Novel biodegradable poly(disulfide amine)s for gene delivery with high efficiency and low cytotoxicity

Novel biodegradable poly(disulfide amine)s with defined structure, high transfection efficiency, and low cytotoxicity were designed and synthesized as nonviral gene delivery carriers. Michael addition between N, N'-cystaminebisacrylamide (CBA) and three N-Boc protected diamines ( N-Boc-1,2-diaminoethane, N-Boc-1,4-diaminobutane, and N-Boc-1,6-diaminohexane) followed by N-Boc deprotection under acidic condition resulted in final cationic polymers with disulfide bonds, tertiary amine groups in main chains, and pendant primary amine groups in side chains. Polymer structures were confirmed by 1H NMR, and their molecular weights were in the range 3.3-4.7 kDa with narrow polydispersity (1.12-1.17) as determined by size exclusion chromatography (SEC). Acid-base titration assay showed that the poly(disulfide amine)s possessed superior buffering capacity to branched PEI 25 kDa in the pH range 7.4-5.1, which may facilitate the escape of DNA from the endosomal compartment. Gel retardation assay demonstrated that significant polyplex dissociation was observed in the presence of 5.0 mM DTT within 1 h, suggesting rapid DNA release in the reduction condition such as cytoplasm due to the cleavage of disulfide bonds. Genetic transfections mediated by these poly(disulfide amine)s were side-chain spacer length dependent. The poly(disulfide amine) with a hexaethylene spacer, poly(CBA-DAH), had comparable transfection efficiency to bPEI 25 kDa in the tested cell lines, i.e., 293T cells, Hela cells, and NIH3T3 cells. This same poly(disulfide amine) mediated 7-fold higher luciferase expression than bPEI 25 kDa in C2C12 cells (mouse myoblast cell line), a cell line difficult to transfect with many cationic polymers. Furthermore, MTT assay indicated that all three poly(disulfide amine)s/pDNA polyplexes were significantly less toxic than bPEI/pDNA complexes.     

In vitro cytotoxicity of poly(amidoamine)s: relevance to DNA delivery

We have examined the cytotoxicity of a number of poly(amidoamine) polymers which have been proposed for use as DNA delivery systems and compared them to the charged polyamino acid polylysine. Most of the poly(amidoamine)s tested were shown to be remarkably non-toxic to both HepG2 and HL60 cell lines. However, one of the structures (NG30, co-monomers methylene bisacrylamide, dimethylethylene diamine) did show cytotoxicity similar to that of polylysine. A second PAA structure (NG37, NG38, NG39, co-monomers bisacryloyl piperazine, 2-methyl piperazine) showed mild cytotoxicity towards both cell lines, related to the degree of polymerisation. The results support the idea that the cytotoxicity of polycations has a strong structural basis rather than being an effect due only to charge. As a consequence of their general reduced level of cytotoxicity, poly(amidoamine)s appear to have possible advantages for complexation with DNA over some other cationic polymers as a key component of DNA delivery systems.     

Hyperbranched poly(amino ester)s with different terminal amine groups for DNA delivery

Hyperbranched poly(amino ester)s containing tertiary amines in the core and primary, secondary, and tertiary amines in the periphery, respectively, were evaluated for DNA delivery in vitro. The same core structure facilitated the investigation on the effects of the terminal amine type on the properties of hyperbranched poly(amino ester)s for DNA delivery. The hydrolysis of the poly(amino ester)s was monitored using (1)H NMR. The results reflected that the terminal amine type had negligible effects on the hydrolysis rate but was much slower than that of linear poly(amino ester)s, probably due to the compact hyperbranched spatial structure preventing the accessibility of water. In comparison with PEI 25 K, the hyperbranched poly(amino ester)s showed much lower cytotoxicity in Cos7, HEK293, and HepG2 cells. Gel electrophoresis indicated that poly(amino ester)s could condense DNA efficiently, and the zeta potentials and sizes of the complexes formed with different weight ratios of hyperbranched poly(amino ester)s and DNA were measured. Remarkably, all the hyperbranched poly(amino ester)s showed DNA transfection efficiency comparable to PEI 25 K in Cos7, HEK293, and HepG2 cells regardless of the terminal amine type. Therefore, the terminal amine type had insignificant effects on the hydrolysis rate, cytotoxicity, DNA condensation capability, and in vitro DNA transfection efficiency of the hyperbranched poly(amino ester)s.     

