Chitosan cross-linked with poly(ethylene glycol)dialdehyde via reductive amination as effective controlled release carriers for oral protein drug delivery.

The covalently cross-linked chitosan-poly(ethylene glycol)1540 derivatives have been developed as a controlled release system with potential for the delivery of protein drug. The swelling characteristics of the hydrogels based on these derivatives as the function of different PEG content and the release profiles of a model protein (bovine serum albumin, BSA) from the hydrogels were evaluated in simulated gastric fluid with or without enzyme in order to simulate the gastrointestinal tract conditions. The derivatives cross-linked with difunctional PEG1540-dialdehyde via reductive amination can swell in alkaline pH and remain insoluble in acidic medium. The cumulative release amount of BSA was relatively low in the initial 2h and increased significantly at pH 7.4 with intestinal lysozyme for additional 12h. The results proved that the release-and-hold behavior of the cross-linked CS-PEG1540H-CS hydrogel provided a swell and intestinal enzyme controlled release carrier system, which is suitable for oral protein drug delivery.

Design and application of cationic amphiphilic ß-cyclodextrin derivatives as gene delivery vectors.

The nano self-assembly profiles of amphiphilic gene delivery vectors could improve the density of local cationic head groups to promote their DNA condensation capability and enhance the interaction between cell membrane and hydrophobic tails, thus increasing cellular uptake and gene transfection. In this paper, two series of cationic amphiphilic ß-cyclodextrin (ß-CD) derivatives were designed and synthesized by using 6-mono-OTs-ß-CD (1) as the precursor to construct amphiphilic gene vectors with different building blocks in a selective and controlled manner. The effect of different type and degree of cationic head groups on transfection and the endocytic mechanism of ß-CD derivatives/DNA nanocomplexes were also investigated. The results demonstrated that the designed ß-cyclodextrin derivatives were able to compact DNA to form stable nanocomplexes and exhibited low cytotoxicity. Among them, PEI-1 with PEI head group showed enhanced transfection activity, significantly higher than commercially available agent PEI25000 especially in the presence of serum, showing potential application prospects in clinical trials. Moreover, the endocytic uptake mechanism involved in the gene transfection of PEI-1 was mainly through caveolae-mediated endocytosis, which could avoid the lysosomal degradation of loaded gene, and had great importance for improving gene transfection activity.

Enhanced gene transfection performance and biocompatibility of polyethylenimine through pseudopolyrotaxane formation with α-cyclodextrin.

Polyethylenimine (PEI), a commercially available gene transfection reagent, is a promising nonviral vector due to its inherent ability to efficiently condense genetic materials and its successful transfection performance in vitro. However, its low transfection efficiency in vivo, along with its high cytotoxicity, limit any further applications in gene therapy. To enhance the gene transfection performance and reduce the cytotoxicity of linear polyethylenimine, pseudopolyrotaxane PEI25k/CD and the polyrotaxanes PEI25k/CD-PA and PEI25k/CD-PB were prepared and their transfection efficiencies were then evaluated. The pseudopolyrotaxane PEI25k/CD exhibited better transfection efficiency and lower cytotoxicity than the transfection reagent linear PEI25k, even in the presence of serum. It also showed a remarkably higher cell viability, similar DNA protecting capability, and better DNA decondensation and release ability, and could be useful for the development of novel and safe nonviral gene delivery vectors for gene therapy.

Cholesterol derived cationic lipids as potential non-viral gene delivery vectors and their serum compatibility.

