Low Molecular Weight Polyethylenimine Research Articles

Addition of free poloxamer 407 to a new gene vector P407-PEI-K12 solution forms a sustained-release in situ hypergel that enhances cell transfection and extends gene expression.

To address the concern around the efficiency/cytotoxicity ratio and the tumor-targeting effects of polyethylenimine (PEI), is a non-viral gene vector used for the delivery of the cancer therapy gene, poloxamer 407 (P407)-PEI-K12, was synthesized by cross-linking low-molecular weight PEI with P407 and further coupling a bifunctional peptide, K12, which is comprised of the tumor-targeting peptide tLyP-1 and the nuclear localization sequence. Furthermore, the addition of free P407 into the polymer/DNA complex solution produced a temperature-sensitive in situ gel-P407/P407-PEI-K12/DNA complex, which improved the effects of sustained-release gene delivery and transfection efficiency. The specificity, cytotoxicity and gene transfection efficiency of P407-PEI-K12 was investigated in Hela cells in vitro. The polymer efficiently prevented the degradation of plasmid DNA by DNase I and had a marked ability for serum tolerance. Agarose gel electrophoresis revealed that plasmid DNA was efficiently condensed and protected. The higher transfection efficiency of P407-PEI-K12h (the molar ratio of P407-PEI and K12 is 1:10) was achieved with a polymer and plasmid DNA ratio (w/w) of 20:1. The ability of free P407 to promote the transfection of the polymer/DNA complex was high (0.09%). The half-life of the P407/P407-PEI-K12-h/DNA gel complex was 228 min, and the transfection efficiency of the P407/P407-PEI-K12-h/DNA complex was markedly higher compared to that of the P407-PEI-K12-h/DNA complex at various release times.

MicroRNA Delivery with Bioreducible Polyethylenimine as a Non-Viral Vector for Breast Cancer Gene Therapy.

Polyethylenimines (PEIs) are outstanding macromolecules belonging to the polycations used in gene transfection. The transfection efficiency and cytotoxicity of PEIs increase with the increase in their molecular weight. To break up the correlation between transfection efficiency and cytotoxicity for non-viral gene delivery, disulfide cross-linked polyethylenimine (PEI-SS) has been widely employed as highly efficient gene vectors for DNA/siRNA delivery in numerous efforts. In this work, PEI-SS is described as a non-viral vector for miRNA delivery for the first time. PEI-SS is synthesized via cross-linking using disulfide bonds as the cross-linker from low molecular weight PEI. PEI-SS can efficiently bind anti-miR-155 to form the polyplex with nano-sized spherical structures in the size range of 10-100 nm. The polyplex is degraded by glutathione (GSH, a reducing agent) in cancer cells. Anti-miR-155 is then released to efficiently inhibit tumor growth.

Development of a Biocompatible Copolymer Nanocomplex to Deliver VEGF siRNA for Triple Negative Breast Cancer.

Triple negative breast cancer (TNBC) is the most difficult breast cancer subtype to treat. TNBC patients have significantly higher expression of vascular endothelial growth factor (VEGF) in tumors compared to non-TNBC patients. VEGF not only exerts its pro-angiogenic effects on endothelial cells but also acts as a survival and autocrine growth factor for VEGF receptor (VEGFR) expressing cancer cells. Silencing the expression of VEGF is therefore a potential therapy for TNBC.Methods: A novel biocompatible linear copolymer poly[bis(ε-Lys-PEI)Glut-PEG] (PLEGP) was developed to deliver VEGF siRNA for TNBC therapy. The copolymer is composed of lysine and glutaric acid, a natural metabolite of amino acids in the body. Low-molecular weight polyethyleneimine (PEI) was grafted to the copolymer to efficiently condense siRNA into nanocomplex without inducing cytotoxicity. Various in vitro studies were performed to evaluate the stability, cellular uptake, tumor penetration, and biological activities of the VEGF siRNA nanocomplex. The anti-tumor activities of the nanocomplex was also evaluated in an orthotopic TNBC mouse model.Results: PEIs with different molecular weights were evaluated, and the copolymer PLEGP1800 was able to easily form a stable nanocomplex with siRNAs and protect them from serum degradation. The siRNA/PLEGP1800 nanocomplex exhibited negligible cytotoxicity but showed high cellular uptake, high transfection efficiency, and high tumor penetration. In vitro activity studies showed that the siRNA nanocomplex significantly inhibited migration and invasion of TNBC cells. Moreover, the VEGF siRNA nanocomplex efficiently inhibited tumor growth in an orthotopic TNBC mouse model and down-regulated VEGF expression in the tumor.Conclusion: PLEGP1800 is a safe and efficient copolymer to deliver siRNAs for TNBC therapy. It could potentially be applied to other cancers by changing the cargo and incorporating tumor-specific ligands.

