Potential Gene Carriers Research Articles

Inclusion of TAT and NLS sequences in lipopeptide molecules generates homogenous nanoparticles for gene delivery applications

PurposesThe objective of this study is to develop a versatile gene carrier based on lipopeptides capable of delivering genetic material into target cells with minimal cytotoxicity. MethodsTwo lipopeptide molecules, palmitoyl-CKKHH and palmitoyl-CKKHH-YGRKKRRQRRR-PKKKRKV, were synthesized using solid phase peptide synthesis and evaluated as transfection agents. Physicochemical characterization of the lipopeptides included a DNA shift mobility assay, particle size measurement, and transmission electron microscopy (TEM) analysis. Cytotoxicity was assessed in CHO-K1 and HepG2 cells using the MTT assay, while transfection efficiency was determined by evaluating the expression of the green fluorescent protein-encoding gene. ResultsOur findings demonstrate that the lipopeptides can bind, condense, and shield DNA from DNase degradation. The inclusion of the YGRKKRRQRRR sequence, a transcription trans activator, and the PKKKRKV sequence, a nuclear localization signal, imparts desirable properties. Lipopeptide-based TAT-NLS/DNA nanoparticles exhibited stability for up to 20 days when stored at 6–8 °C, displaying uniformity with a compact size of approximately 120 nm. Furthermore, the lipopeptides exhibited lower cytotoxicity compared to the poly-L-lysine. Transfection experiments revealed that protein expression mediated by the lipopeptide occurred at a charge ratio ranging from 4.0 to 8.0. ConclusionThese results indicate that the lipopeptide, composed of a palmitoyl alkyl chain and TAT and NLS sequences, can efficiently condense and protect DNA, form stable and uniform nanoparticles, and exhibit promising characteristics as a potential gene carrier with minimal cytotoxicity.

Deterioration of sludge characteristics and promotion of antibiotic resistance genes spread with the co-existing of polyvinylchloride microplastics and tetracycline in the sequencing batch reactor

With the continuous increase in microplastics (MPs) and tetracycline (TC) entering wastewater treatment plants (WWTPs) along with sewage, the co-existence of MPs and TC in the biological treatment of wastewater has attracted extensive attention. This study investigated the effect of 1 mg/L polyvinyl chloride (PVC) MPs and 100 ng/L TC co-existing on sequencing batch reactors (SBRs) (S2) treating phenol wastewater in contrast to the control with TC alone (S1). The phenol removal efficiency was significantly inhibited by the co-existence of PVC MPs and TC. Sludge characteristics were also distinctively influenced. The decreased zone sludge velocity (ZSV) and increased sludge volume index (SVI) indicated that the combined effect of PVC MPs and TC deteriorated sludge settleability, which had positive and negative linear correlations with extracellular polymeric substances (EPS) content and the protein (PN)/polysaccharide (PS) ratio, respectively. Moreover, the decreased and increased relative abundances of potential phenol-degraders and antibiotic resistance gene (ARG) carriers may elucidate the inhibition of phenol removal and promotion of ARGs propagation with the co-occurrence of PVC MPs and TC. In addition, the enhanced potential ARGs hosts, loss of the EPS protective effect, and increased membrane permeability induced by reactive oxygen species (ROS) jointly promoted ARGs dissemination in the co-existence of PVC MPs and TC. Notably, the co-occurrence of ARGs and mobile genetic element (MGEs) indicated that the co-existence of PVC MPs and TC promoted the spread of some transposase-associated ARGs mediated by horizontal gene transfer (HGT).

Metal-Organic Frameworks: Versatile Platforms for Biomedical Innovations.

This review article explores the multiple applications and potential of metal-organic frameworks (MOFs) in the biomedical field. With their highly versatile and tunable properties, MOFs present many possibilities, including drug delivery, biomolecule recognition, biosensors, and immunotherapy. Their crystal structure allows precise tuning, with the ligand typology and metal geometry playing critical roles. MOFs' ability to encapsulate drugs and exhibit pH-triggered release makes them ideal candidates for precision medicine, including cancer treatment. They are also potential gene carriers for genetic disorders and have been used in biosensors and as contrast agents for magnetic resonance imaging. Despite the complexities encountered in modulating properties and interactions with biological systems, further research on MOFs is imperative. The primary focus of this review is to provide a comprehensive examination of MOFs in these applications, highlighting the current achievements and complexities encountered. Such efforts will uncover their untapped potential in creating innovative tools for biomedical applications, emphasizing the need to invest in the continued exploration of this promising field.

