarkably increases the gene transfection efficacy in an ROS-dependent manner. Importantly, SAHA synergizes with B-PDEAEA mediated therapeutic gene TNF-related apoptosis-inducing ligand (TRAIL) delivery in inducing apoptosis of cancer cells. These findings support the first concept of improving the gene delivery efficacy of stimuli-responsive vectors through upregulating the cellular ROS via an FDA approved anticancer agent. Additionally, combination of SAHA and TRAIL gene therapy could be a potential strategy for cancer treatment.     

Barriers to Non-Viral Vector-Mediated Gene Delivery in the Nervous System

Efficient methods for cell line transfection are well described, but, for primary neurons, a high-yield method different from those relying on viral vectors is lacking. Viral transfection has several drawbacks, such as the complexity of vector preparation, safety concerns, and the generation of immune and inflammatory responses when used in vivo. However, one of the main problems for the use of non-viral gene vectors for neuronal transfection is their low efficiency when compared with viral vectors. Transgene expression, or siRNA delivery mediated by non-viral vectors, is the result of multiple processes related to cellular membrane crossing, intracellular traffic, and/or nuclear delivery of the genetic material cargo. This review will deal with the barriers that different nanoparticles (cationic lipids, polyethyleneimine, dendrimers and carbon nanotubes) must overcome to efficiently deliver their cargo to central nervous system cells, including internalization into the neurons, interaction with intracellular organelles such as lysosomes, and transport across the nuclear membrane of the neuron in the case of DNA transfection. Furthermore, when used in vivo, the nanoparticles should efficiently cross the blood-brain barrier to reach the target cells in the brain.     

In vivo characteristics of cationic liposomes as delivery vectors for gene therapy

After a decade of clinical trials, gene therapy seems to have found its place between excessive ambitions and feasible aims, with encouraging results obtained in recent years. Intracellular delivery of genetic material is the key step in gene therapy. Optimization of delivery vectors is of major importance for turning gene therapy into a successful therapeutic method. Nonviral gene delivery relies mainly on the complexes formed from cationic liposomes (or cationic polymers) and DNA, i.e., lipoplexes (or polyplexes). Many lipoplex formulations have been studied, but in vivo activity is generally low compared to that of viral systems. This review gives a concise overview of studies on the application of cationic liposomes in vivo in animal models of diseases and in clinical studies. The transfection efficiency, the pharmacokinetic and pharmacodynamic properties of the lipid-DNA complexes, and potentially relevant applications for cationic liposomes are discussed. Furthermore, the toxicity of, and the induction of an inflammatory response in association with the administration of lipoplexes are described. Increasing understanding of lipoplex behavior and gene transfer capacities in vivo offers new possibilities to enhance their efficiency and paves the path to more extensive clinical applications in the future.     

Temperature-responsive cationic block copolymers as nanocarriers for gene delivery

Cationic block copolymers have been regarded as promising alternatives to the use of viral vectors for gene delivery. In this work, poly(N-isopropylacrylamide)_n-block-poly((3-acrylamidopropyl)trimethylammonium chloride)_m (PNIPAAM_n-b-PAMPTMA(+)_m) block copolymers with n = 48 or 65 and m = 6, 10 or 20 were synthesized and evaluated in terms of their potential for in vitro transfection of HeLa cells. These block copolymers collapse above a phase transition temperature, allowing the entrapment of the DNA molecules they are adsorbed to. The transfection efficiency increased with polymer concentration and was higher in the presence of a long PNIPAAM block and for a short charged block. In general, increasing the length of the charged block decreased the transfection efficiency. Additionally, polymer–DNA complexes (polyplexes) formed at lower polymer/DNA charge ratios caused lower cell toxicity levels. All polymers were shown to efficiently protect the DNA, even when they were present at low concentrations. At 37 °C, the polyplexes mostly formed structures with size ranging from 100 to 500 nm. The results also showed that the thermoresponsive contraction of PNIPAAM causes the charged block segments to be pressed out to the surface. The formation of compact structures seems to be a key factor in achieving high transfection efficiency.     

Comprehensive comparison of two new biodegradable gene carriers

Safety and high transfection efficiency are the prerequisites for an ideal gene vector. Polyethylenimine (PEI), especially PEI 25k (25 kDa), is a well-known cationic gene carrier with high transfection efficiency. However, the high toxicity, depended on its molecular weight, has limited its use as a potential gene carrier. In our research, for the purpose of reducing the toxicity and increasing the transfection efficiency and further to inspect where the degradation of these biodegradable polymers take place would be more beneficial, in cytoplasm or in endocytic vesicles, two kinds of degradable polymers were synthesized. One is an acid-liable PEI derivate (PEI-GA) which was cross-linked by PEI 2k with glutadialdehyde (GA) through imine linkages and the other one (PEI-TEG) was cross-linked PEI 2k with modified triethyleneglycol (TEG) through biscarbamate linkages and can be degraded at neutral environment. By the use of a series of assay methods both in vitro and in vivo, the results showed that PEI-TEG was found to be biodegradable at neutral environment and exhibit high transfection ability with low toxicity, which indicated its potential as a candidate carrier for gene therapy.