Zinc ion coordination significantly improved the transfection efficiency of low molecular weight polyethylenimine.

Polycationic gene carriers are attracting increasing attention for their use as gene delivery systems. Herein, a novel and high-performance polycationic gene carrier (i.e., Zn-DPPA2) was constructed by introducing a dipicolylamine compound along with zinc ion coordination onto polyethylenimine (PEI1.8k) via Schiff base bonds. Zn-DPPA2 exhibited efficient transfection and negligible cytotoxicity. Zn-DPPA2/DNA complexes possessed significant endocytosis efficiency and excellent endosomal escape ability. Additionally, this polycaitionic gene carrier showed an excellent ability as an antiserum. The nanoscale size and good size stability ensured that Zn-DPPA2/DNA could accumulate in tumor tissue through the enhanced permeability and retention (EPR) effect. Moreover, Zn-DPPA2/DNA complexes exhibited efficient transfection in tumor tissue after intravenous injection. This strategy provided a new idea for polycationic gene carrier construction, which has prospects for widespread application in the field of gene therapy.

Glutathione modified low molecular weight PEI for highly improved gene transfection ability and biocompatibility

A versatile oligopeptide, glutathione, was introduced to construct novel cationic gene vectors with further excellent transfection efficiency and serum tolerance.

ATP-Responsive Low-Molecular-Weight Polyethylenimine-Based Supramolecular Assembly via Host–Guest Interaction for Gene Delivery

In this work, we report on an ATP-responsive low-molecular-weight polyethylenimine (LMW-PEI)-based supramolecular assembly. It formed via host-guest interaction between PEI (MW = 1.8 kDa)-α-cyclodextrin (α-CD) conjugates and PEI1.8k-phenylboronic acid (PBA) conjugates. The host-guest interaction between PEI1.8k-α-CD and PEI1.8k-PBA was confirmed by the 2D-NOESY chromatogram experiment and competition test. The ATP-responsive property of the supramolecular assembly was evaluated by a series of ATP-triggered degradation and siRNA release studies in terms of fluorescence resonance energy transfer, agarose gel electrophoresis assay, and the time course monitoring of the particle size and morphology. Confocal laser scanning microscopy confirmed the intracellular disassembly of the supramolecular polymer and the release of siRNA. The supramolecular assembly showed high buffering capability and was capable of protecting siRNA from RNase degradation. It had high cytocompatibility according to in vitro cytotoxicity and hemolysis assays. LMW-PEI-based supramolecular assembly facilitated cellular entry of siRNA via energy-dependent endocytosis. Moreover, the assembly/SR-A siRNA polyplexes at N/P ratio of 30 was most effective in knocking down SR-A mRNA and inhibiting uptake of modified LDL. Taken together, this work shows that ATP-responsive LMW-PEI-based supramolecular assembly is a promising gene vector and has potential application in treating atherosclerosis.

Low molecular weight PEI-based fluorinated polymers for efficient gene delivery

Fluorinated biomaterials have been reported to have promising features as non-viral gene carriers. In this study, a series of fluorinated polymeric gene carriers were synthesized via Michael addition from low molecular weight polyethyleneimine (PEI) and fluorobenzoic acids (FBAs)-based linking compounds with different numbers of fluorine atoms. The structure-activity relationship (SAR) of these materials was systematically investigated. SAR studies showed that fluorine could screen the positive charge of these polymers. However, this shielding effect of fluorine would endow fluorinated polymers with good balance between DNA condensation and release. In vitro transfection results suggested that these fluorinated polymers could mediate efficient gene delivery. Flow cytometry and confocal microscopy studies demonstrated that more efficient cell uptake could be achieved by fluorinated materials with more fluorine atoms. Cytotoxicity assays showed that these fluorinated materials exhibited very low cytotoxicity even at high mass ratios. This study demonstrates that FBA-based fluorinated biopolymers have the potential for practical application.

Facile Gene Delivery Derived from Branched Low Molecular Weight Polyethylenimine by High Efficient Chemistry.