Biodistribution of nanodiamonds is determined by surface functionalization

Solid-core nanoparticles made of carbon, metals and other inorganic materials are gaining interest as nanocarriers to facilitate nucleic acid therapeutics. Our group designed amino acid-functionalized nanodiamonds (NDs, collectively fNDs) to serve this purpose via covalent conjugation of basic amino acid lysine and di-peptide lysyl-histidine on the ND surface. The purpose of this study is to elucidate the functionalization-dependent biodistribution profiles of fNDs, a key step in the process of developing these novel nano-systems. Here the aim was two-fold i.e., to establish an optimized protocol for radiolabeling of fNDs and to study the functionalization-dependent differences in pharmacokinetic behavior of NDs. Both types of fNDs yielded a surface loading of 1.94 and 14.45 mmol/g of lysine and lysyl-histidine amino acid conjugates respectively. Deferoxamine (DFO) was conjugated directly on the ND surface for the first time to chelate zirconium (89Zr), the radiolabel of interest for in vivo tracking. To achieve an effective balance between radiolabeling efficiency, surface chemistry and colloidal stability of DFO-conjugated fNDs, attachment of DFO was evaluated in the range of 1–8 %. A 3 % loading with DFO was sufficient to chelate 2 MBq 89Zr with good radiochemical yield (≥90 %) while maintaining the physicochemical characteristics of the fNDs, namely particle size ≤300 nm and overall positive zeta potential of higher than 15 mV. Furthermore, 89Zr-labeled fNDs remain stable in phosphate buffered saline and mouse serum for over 96 h maintaining a radiochemical stability of ≥97 %. Following process optimization, we evaluated differential organ distribution and pharmacokinetic profile of 89Zr-labeled fNDs. All NDs showed the highest uptake in the liver, followed by the spleen. However, the degree of uptake and retention time in the liver varied based on the surface functionalization. The lysyl-histidine-NDs showed the highest AUC, followed by the carboxylated NDs and the lysine-NDs. Unlike lysine-NDs, which depleted quickly from the liver (marked decline in ND-associated radioactivity from 5.6 to 2.3 %), the lysyl-histidine-NDs maintained a longer residence for at least 72 h in the liver marked by ND-associated radioactivity of 8.9 % after 6 h that increased further to 11.7 % after 72 hours post injection. Overall, this work presents a proof-of-concept to study the effect of surface characteristics on the in vivo behavior and organ deposition of NDs. Here, we successfully established an optimized protocol to achieve radiolabeling of NDs while maintaining the optimum physicochemical traits of fNDs to act as potential gene carriers. Using these approaches, we elucidated that the biodistribution and the pharmacokinetic behavior of NDs varies based on the functionalization design.

Fish scales : Waste processing as a potential source of bioactive compounds

The rise of fish processing over the years has led to an upsurge of by-products like fish scales, bones and skin which are often considered as trash and discarded. Study on two most common carp fish in Indian freshwater, Catla catla and Labeo rohita revealed some valuable by- products from nonedible fish scales such as type I collagen, gelatin, hydroxyapatite, chitin and chitosan. The remarkable antioxidant effects of fish gelatin isolated from fish scale is of great pharmaceutical importance in addition to type I collagen which is utilized for implants to culture skin cells or as drug carrier for restoration of skin. Collagen has haemostatic characteristic and facilitate blood coagulation as well as tissue repair. A natural biocompatible component of fish scale that coexists with collagen protein is hydroxyapatite. Hydroxyapatite is also used in controlled drug release by pharmaceutical industry and hip replacement surgery. Currently chitosan obtained from chitin present in fish by-product is gaining popularity as a potential drug candidate and gene carrier for anticancer chemotherapy. Taken altogether, our study highlights the importance and method of extraction of different by-products of Indian fresh water fishes in health science.

Dermoscopy in the Diagnostics of Incontinentia Pigmenti Skin Lesions.