Evaluation of hyperbranched poly(amino ester)s of amine constitutions similar to polyethylenimine for DNA delivery

New hyperbranched poly(amino ester)s were synthesized via A3 + 2BB'B' ' approach, represented by the Michael addition polymerization of trimethylol-propane triacrylate (TMPTA) (A3-type monomers) with a double molar 1-(2-aminoethyl)piperazine (AEPZ) (BB'B'-type monomer) performed in chloroform at ambient temperature. The results obtained by in situ monitoring the polymerization using NMR and MS indicated that hyperbranched poly(TMPTA1-AEPZ2) was formed via a A(B'B')2 intermediate, and the B' ' (the formed 2 degrees amine) was kept intact in the reaction. Therefore, poly(TMPTA1-AEPZ2) contained secondary and tertiary amines in the core and primary amines in the periphery similar to polyethylenimine (PEI). The chemistry of protonated poly(TMPTA1-AEPZ2) was further confirmed by 13C NMR, and the molecular weight, the radius of gyration (Rg), and the hydrodynamic radius (Rh) were determined using GPC, small-angle X-ray scattering (SAXS), and laser dynamic light scattering (LDLS), respectively. The ratio of Rg/Rh of ca. 1.1 verified the hyperbranched structure. Protonated hyperbranched poly(TMPTA1-AEPZ2) is degradable and less cytotoxic as compared with PEI (25 K). Gel electrophoresis reflected that stable complexes could be formed from protonated hyperbranched poly(TMPTA1-AEPZ2) and DNA, and the size and xi-potential of the complexes were characterized. Remarkably, protonated hyperbranched poly(TMPTA1-AEPZ2) showed transfection efficiency comparable to PEI (25 k) for in vitro DNA delivery.     

Novel bioreducible poly(amido amine)s for highly efficient gene delivery

A series of novel bioreducible poly(amido amine)s containing multiple disulfide linkages (SS-PAAs) were synthesized and evaluated as nonviral gene vectors. These linear SS-PAAs could be easily obtained by Michael-type polyaddition of various primary amines to the disulfide-containing cystamine bisacrylamide. The SS-PAA polymers are relatively stable in medium mimicking physiological conditions (pH 7.4, 150 mM PBS, 37 degrees C), but are rapidly degraded in the presence of 2.5 mM DTT, mimicking the intracellular reductive environment (pH 7.4, [R-SH] = 5 mM, 37 degrees C). The polymers efficiently condense DNA into nanoscaled (<200 nm) and positively charged (>+20 mV) polyplexes that are stable under neutral conditions but are rapidly destabilized in a reductive environment, as was revealed by both dynamic light scatting measurement and agarose gel assays. Moreover, most of the poly(amido amine)s possess buffer capacities in the pH range pH 7.4-5.1 that are even higher than polyethylenimine (pEI), a property that may favorably contribute to the endosomal escape of the polyplexes. Polyplexes of four of the seven SS-PAAs studied were able to transfect COS-7 cells in vitro with transfection efficiencies significantly higher than those of branched pEI, being one of the most effective polymeric gene carriers reported to date. Importantly, also in the presence of serum, a high level of gene expression could be observed when the incubation time was elongated from 1 h to 4 h. XTT assays showed that SS-PAAs and their polyplexes possess essentially no or only very low cytotoxicity at concentrations where the highest transfection activity is observed. The results indicate that bioreducible poly(amido amine)s have excellent properties for the development of highly potent and nontoxic polymeric gene carriers.     