Convenient methods for the preparation of gene delivery platforms based on branched low molecular weight polyethylenimine (PEI) were described. Firstly, PEI lipids, with a low molecular weight PEI headgroup and hexadecyl chain tail group, were prepared through a highly efficient ring-opening reaction of glycidyl hexadecyl ether (EpoxyC16) by amine from PEI. Then, the PEI lipids were used as a component of cationic liposomes and as a surfactant for the preparation of poly(D,L-lactide-co-glycolide) (PLGA) nanoparticle (NP) via solvent extraction/evaporation method. As potential effective gene delivery platforms, their preparation, size, size distribution, toxicities, plasmid DNA loading, in vitro transfection and intracellular trafficking were studied. Both facile platforms showed less toxicity and higher transfection efficacy when compared to high molecular weight PEI in vitro, and may have further versatile applications in the gene delivery field.

PH-Responsive Cross-Linked Low Molecular Weight Polyethylenimine as an Efficient Gene Vector for Delivery of Plasmid DNA Encoding Anti-VEGF-shRNA for Tumor Treatment.

RNA interference (RNAi) is a biological process through which gene expression can be inhibited by RNA molecules with high selectivity and specificity, providing a promising tool for tumor treatment. Two types of molecules are often applied to inactivate target gene expression: synthetic double stranded small interfering RNA (siRNA) and plasmid DNA encoding short hairpin RNA (shRNA). Vectors with high transfection efficiency and low toxicity are essential for the delivery of siRNA and shRNA. In this study, TDAPEI, the synthetic derivative of low-molecular-weight polyethylenimine (PEI), was cross-linked with imine bonds by the conjugation of branched PEI (1.8 kDa) and 2,5-thiophenedicarboxaldehyde (TDA). This biodegradable cationic polymer was utilized as the vector for the delivery of plasmid DNA expressing anti-VEGF-shRNA. Compared to PEI (25 kDa), TDAPEI had a better performance since experimental results suggest its higher transfection efficiency as well as lower toxicity both in cell and animal studies. TDAPEI did not stimulate innate immune response, which is a significant factor that should be considered in vector design for gene delivery. All the results suggested that TDAPEI delivering anti-VEGF-shRNA may provide a promising method for tumor treatment.

Charge and Assembly Reversible Micelles Fueled by Intracellular ATP for Improved siRNA Transfection.

Hydrophobic modification on polycations were commonly used to improve the stability and transfection efficiency of polyplexes. However, the improved stability often means undesired release of the encapsulated siRNA, limiting the application of cationic micelles for siRNA delivery. The current strategy of preparing bioresponsive micelles based on the cleavage of sensitive linkages between polycation and hydrophobic part was far from sufficient, owing to the siRNA binding of the separated polycations from micelles leading to the incomplete release of siRNA. In this study, we propose a new strategy by the combination of micelles disassembly and separated polycations charge reversal. FPBA (3-fluoro-4-carboxyphenylboronic acid) grafted PEI 1.8 k (polyethylenimine) as the polycations of PEI-FPBA and dopamine (with diol-containing moiety) conjugated with cholesterol as the hydrophobic part (Chol-Dopa). The PFCDM micelles was assembled by PEI-FPBA and Chol-Dopa, based on the FPBA-Dopa conjugation. The prepared PFCDM showed strong siRNA loading ability and superior stability in the presence of PBS or serum. Besides, the PFCDM exhibited excellent ATP sensibility. The intracellular ATP could effectively trigger the disassembly of micelles and charge reversal of PEI-FPBA, resulting in the burst release of siRNA in the cytosol. With the property of extracellular stability and intracellular instability, PFCDM displayed good performance on in vitro and in vivo luciferase silencing on 4T1 cells. It should also be noted that the assembly of low molecular weight PEI was relatively safe to cells compared with 25 k PEI. To sum up, the ATP-fueled assembly and charge reversible micelles gave examples for polyplexes to achieve better stability and on demand cargo release at the same time and shows potential to be used for in vitro and in vivo siRNA transfection.

Inhibition of Tumor Growth via Systemic siRNA Delivery Using Reducible Bile Acid-Conjugated Polyethylenimine.

RNA interference (RNAi), mediated by small interfering RNA (siRNA), has been considered as a potential therapeutic agent for cancer owing to its ability to suppress target genes in a sequence-specific manner. In this study, a conjugate of the low molecular weight (MW) polyethylenimine (PEI) (MW 1800) and deoxycholic acid (DA) was further modified with 4-fluorothiophenol (FTP) (TP-DA-PEI) to achieve systemic siRNA delivery. The thiophenol group would be involved with disulfide bonds between the polymer chains and siRNA modified with free thiols (thiol-siRNA) to form and π–π interactions between the pendent aromatic groups and coprostane ring of the bile acid. The TP-DA-PEI conjugates could generate stable nanoparticles with thiol-siRNA. The TP-DA-PEI conjugate not only achieved enhanced intracellular uptake, serum stability, and transfection efficiency, but also showed high accumulation of TP-DA-PEI/thiol-siRNA polyplexes and significant tumor growth inhibition effect in tumor-bearing mice after systemic administration.