IntroductionIncontinentia pigmenti (IP) is a rare X-linked geno-dermatosis characterized by numerous findings. Skin biopsy and histopathological analysis are considered as minor criteria for the diagnosis of IP. We assume that dermoscopy can assist the earlier diagnosis of IP.ObjectivesTo gain experience in earlier diagnosis of IP by observing dermoscopic findings of cutaneous changes.MethodsWe revised confirmed cases of IP and examined them using dermoscopy, comparing histopathological and dermoscopic results.ResultsStage I presented solitary and grouped vesicles in linear arrangement on erythematous skin. Early stage II presented star-shaped verrucous lesions on erythematous or pigmented skin. In well-developed lesions, dotted vessels surround keratotic part, some with thrombosed capillaries, resembling a viral wart. Stage III presented linear brown dots on the pigmented areas. Dermoscopic image was uniform in all the examined pigmented Blaschko linear changes. Stage IV presented numerous dotted vessels on the hypopigmented skin. Terminal hair was scarce or absent in all four stages. The surrounding normal skin had perifollicular depigmentations in stages III and IV.ConclusionsDermoscopy of all four stages is very specific compared to the dermoscopy of inflammatory dermatoses and pigmentations. Stage III has very close clinical, histological and dermoscopic mimickers and needs to be carefully examined with obligatory genetic testing. Dermoscopy of the stage IV closely corresponds to histopathological findings and may be crucial as a quick tool in revealing potential IP gene carriers. Dermoscopy should be used in addition to clinical examination since the two methods are complementary.

Preparation and characterization of polyamidoamine dendrimers conjugated with cholesteryl-dipeptide as gene carriers in HeLa cells

Improving the transfection efficiency of non-viral carriers by using cationic polymers is a useful approach to addressing several challenges in gene therapy, such as cellular uptake, endosomal escape, and toxicity. Among the various cationic polymers, polyamidoamine (PAMAM) dendrimers have been widely utilized because of the abundance of terminal functional groups, thereby enabling further functionalization and enhancing DNA condensation and internalization into cells. The combination of various functional groups is required for these PAMAM dendrimer derivatives to function appropriately for gene delivery. Herein, we synthesized PAMAM G2-HRChol by conjugating dipeptide (histidine-arginine) and cholesterol at different ratios (6% or 23%) on the surface of PAMAM dendrimer generation 2 (PAMAM G2). Both PAMAM G2-HRChol 6% and PAMAM G2-HRChol 23% have buffering capacity, leading to improved endosomal escape after entering the cells. PAMAM G2-HRChol 6% and PAMAM G2-HRChol 23% dendrimers were condensed with pDNA to form nano-polyplexes at a weight ratio of 4 (polymer/pDNA). Polyplexes are positively charged, which facilitates cellular uptake. The transfection efficiency of PAMAM G2-HRChol 6% and PAMAM G2-HRChol 23% dendrimers was similar to that of PEI 25 kDa under optimum conditions, and the cytotoxicity was much lower than that of PEI 25 kDa in HeLa cells. In addition, after apoptin gene transfection was performed, cell death ratios of 34.47% and 22.47% were observed for PAMAM G2-HRChol 6% and PAMAM G2-HRChol 23%, respectively. The results show that a suitable amount of cholesterol can improve gene transfection efficiency, and the PAMAM G2-HRChol 6% dendrimer could be a potential gene carrier in HeLa cells.

In Vitro Cellular Uptake and Transfection of Oligoarginine-Conjugated Glycol Chitosan/siRNA Nanoparticles.

Chitosan and its derivatives have been extensively utilized in gene delivery applications because of their low toxicity and positively charged characteristics. However, their low solubility under physiological conditions often limits their application. Glycol chitosan (GC) is a derivative of chitosan that exhibits excellent solubility in physiological buffer solutions. However, it lacks the positive characteristics of a gene carrier. Thus, we hypothesized that the introduction of oligoarginine peptide to GC could improve the formation of complexes with siRNA, resulting in enhanced uptake by cells and increased transfection efficiency in vitro. A peptide with nine arginine residues and 10 glycine units (R9G10) was successfully conjugated to GC, which was confirmed by infrared spectroscopy, 1H NMR spectroscopy, and elemental analysis. The physicochemical characteristics of R9G10-GC/siRNA complexes were also investigated. The size and surface charge of the R9G10-GC/siRNA nanoparticles depended on the amount of R9G10 coupled to the GC. In addition, the R9G10-GC/siRNA nanoparticles showed improved uptake in HeLa cells and enhanced in vitro transfection efficiency while maintaining low cytotoxicity determined by the MTT assay. Oligoarginine-modified glycol chitosan may be useful as a potential gene carrier in many therapeutic applications.