Poly(glycoamidoamine)s for gene delivery: stability of polyplexes and efficacy with cardiomyoblast cells

Polymeric vectors have potential as nucleic acid delivery vehicles for novel gene therapy and oligonucleotide treatments for cardiovascular disease. In this report, poly(glycoamidoamine)s that contain four secondary amines and either two or four hydroxyl units in the repeat unit with D-glucarate (D4), meso-galactarate (G4), D-mannarate (M4), and l-tartarate (T4) stereochemistry have been investigated for their pDNA-binding affinity, DNase protection effect, and polyplex stability in the presence of salt and serum. Also, the luciferase gene delivery and cellular internalization of polyplexes formed with these polymers have been investigated with rat cardiomyoblast [H9c2(2-1)] cells. The results demonstrate that the number of hydroxyl groups and the stereochemistry affect the biological properties. Polymers T4 and G4 have higher pDNA binding affinity, protect pDNA from nuclease degradation, and do not release pDNA in the presence of serum. Polymers D4 and M4 bind pDNA with lower affinity, which allows for some pDNA degradation and release in the presence of serum. Although T4 forms the most stable polyplexes, vector G4 reveals the highest luciferase gene expression in serum-free media and the greatest cellular internalization of fluorescein-labeled pDNA both in serum-free and serum-supplemented media. The results of these studies indicate that the polymer-DNA binding affinity, nuclease protection capability, and polyplex stability are important parameters to facilitate effective pDNA delivery with poly(glycoamidoamine)s in cultured cardiomyoblast cells. The carbohydrate type also plays an important role to increase cellular uptake and gene expression where the polymer with the galactarate stereochemistry (in G4) is found to be the most effective vector for pDNA delivery to cardiomyoblast cells in vitro.     

Poly(glycoamidoamine)s: a broad class of carbohydrate-containing polycations for nucleic acid delivery

In the era of nucleic acid therapeutics, there is an urgent need for non-viral delivery vehicles that can cross the extracellular and intracellular barriers and deliver nucleic acids to specific intracellular regions. This paper reviews the development of a subclass of polymer-based delivery vehicles termed poly(glycoamidoamine)s (PGAAs). The general design of this family consists of carbohydrate residues copolymerized with oligoethyleneamine units, which have proven to be an effective motif that promotes polyplex formation, efficient cellular internalization, high gene expression and low cytotoxicity with cultured cell lines and primary cell types. We then discuss the structure-property relationships of the PGAA class of delivery vehicles and studies aimed at understanding the mechanisms involved in cellular internalization and trafficking.     

Dual bio-responsive gene delivery via reducible poly(amido amine) and survivin-inducible plasmid DNA

A bioreducible poly(amido amine) (SS-PAA) gene carrier, known as poly (amido-butanol) (pABOL), was used to transfect a variety of cancer and non-cancer cell lines. To obtain cancer-specific transgene expression for therapeutic efficiency in cancer treatment, we constructed survivin-inducible plasmid DNA expressing the soluble VEGF receptor, sFlt-1, downstream of the survivin promoter (pSUR-sFlt-1). Cancer-specific expression of sFlt-1 was observed in the mouse renal carcinoma (RENCA) cell line. pABOL enhanced the efficiency of gene delivery compared to traditional carriers used in the past. Thus, a dual bio-responsive gene delivery system was developed by using bioreducible p(ABOL) for enhanced intracellular gene delivery and survivin-inducible gene expression system (pSUR-sFlt-1 or pSUR-Luc reporter gene) that demonstrates increased gene expression in cancer that has advantages over current gene delivery systems.     

Bioreducible poly(amido amine)s with different branching degrees as gene delivery vectors

Based on the knowledge that cationic polymers with different topographical structures behave differently in gene transfection process, herein, we synthesized three biodegradable poly(amido amine)s (PAAs) with the same repeating units and molecular weights except for degree of branching: linear PAA (LPAA), low‐branched PAA (LBPAA), and high‐branched PAA (HBPAA). We found that LBPAA could more effectively compact pDNA into positively charged nanoparticles than both HBPAA and LPAA. LBPAA polyplexes had the highest transfection efficiency among the three PAA polyplexes, and the difference in transfection efficiency is mainly attributed to the endocytosis rate. The cytotoxicity of PAAs was negligible at the transfection doses, probably due to the degradable disulfide bonds. Therefore, we could use branching as a parameter to simply tune a polymer's cellular uptake behavior and transfection efficiency. Biotechnol. Bioeng. 2013; 110: 990–998. © 2012 Wiley Periodicals, Inc.     