Aromatic Thioacetal-Bridged ROS-Responsive Nanoparticles as Novel Gene Delivery Vehicles.

In this report, a series of polycations are designed and synthesized by conjugating reactive oxygen species (ROS)-responsive thioacetal-linkers to low molecular weight (LMW) polyethylenimine (PEI) via ring-opening polymerization. Their structure–activity relationships (SARs) as gene delivery vectors are systematically studied. Although the MWs of the target polymers are only ~9 KDa, they show good DNA binding ability. The formed polyplexes, which are stable toward serum but decomposed under ROS-conditions, have appropriate sizes (180~300 nm) and positive zeta-potentials (+35~50 mV). In vitro experiments reveal that these materials have low cytotoxicity, and higher transfection efficiency (TE) than controls. Furthermore, the title polymers exhibit excellent serum tolerance. With the present of 10% serum, the TE of the polymers even increases up to 10 times higher than 25 KDa PEI and 9 times higher than Lipofectamine 2000. The SAR studies also reveal that electron-withdrawing groups on the aromatic ring in 4a may benefit to balance between the DNA condensation and release for efficient gene transfection.

Amine-impregnated porous nanofiber membranes for CO2 capture

Global warming is a worldwide issue, mainly caused by excessive emission of carbon dioxide (CO2), thus, demands the development of robust and flexible sorbents with consider CO2 capturing capacity. Herein, we report an easy and facile two-step fabrication of hierarchically structured and highly porous polystyrene (PS)/polyurethane (PU) electrospun nanofiber membrane impregnated with various amines for post-combustion CO2 capture. Synthesized porous nanofiber membranes have highly flexible and strong mechanical characteristics. The comparative analysis of porous membranes functionalized with three functional amines shows that low molecular weight polyethyleneimine (PEIL) impregnated porous nanofiber membranes offer relatively higher CO2 adsorption (1.64 mmol/g) at 40 °C. In addition, optimized samples exhibit excellent regenerability, (≥ 90% of their original value) even after 19 adsorption-desorption cycles showing a stable and durable CO2 capture performance. This work paves the way for developing electrospun nanofiber membranes capable of efficient CO2 adsorption.

SiRNA/lipopolymer nanoparticles to arrest growth of chronic myeloid leukemia cells in vitro and in vivo

Therapies for the treatment of Chronic Myeloid Leukemia and other leukemias are still limited for patients at advanced stages, which allow development of point mutations in the BCR-ABL fusion gene that render CML cells insensitive to therapies. An effective non-viral delivery system based on lipopolymers is described in this study to deliver specific siRNAs to CML cells for therapeutic gene silencing. The lipopolymer, based on the lipid α-linolenic acid (αLA) substitution on low molecular weight polyethyleneimine (PEI), was used to deliver siRNA against the BCR-ABL gene and, the resultant therapeutic effect was evaluated in in vitro and in vivo CML models. The study concluded that siRNA/PEI-αLA nanoparticles enabled silencing of the BCR-ABL gene and BCR-ABL protein, which consequently reduced growth on CML K562 cells in vitro and arrested the growth of localized tumors in a localized CML mouse model. The results from this study confirmed the potential use of lipopolymers as delivery systems and are encouraging for the future design of non-viral delivery systems for the treatment of CML and other hematological malignancies resulting from gene fusions.

Calcium enhances gene expression when using low molecular weight poly-l-lysine delivery vehicles

Four molecular weights of poly-l-lysine (PLL) [1000–5000 Da, 1500–8000 Da, 4000–15,000 Da and 15,000–30,000 Da] and three molecular weights of polyethyleneimine (PEI) [800 Da, 2,000 Da, and 25,000 Da] were used to systematically study the effect of calcium (Ca2+) to improve transfection efficiency of polyelectrolyte complexes. Complexes made using different molecular weights of PLL or PEI polymer showed clear differences in the levels of gene expression in the presence and absence of calcium chloride when tested using A549 human lung carcinoma cells. Complexes formed from PLL or PEI 800 Da were exhibited negligible expression of pDNA according to a luciferase reporter assay. Low molecular weight PLL and PEI 800 Da, however, became highly efficient gene delivery vehicles when calcium was added to the nascent complexes while maintaining the low cytotoxicity of low molecular weight polyamines. Additional analyses indicated that the most effective formulations utilized a threshold level of calcium, which created small, stable particles, but also facilitated unpackaging of the gene complexes and release of pDNA.