Metal-free multicomponent polymerization toward cationic polyamidines

Cationic polymers, also known as polycations, are considered to be the most potential non-viral gene carriers due to their unique advantages such as the ability to bind the negative charge of nucleic acid molecules. Multicomponent polymerization (MCP) is a one-step, tandem strategy to construct complex structures based on multicomponent reactions. Herein, we developed a metal-free MCP method based on three monomers of p-dinitrovinylbenzene (p-DNVB), 1,1-dimethylethyl N,N-dibromocarbamate (BocNBr2), and bis-secondary-amines with a ratio of 1:2:1, to access a library of Boc-substituted polyamidines with well-defined structures and suitable molecular weights (Mw ranging from 4400 Da to 11,000 Da) in high yields (up to 85%) under mild conditions. Upon the removal of Boc groups, a series of water-soluble polymers with cationic property were prepared and their gene binding capability was further evaluated.

Vimentin Targeted Nano-gene Carrier for Treatment of Renal Diseases.

BackgroundChronic kidney disease (CKD) is a global health problem, and there is no permanent treatment for reversing kidney failure; thus, early diagnosis and effective treatment are required. Gene therapy has outstanding potential; however, the lack of safe gene delivery vectors, a reasonable transfection rate, and kidney targeting ability limit its application. Nanoparticles can offer innovative ways to diagnose and treat kidney diseases as they facilitate targetability and therapeutic efficacy.MethodsHerein, we developed a proximal renal tubule-targeting gene delivery system based on alternative copolymer (PS) of sorbitol and polyethyleneimine (PEI), modified with vimentin-specific chitobionic acid (CA), producing PS-conjugated CA (PSC) for targeting toward vimentin-expressing cells in the kidneys. In vitro studies were used to determine cell viability, transfection efficiency, serum influence, and specific uptake in the human proximal renal tubular epithelial cell line (HK-2). Finally, the targeting efficiency of the prepared PSC gene carriers was checked in a murine model of Alport syndrome.ResultsOur results suggested that the prepared polyplex showed low cytotoxicity, enhanced transfection efficiency, specific uptake toward HK-2 cells, and excellent targeting efficiency toward the kidneys.ConclusionCollectively, from these results it can be inferred that the PSC can be further evaluated as a potential gene carrier for the kidney-targeted delivery of therapeutic genes for treating diseases.

Metallacarborane Complex Boosts the Rate of DNA Oligonucleotide Hydrolysis in the Reaction Catalyzed by Snake Venom Phosphodiesterase.

Antisense oligonucleotides conjugated with boron clusters (B-ASOs) have been described as potential gene expression inhibitors and carriers of boron for boron neutron capture therapy (BNCT), providing a dual-action therapeutic platform. In this study, we tested the nucleolytic stability of DNA oligonucleotides labeled with metallacarborane [(3,3’-iron-1,2,1’,2’-dicarbollide)(−1)]ate [Fe(C2B9H11)2] (FESAN) against snake venom phosphodiesterase (svPDE, 3’→5’-exonuclease). Contrary to the previously observed protective effect of carborane (C2B10H12) modifications, the B-ASOs containing a metallacarborane moiety at the 5’-end of the oligonucleotide chain were hydrolyzed faster than their parent nonmodified oligomers. Interestingly, an enhancement in the hydrolysis rate was also observed in the presence of free metallacarborane, and this reaction was dependent on the concentration of the metallacarborane. Microscale thermophoresis (MST) analysis confirmed the high affinity (Kd nM range) of the binding of the metallacarborane to the proteins of crude snake venom and the moderate affinity (Kd µM range) between the metallacarborane and the short single-stranded DNA. We hypothesize that the metallacarborane complex covalently bound to B-ASO holds DNA molecules close to the protein surface, facilitating enzymatic cleavage. The addition of metallacarborane alone to the ASO/svPDE reaction mixture provides the interface to attract freely floating DNA molecules. In both cases, the local DNA concentration around the enzymes increases, giving rise to faster hydrolysis. It was experimentally shown that an allosteric effect, possibly attributable to the observed boost in the 3’→5’-exonucleolytic activity of snake venom phosphodiesterase, is much less plausible.