Poly(glycoamidoamine)s for gene delivery. structural effects on cellular internalization, buffering capacity, and gene expression

The study of polymeric nucleic acid delivery vehicles has recently grown because of their promise for many biomedical applications. In an effort to understand how the chemical traits of polymers affect the biological mechanisms of nucleic acid delivery, we have calculated the buffering capacity in the physiological pH range of a series of 10 poly(glycoamidoamine)s with systematic structural variations in the amine stoichiometry (from 1 to 4), carbohydrate moiety (d-glucarate or l-tartarate), and amine spacer (ethylene or butylene) within their repeat units. In addition, we have compared the buffering capacity of these polymeric vectors to their polyplex (polymer-DNA complex) stability, cellular internalization, and gene expression profiles to understand the parameters that are important for increasing gene delivery efficiency. The results indicate that the buffering capacity is not always the primary characteristic that determines the gene delivery efficiency for all the poly(glycoamidoamine)s. We have found that the buffering capacity may affect the gene delivery efficiency only when analogous structures containing the same number of amines but different carbohydrates are compared. We reveal that the cellular internalization is the key step in the gene delivery process with systems containing different amine stoichiometry. Also, increasing the number of methylene groups between the secondary amines increases toxicity to a large degree. This systematic and heuristic approach of studying the correlations between structural variables and gene delivery efficiency will facilitate the development of effective synthetic vectors for specific nucleic acid delivery applications.     

Cell specific cytotoxicity and structure-activity relationship of lipophilic 1-B-D-arabinofuranosylcytosine (ara-C) derivatives.

Lipophilic derivatives of ara-C were developed with the aim to improve drug penetration and retention in solid tumors. Ara-C was esterified at the 5'-position with fatty acids (16-22 C-atoms, 0-3 double bonds). The derivatives were inactive in cell lines with various forms of ara-C and 2',2'-difluorodeoxycytidine (dFdC, gemcitabine) resistance, including deoxycytidine kinase (dCK) deficiency. The activity in the parent cell lines correlated negatively with chain length and positively with double bonds.     

Formulation and in vitro transfection efficiency of poly (D, L-lactideco-glycolide) microspheres containing plasmid DNA for gene delivery

The stability, in vitro release, and in vitro cell transfection efficiency of plasmid DNA (pDNA) poly (D,L.-lactide-co-glycolide) (PLGA) microsphere formulations were investigated. PLGA microspheres containing free and polylysine (PLL)-complexed pDNA were prepared by a water-oil-water solvent extraction/evaporation technique. Encapsulation enhanced the retention of the supereoiled structure of pDNA as determined by gel electrophoresis. PLL complexation of pDNA prior to encapsulation increased both the stability of the supercoiled form and the encapsulation efficiency. Free pDNA was completely degraded after exposure to DNase while encapsulation protected the pDNA from enzymatic degradation. Rapid initial in vitro release of pDNA was obtained from microspheres containing free pDNA. while the release from microspheres containing PLL-complexed pDNA was sustained for more than 42 days. Bioactivity of encapsulated pDNA determined by in vitro cell transfection using Chinese hamster ovary cells (CHO) showed that the bioactivity of encapsulated pDNA was retained in both formulations but to a greater extent with PLL-complexed pDNA microspheres. These results demonstrated that PLGA microspheres could be used to formulate a controlledrelease delivery system for pDNA that can protect the pDNA from DNase degradation without loss of functional activity.     