Effects of extracts prepared from modified porous poly(ether imide) microparticulate absorbers on cytotoxicity, macrophage differentiation and proinflammatory behavior of human monocytic (THP-1) cells.

Remaining uremic toxins in the blood of chronic renal failure patients represent one central challenge in hemodialysis therapies. Highly porous poly(ether imide) (PEI) microparticles have been recently introduced as candidate absorber materials, which show a high absorption capacity for uremic toxins and allow hydrophilic surface modification suitable for minimization of serum protein absorption. In this work, the effects of extracts prepared from PEI microparticles modified by nucleophilic reaction with low molecular weight polyethylene imine (Pei) or potassium hydroxide (KOH), on human monocytic (THP-1) cells are studied. The obtained results suggested that the extracts of Pei and KOH modified PEI absorbers have no negative effect on THP-1 cell viability and do not initiate the critical differentiation towards macrophages. The extracts did not enhance transcript or protein levels of investigated proinflammatory markers in THP-1 cells, namely, TNFμ, MCP1, IL6 and IL8. Based on these findings such modified PEI microparticles should be qualified for further pre-clinical evaluation i.e. in an in vivo animal experiment.

Lower-Molecular-Weight Chitosan-Treated Polyethyleneimine: a Practical Strategy For Gene Delivery to Mesenchymal Stem Cells

Background/Aims: Genetic modification of mesenchymal stem cells (MSCs) is an essential requirement for their use as a delivery vehicle. To achieve higher transfection efficiency and better reproducibility than previously synthesized chitosan (100 kDa)-polyethylenimine (PEI; 1200 Da), we synthesized a low molecular weight PEI (1200 Da)-grafted chitosan (50 kDa) (CP). Methods: Safety of CP/DNA or PEI (25 kDa)/DNA was evaluated by an MTT assay using A549 cells or MSCs and a zebrafish embryo model. Effects of CP/DNA on the characteristics of MSCs were evaluated using flow cytometry. Additionally, a pGL3 plasmid was used to investigate the transfection efficiency of PEI (25 kDa), chitosan (100 kDa)-PEI (1200 Da), and CP with different N/P mass ratios on A549 cells and MSCs. Furthermore, CP/pGL3 was used to investigate the effect of serum on transfection, and intracellular transport was assessed by observing the intracellular location of DNA using laser scanning confocal microscopy. In addition, the effect of endocytosis on transfection efficiency was evaluated using A549 cells pre-treated with different inhibitors. Investigations related to analysis of transfection efficiency were all performed using the BCA protein assay to standardize the data. Furthermore, TGF-β1-and CXCR4-expressing plasmids were applied to evaluate the gene transfer efficiency of CP, including its effects on the osteogenic differentiation and migratory ability of MSCs. Results: The safety evaluation demonstrated that CP/DNA had significantly lower toxicity than PEI (25 kDa)/DNA. Additionally, DNA entered MSCs transfected by CP without changing their properties, while the examination of intracellular transport demonstrated that CP/pGL3 was internalized rapidly into MSCs. Furthermore, studies of the internalization mechanism showed that CP/pGL3 complexes entered the cells through caveolae-mediated endocytosis, thereby suggesting that the CP coating helped DNA enter A549 cells without the requirement for receptors. Compared to PEI (25 kDa), the interference of serum on transfection was reduced significantly with the use of CP in both A549 cells and MSCs. To evaluate the effects of gene delivery using the constructed CP complex and the possibility of obtaining gene-engineered MSCs, TGF-β1- and CXCR4-expressing plasmids were successfully delivered into MSCs, confirming their ability to induce osteogenesis and change the migratory ability of MSCs, respectively. Conclusion: These results demonstrated that CP could be used to deliver genes into MSCs and could potentially be used in gene therapy based on MSCs.