Chiral Ru(ii) complexes act as a potential non-viral gene carrier for directional transportation to the nucleus and cytoplasm.

Guanine-rich DNA sequences can spontaneously fold into four-stranded structures called G-quadruplexes (G4s). G4s have been identified extensively in the promoter regions of several proto-oncogenes, including c-myc, as well as telomeres. G4s have attracted an increasing amount of attention in the field of nanotechnology because of their use as versatile building blocks of DNA-based nanostructures. In this study, we report the self-assembly of c-myc G-quadruplex DNA controlled by a pair of chiral ruthenium(ii) complexes coordinated by 2-(4-phenyacetylenephenyl)-1H-imidazo[4,5f][1,10]phenanthroline (PBEPIP), Λ-[Ru(bpy)2(PBEPIP)](ClO4)2 (Λ-RM0627, bpy = bipyridine) and Δ-[Ru(bpy)2(PBEPIP)](ClO4)2 (Δ-RM0627). Λ-RM0627 could promote the high-order self-assembly of c-myc G-quadruplex DNA into a nanowire structure, whereas Δ-RM0627 could induce DNA condensation into G-quadruplex aggregates. Moreover, in vitro studies on human liver carcinoma HepG2 cells showed that the nanowire of c-myc G-quadruplex DNA promoted by Λ-RM0627 could be localized in the nuclei of cells, whereas the nanoparticle of c-myc G-quadruplex DNA generated by Δ-RM0627 was taken up and localized in the cytoplasm. This study provides examples of the enantioselective self-assembly of G4 DNA molecules controlled by chiral ruthenium(ii) complexes and suggests the potential applications of assembled nanostructures as non-viral DNA vectors for gene therapy.

Dextran-based coacervate nanodroplets as potential gene carriers for efficient cancer therapy

The intractable toxicity of cationic polymers limits their applicability in gene transport and controlled release. In consideration of the good biocompatibility and biofunctionality of dextran, herein we design and synthesize two types of amino group-containing cationic copolymers based on dextran by the copolymerization of cationic monomers from dextran backbones. Additionally, allyl crosslinkers containing disulfide bonds were introduced into polymerization, that made the copolymer crosslinked by disulfide. The resultant coacervates were formed from the self-assembly of cationic coplymers and anionic genes, and redox-responsive disulfide branch points endow coacervates with reducing environment responsiveness. The in vitro experiments showed that the dextran-based coacervates were sensitive to the reducing environment and underwent cleavage, which resulted in an effective release, uptake, and transfection of the genes by 293T cells. In addition, dextran-based coacervates can be used to carry siRNA into cancer cells with a high transfection efficiency, demonstrating their potential applicability in treatment against cancer.

Development of a novel DNA delivery system based on rice bran polysaccharide-Fe(III) complexes

Metal complexes, as a type of potential non-virus gene carriers, have gained much attention due to their properties of high charge density and unique three-dimensional structure. This study investigated the potential of polyethyleneimine (PEI)-modified rice bran polysaccharide-Fe(III) complex (PEI-PI) as a safe gene delivery system and explored the effect of Fe(III) on the efficiency of gene transfection mediated by PEI modified rice bran polysaccharide (PEI-P) and PEI-PI. Gel retardation assay was used to study the DNA binding and protection capability, MTT assay was performed to evaluate the biocompatibility, and PEI-PI complex-mediated EGFP gene transfection was studied in vitro. Results showed the PEI-PI could induce DNA condensation and protect DNA from degradation by DNase I at a low weight ratio (vector/DNA) of 2. At the same weight ratio, PEI exhibited the strongest DNA binding capability but PEI-PI exhibited the highest gene transfection efficiency among all carrier systems. Compared with the PEI-P + Fe(III) system, PEI-PI not only had a more significant capability to condense DNA but also presented higher gene transfection efficiency. Moreover, PEI-PI exhibited no obvious cytotoxicity to cells. This work provides a strategy for the design and development of gene vectors based on PI complexes.