Cross-linked polyethylenimine as potential DNA vector for gene delivery with high efficiency and low cytotoxicity

Polyethylenimine (PEI) has been known as an efficient gene carrier with the highest cationiccharge potential.High transfection efficiency of PEI,along with its cytotoxicity,strongly depends on itsmolecular weight.To enhance its gene delivery efficiency and minimize cytotoxicity,we have synthesizedsmall cross-linked PEI with biodegradable linkages and evaluated their transfection efficiencies in vitro.Inthis study,branched PEI with a molecular weight of 800 Da was cross-linked by small diacrylate[1,4-butanediol diacrylate or ethyleneglycol dimethacrylate (EGDMA)] for 2-6 h.The efficiencies of thecross-linked PEI in in vitro transfection of plasmid DNA containing enhanced green fluorescent protein(EGFP) reporter gene were assessed in melanoma B 16F10 cell line and other cell lines.Flow cytometrywas used to quantify the cellular entry efficiency of plasmid and the transgene expression level.Thecytotoxicities of the cross-linked PEI in these cells were evaluated by MTT assay.EGDMA-PEI 800-4h,atypical cross-linked PEI reported here,mediated a more efficient expression of reporter gene than thecommercially available 25-kDa branched PEI control,and resulted in a 9-fold increase in gene deliveryin B16F10 cells and a 16-fold increase in 293T cells,while no cytotoxicity was found at the optimizedcondition for gene delivery.Furthermore,the transfection activity of polyplexes was preserved in thepresence of serum proteins.     

Gene delivery properties of end-modified poly(beta-amino ester)s

Here, we present the synthesis of a library of end-modified poly(beta-amino ester)s and assess their utility as gene delivery vehicles. Polymers were synthesized using a rapid, two-step approach that involves initial preparation of an acrylate-terminated polymer followed by a postpolymerization amine-capping step to generate end-functionalized polymers. Using a highly efficient poly(beta-amino ester), C32, we show that the terminal amine can greatly affect and improve polymer properties relevant to gene delivery. Specifically, the in vitro transfection levels can be increased by 30% and the optimal polymer:DNA ratio lowered 5-fold by conjugation of the appropriate end group. The most effective modifications were made by grafting primary diamine molecules to the chain termini. The added charge and hydrophobicity of some derivatives enhanced DNA binding and resulted in the formation of polymer-DNA complexes less than 100 nm in diameter. In addition, cellular uptake was improved 5-fold over unmodified C32. The end-modified poly(beta-amino ester)s presented here are some of the most effective gene-delivery polycations, superior to polyethylenimine and previously reported poly(beta-amino ester)s. These results show that the end-modification of poly(beta-amino ester)s is a general strategy to alter functionality and improve the delivery performance of these materials.     

Reducible poly(amido ethylenimine)s designed for triggered intracellular gene delivery

Poly(amido ethylenimine) polymers, a new type of peptidomimetic polymer, containing multiple disulfide bonds (SS-PAEIs) designed to degrade after delivery of plasmid DNA (pDNA) into the cell were synthesized and investigated as new carriers for triggered intracellular gene delivery. More specifically, three SS-PAEIs were synthesized from Michael addition reactions between cystamine bisacrylamide (CBA) and three different ethylene amine monomers, i.e., ethylenediamine (EDA), diethylenetriamine (DETA), or triethylenetetramine (TETA). Complete addition reactions were confirmed by (1)H NMR. The molecular weight, buffer capacity, and relative degree of branching for each SS-PAEI was determined by gel permeation chromatography (GPC), acid-base titration, and liquid chromatography-mass spectroscopy (LC-MS), respectively. Physicochemical characteristics of polymer/pDNA complexes (polyplexes) were analyzed by gel electrophoresis, particle size, and zeta-potential measurements. All three SS-PAEIs effectively complex pDNA to form nanoparticles with diameters less than 200 nm and positive surface charges of approximately 32 mV. The in vitro gene transfer properties of SS-PAEIs were evaluated using mouse embryonic fibroblast cell (NIH3T3), primary bovine aortic endothelial cell (BAEC), and rat aortic smooth muscle cell (A7R5) lines. Interestingly, polyplexes based on all three SS-PAEIs exhibited remarkably high levels of reporter gene expression with nearly 20x higher transfection efficiency than polyethylenimine 25k. The high transfection efficiency was maintained in the presence of 10% serum in the transfection medium. Furthermore, confocal microscopy experiments using labeled pDNA indicated that polyplexes of SS-PAEI displayed greater intracellular distribution of pDNA as compared to PEI, most likely due to environmentally triggered release. Therefore, SS-PAEIs are a new class of transfection agents that facilitate high gene expression while maintaining a low level of toxicity.     