Targeting death receptors for drug-resistant cancer therapy: Codelivery of pTRAIL and monensin using dual-targeting and stimuli-responsive self-assembling nanocomposites

Chemoresistance remains a formidable hurdle against cancer therapy. Seeking for novel therapy strategies is an urgent need for those who no longer benefit from chemotherapy. Chemoresistance is usually associated with the dysfunction of intrinsic apoptosis. Targeting extrinsic apoptosis via TRAIL signaling and the death receptors could be a potential solution to treat chemoresistant cancer. A highly biocompatible nano system for codelivery of the TRAIL DNA and the death receptor sensitizer monensin was developed, in which low-molecular-weight PEI (LMW-PEI) was crosslinked by the sulfhydryl cyclodextrin via disulfide bonds, and then bound with DNA, thus forming the bioreducible polyplex cores. In addition, the cyclodextrin also functioned as a carrier for the hydrophobic monensin via host-guest inclusion. Poly-γ-glutamic acid (γ-PGA) was used to modify the polyplex core via charge interaction. The γ-PGA corona can specifically bind with the tumor-associated gamma-glutamyl transpeptidase (GGT) overexpressed on the tumor cells, and achieve tumor-targeting delivery. Moreover, the tumor-homing peptide RGD-modified γ-PGA was also prepared as the surface coating materials for further improving gene delivery efficiency. This gene delivery system was characterized by the dual ligand-targeting, dual stimuli-responsive features. The ligands of RGD and γ-PGA can target the tumor-associated receptors (i.e., integrin and GGT). The conformation of γ-PGA is pH-sensitive, and the tumor acidic micro environments could trigger the detachment of surface-coating γ-PGA. The disulfide crosslinking LMW-PEI is redox-sensitive, and its fast disassembling in the tumor cells could favor the efficient gene delivery. The anti-tumor efficacy was demonstrated both in vitro and in vivo. Moreover, MYC-mediated synthetic lethality could be an important mechanism for overcoming the drug resistance. An important finding of our studies is the demonstration of the in vivo treatment efficacy of TRAIL/monensin, thus providing a potential novel therapeutic strategy for overcoming drug-resistant cancer.

Ring-opening polymerization of diepoxides as an alternative method to overcome PEG dilemma in gene delivery

Gene therapy has shown great potential in the treatment of various diseases, and the development of low-toxic and efficient gene carriers is still one of the burning issues for gene therapy. PEGylation is generally regarded as an efficient strategy to improve the biocompatibility of non-viral vectors. However, PEGylation also led to several problems such as lower cellular uptake, difficult endosomal escape and much reduced transfection efficiency, which was described as “PEG dilemma”. We herein devote to the development of polymeric gene vectors that can overcome such dilemma via diepoxide ring-opening polymerization (DEROP) of low molecular weight PEI, and unmodified PEI and PEG-grafted PEI were used for comparison. Transfection experiments with various concentrations of serum revealed that the DEROP polymers gave much higher transfection efficiency together with better serum tolerance than PEI and PEGylated ones. Protein adsorption and flow cytometry assays further proved their better serum resistance. Besides, confocal microscopy suggested that the polyplexes formed from these polymers could escape from endosome/lysosome more efficiently than those derived from PEGylated material. In vivo biochemical studies also reflect good biocompatibility of the DEROP polymers. Results demonstrate that such polymer construction strategy gave cationic materials with both high transfection efficiency and improved biocompatibility, and afforded an effective way to overcome PEG dilemma.

Tween 85-Modified Low Molecular Weight PEI Enhances Exon-Skipping of Antisense Morpholino Oligomer InVitro and in mdx Mice.

We investigated a series of Tween 85 modified low molecular weight polyethylenimine (LPEI, 0.8k/1.2k/2.0k)-copolymers (Zs) through simple formulation and covalent conjugation with phosphorodiamidate morpholino oligomer (PMO) for their potential to enhance delivery in vitro and in dystrophic mdx mice. Z polymers significantly enhanced PMO-induced exon-skipping in a GFP reporter-based cell culture system. Application of optimized formulations of Zs with PMO targeted to dystrophin exon 23 demonstrated a significant increase in exon-skipping efficiency in mdx mice. Consistent with our observations in vitro, optimization of molecular size and hydropholic-lipopholic balance (HLB) of polymers are important factors to achieve enhanced PMO delivery in vivo. The best formulation of Zs enhanced PMO delivery with 20- and 6-fold over PMO alone in vitro and in vivo, respectively. Further, chemical conjugation of the polymer and PMO exhibits greater benefit than polymer/PMO simple formulation in PMO delivery efficiency. Observed cytotoxicity of the Zs was lower than Endo-porter and PEI 25k in vitro, and no tissue toxicity was clearly detected with the Zs at the dosage tested. These results indicate the potential of the Zs as effective and safe PMO delivery carriers for treating diseases such as muscular dystrophy.