Efficient gene delivery by oligochitosan conjugated serum albumin: Facile synthesis, polyplex stability, and transfection

Chitosan and its derivatives have shown to be potential gene carriers with biocompatiblility and safety. However, their practical delivery is far from being ideal because of the low transfection efficiency. The present work describes the potential of a natural protein, bovine serum albumin (BSA), conjugated with a natural oligosaccharide, oligochitosan (OC), as a considerable promising approach for a safe and efficient non-viral gene delivery vector. The FTIR spectra proved the effective conjugation of BSA with OC through covalent bond. The condensation ability of plasmid DNA (pDNA) with a BSA-OC biopolymer was analyzed by gel retardation assay, competition binding assay, and dynamic light scattering used to measure the nanoparticle size. In addition, the BSA-OC biopolymer showed the protection of pDNA from enzymatic degradation by DNase I and showed good stability when exposed to 50% fetal bovine serum. The transfection efficiency was evaluated in the presence of 10% serum-supplemented media or serum-free media on three kinds of mammalian cells. Our results showed that the BSA-OC biopolymer is a good non-viral vehicle for gene delivery. We investigated the parameters such as the pDNA payload, temperature, incubating duration, and biopolymer/pDNA ratio on the transfection efficiency. This hybrid vehicle had the ability to transfect 90% of cells and to maintain 80% of cell viability. The aforementioned results suggest that the facile synthesis of the BSA-OC biopolymer could overcome the cytotoxicity problem and transfection barriers during in vitro gene delivery.

Cationic starch/pDNA nanocomplexes assembly and their nanostructure changes on gene transfection efficiency

This study aims at developing biocompatible starch based gene carriers with good gene delivery and transfection efficacy. By controlling the molecular weight and aggregation behavior of spermine modified cationic starch (CS) molecules, nanocomplexes spontaneously formed through electrostatic interaction using CS and plasmid pAcGFP1-C1 (pDNA) displaying different structural changes (particle size, zeta potential, shape, compactness) response to the simulated intracellular pH variation. Results indicated that CS2 with weight average molecular weight (Mw) of 6.337 × 104 g/mol displayed relatively higher transfection efficacy (~30%) in HepG2 cells than others and revealed significantly low cytotoxicity. By simulating the intracellular pH variation, Dynamic Light Scattering (DLS) and Small Angle X-ray Scattering (SAXS) results demonstrated that CS2 could bind to pDNA tightly and form nanocomplexes with smaller and compact internal aggregate structure at acidic conditions, which facilitated the effective pDNA protection under endosome pH change, while larger and loose internal aggregate structure at physiological pH which promoted the disintegration of CS2/pDNA nanocomplexes. Therefore, CS with suitable Mw of around 6.0 × 104 g/mol represents a potential gene carrier for gene delivery. This study also demonstrated that controlling the internal nanostructure change of polymer/gene nanocomplexes could provide guidance in designing effective starch based gene carriers.

Drug Carrier Systems Based on Cyclodextrin Supramolecular Assemblies and Polymers: Present and Perspectives.

The pharmaceutical applications of cyclodextrins (CDs), cyclic oligosaccharides capable of including hydrophobic molecules inside their cavities, have been known for decades. Besides the solubilising and encapsulating abilities of natural and modified CDs due to the formation of inclusion complexes, there is an increasing interest in organized macrostructures based on CDs as potential drug delivery devices and gene carrier systems. The present review discusses first the case of drug carriers based on monomeric modified CDs (amphiphilic and CD core-star polymers), in which self-assembly plays a major role. Polyrotaxanes, i.e., CDs threaded onto a polymer chain, are then reviewed in relation to their pharmaceutical applications. Finally, covalently linked CDs, either by grafting or crosslinking, are analyzed, including more complex structures formed by assembling CDcontaining networks or chains. We have tried along this review to cover the most recent developments on these structures for drug delivery in a "beyond the cyclodextrin" approach. The review will be helpful, both for readers who want to be introduced into the world of these remarkable structures, or for specialists who are doing research in this field.