Synthesis, cytotoxicity, and structure-activity relationship (SAR) studies of andrographolide analogues as anti-cancer agent

A series of analogues of andrographolide, prepared through chemo-selective functionalization at C14 hydroxy, have been evaluated for in vitro cytotoxicities against human leukemic cell lines. Two of the analogues (6a, 9b) exhibited significant potency. Preliminary studies on structure-activity relationship (SAR) revealed that the α-alkylidene-γ-butyrolactone moiety of andrographolide played a major role in the activity profile. The structures of the analogues were established through spectroscopic and analytical data.     

Trans-Pd/Pt(II) saccharinate complexes with a phosphine ligand: Synthesis,cytotoxicity and structure-activity relationship

New trans-[Pd(sac)₂(PPhMe₂)(DMSO)]·H₂O (Pd) and trans-[Pt(sac)₂(PPhMe₂)₂]·H₂O (Pt) complexes (sac = saccharinate and PPhMe₂ = dimethylphenylphosphine) were synthesized and characterized by elemental analysis, IR, NMR, ESI-MS spectral analyses and X-ray diffraction. The complexes were evaluated for their in vitro cytotoxicity against breast (MCF-7), colon (HCT116) and lung (A549) human cancer cell lines. The ATP viability assay displayed that Pd was biologically inactive, but Pt showed significant anticancer potency on MCF-7 cancer cells, similar to cisplatin. The results suggested that Pt targeted DNA, whereas Pd displayed higher binding affinity towards human serum albumin (HSA). Mechanism of action studies of Pt suggested apoptotic cell death due to significant increase in intracellular ROS (reactive oxygen species) levels, mitochondrial damage and formation of DNA double-strand breaks. Finally, this work represents a new example of potent transplatin anticancer complexes.     

Linear cationic click polymers/DNA nanoparticles: in vitro structure-activity relationship and in vivo evaluation for gene delivery

The aim of this work was to explore the structure--activity relationships (SAR) of a series of novel linear cationic click polymers with various structures for in vitro gene delivery and in vivo gene transfer. The experimental results revealed that the minimal structure variation could result in a crucial effect on DNA-binding ability, buffering capacity, and the cellular delivery capacity of polymer, all of which brought about the obvious effects on their transfection efficiencies. The polymer synthesized from diazide monomer containing bis-ethylenediamine unit and dialykene monomer containing bis-ethylene glycol unit (B(2)) could effectively condense DNA into complex nanoparticles (B(2)Ns), which showed the highest in vitro transfection efficiency. The biodistribution and transfection efficiency of B(2)Ns in nude mice bearing tumor demonstrated the ability of effectively delivering DNA into tumor tissue. These results implied that this gene vector based on linear cationic click polymer could be a promising gene delivery system for tumor gene therapy.     

Synthesis of poly(beta-amino ester)s optimized for highly effective gene delivery

Several families of synthetic polymers, including degradable poly(beta-amino ester)s, have been previously shown to effectively mediate gene transfer. However, the combined impact of potentially significant factors-such as polymer molecular weight, polymer chain end-group, and polymer/DNA ratio-on different gene transfer properties has yet to be systematically investigated. The elucidation of these relationships may aid in the design of nonviral vectors with greatly enhanced transfection properties. To examine these factors, two distinct poly(beta-amino ester) structures, Poly-1 and Poly-2, were generated by adding 1,4-butanediol diacrylate and 1,6-hexanediol diacrylate, respectively, to 1-aminobutanol. Twelve unique versions of each structure were synthesized by varying amine/diacrylate stoichiometric ratios, resulting in polymers with either amine or acrylate end-groups and with molecular weights ranging from 3350 to 18000. Using high throughput methods, all polymers were tested in quadruplicate at nine different polymer/DNA ratios ranging from 10:1 w/w to 150:1 w/w. Through the optimization of molecular weight, polymer chain end-group, and polymer/DNA ratio, these polymers successfully mediated gene transfer at levels that surpassed both PEI and Lipofectamine 2000 in vitro.