Development of a gene carrier using a triblock co-polyelectrolyte with poly(ethylene imine)-poly(lactic acid)-poly(ethylene glycol)

The success of gene therapy mainly depends on the carriers for effective gene delivery. A non-viral vector using a cationic block co-polyelectrolyte, PEI-PLA-PEG polyethyleneimine-poly(lactic acid)-poly(ethylene glycol)) was developed as a potential gene carrier. The cationic PEI-PLA-PEG showed less toxicity compared to PEI and formed a gene nanocomplex (termed polyplex) by interaction with plasmid DNA or small interference RNA. The polyplex showed smaller particle size and greater positive zeta potential by increasing the high polymer nitrogen/DNA phosphate ratio. The polyplex with a nitrogen/DNA phosphate ratio of 16 or 32 demonstrated higher gene transfection by fluorescence imaging, flow cytometry measurement, and β-galactosidase activity. In particular, the polyplex with therapeutic histone deacetylase small interference RNA at nitrogen/DNA phosphate ratio 16 showed the most favorable properties with definite tumor growth inhibition. The synthetic PEI-PLA-PEG also showed less toxicity and would, therefore, be a great potential gene carrier, particularly given that small interference RNA delivery does not increase the charge density of small interference RNA due to the formation of a stable complex through conjugation with PLA-PEG.

Poly(L-lysine)-based cylindrical copolypeptide brushes as potential drug and gene carriers

Cylindrical copolypeptide brush PLLF-g-(PLF-b-PLL), consisting of a poly(L-lysine-co-L-phenylalanine) (PLLF) backbone and poly(L-phenylalanine)-b-poly(L-lysine) (PLF-b-PLL) amphiphilic block copolypeptide side chains, has been synthesized, characterized, and evaluated as drug and gene carriers. PLLF-g-(PLF-b-PLL) brushes were synthesized by the multi-step ring-opening polymerization of amino acid N-carboxyanhydrides via a “grafting from” strategy. Transmission electron microscopy measurements and pyrene probe fluorescence study revealed that the synthesized copolypeptide brushes adopted nanosized cylindrical morphologies which resembled unimolecular polymeric micelles with a hydrophobic PLF core and a hydrophilic PLL periphery. An encapsulation study demonstrated that hydrophobic molecules (e.g., pyrene and oil red O) can be solubilized in the core of the copolypeptide brushes, while anionic hydrophilic molecules (e.g., rose bengal and methyl orange) can be entrapped into the brush periphery. Hydrophobic molecules and hydrophilic guests can be encapsulated simultaneously in a site-isolated state. Furthermore, the in vitro gene transfection efficiencies of copolypeptide brushes were evaluated in HEK293 and HeLa cells. The synthesized copolypeptide brushes exhibited low cytotoxicity and mediated efficient gene transfection. Our research suggested that these cylindrical copolypeptide brushes with a brush-like PLL periphery could be used as promising carriers for the codelivery of drugs and genes.

Self-assembly of c-myc DNA promoted by a single enantiomer ruthenium complex as a potential nuclear targeting gene carrier.

Gene therapy has long been limited in the clinic, due in part to the lack of safety and efficacy of the gene carrier. Herein, a single enantiomer ruthenium(II) complex, Λ-[Ru(bpy)2(p-BEPIP)](ClO4)2 (Λ-RM0627, bpy = 4,4′-bipyridine, p-BEPIP = 2-(4-phenylacetylenephenyl)imidazole [4,5f][1, 10] phenanthroline), has been synthesized and investigated as a potential gene carrier that targets the nucleus. In this report, it is shown that Λ-RM0627 promotes self-assembly of c-myc DNA to form a nanowire structure. Further studies showed that the nano-assembly of c-myc DNA that induced Λ-RM0627 could be efficiently taken up and enriched in the nuclei of HepG2 cells. After treatment of the nano-assembly of c-myc DNA with Λ-RM0627, over-expression of c-myc in HepG2 cells was observed. In summary, Λ-RM0627 played a key role in the transfer and release of c-myc into cells, which strongly indicates Λ-RM0627 as a potent carrier of c-myc DNA that targets the nucleus of tumor cells.