Cholesterol derivatives M1-M6 as synthetic cationic lipids were designed and the biological evaluation of the cationic liposomes based on them as non-viral gene delivery vectors were described. Plasmid pEGFP-N1, used as model gene, was transferred into 293T cells by cationic liposomes formed with M1-M6 and transfection efficiency and GFP expression were tested. Cationic liposomes prepared with cationic lipids M1-M6 exhibited good transfection activity, and the transfection activity was parallel (M2 and M4) or superior (M1 and M6) to that of DC-Chol derived from the same backbone. Among them, the transfection efficiency of cationic lipid M6 was parallel to that of the commercially available Lipofectamine2000. The optimal formulation of M1 and M6 were found to be at a mol ratio of 1:0.5 for cationic lipid/DOPE, and at a N/P charge mol ratio of 3:1 for liposome/DNA. Under optimized conditions, the efficiency of M1 and M6 is greater than that of all the tested commercial liposomes DC-Chol and Lipofectamine2000, even in the presence of serum. The results indicated that M1 and M6 exhibited low cytotoxicity, good serum compatibility and efficient transfection performance, having the potential of being excellent non-viral vectors for gene delivery.

Preparation, Physicochemical Properties, and Transfection Activities of Tartaric Acid-Based Cationic Lipids as Effective Nonviral Gene Delivery Vectors.

In this work two novel cationic lipids using natural tartaric acid as linking backbone were synthesized. These cationic lipids were simply constructed by tartaric acid backbone using head group 6-aminocaproic acid and saturated hydrocarbon chains dodecanol (T-C12-AH) or hexadecanol (T-C16-AH). The physicochemical properties, gel electrophoresis, transfection activities, and cytotoxicity of cationic liposomes were tested. The optimum formulation for T-C12-AH and T-C16-AH was at cationic lipid/dioleoylphosphatidylethanolamine (DOPE) molar ratio of 1 : 0.5 and 1 : 2, respectively, and N/P charge molar ratio of 1 : 1 and 1 : 1, respectively. Under optimized conditions, T-C12-AH and T-C16-AH showed effective gene transfection capabilities, superior or comparable to that of commercially available transfecting reagent 3ß-[N-(N',N'-dimethylaminoethyl)carbamoyl]cholesterol (DC-Chol) and N-[2,3-dioleoyloxypropyl]-N,N,N-trimethylammonium chloride (DOTAP). The results demonstrated that the two novel tartaric acid-based cationic lipids exhibited low toxicity and efficient transfection performance, offering an excellent prospect as nonviral vectors for gene delivery.

Construction of α-Cyclodextrin-Based Stimuli-Responsive Gene Delivery Nanovectors: In Vitro, Ex Vivo, and In Vivo Investigations.

Stimuli-responsive materials are promising paradigm applied to construct diagnostic and therapeutic intracellular controlled release vectors, while highlighting many challenges and opportunities. In this paper, six α-cyclodextrin-based supramolecular nanovectors were constructed and the efficacy of amine groups, stimuli-responsive profiles and endocytic mechanisms were investigated. The results indicated that the designed supermolecules can compact DNA to form stable complexes and display low cytotoxicity. Among them, PRPEI-2 with suitable PEI amine group exhibited enhanced transfecting performance, high dilution stability, nice serum compatibility, and good acid-responsive profiles to enable endosome escape, significantly higher than commercially available transfecting agent PEI25000, the most effective vector studied to date. The endocytic uptake mechanisms involved in the transfection was mainly through clathrin-mediated pathway, which is closely associated with and can be improved by endosome escape. Moreover, PRPEI-2/DNA polyplex can be effectively expressed in vivo even after 48 h via only single tail-vein injection, and the gene expression and main tissue distribution appeared in the testis, liver, brain and spleen. These excellent characteristics demonstrated that the supramolecular PRPEI-2 represents an excellent prospect as stimuli-responsive nanovectors for gene diagnosis and therapy.

Microbial Poly-3-Hydroxybutyrate (PHB) as a Feed Additive for Fishes and Piglets.

The large-scale use of petrochemical-based plastics is damaging our environment. Discarded plastics are harmful to both marine and land animals, sometimes causing death when ingested. Biodegradable plastics have gained attentions from the public and the academia to reduce environmental burdens. Poly-3-hydroxybutyrate (PHB), the simplest and the best-studied bioplastic member of the polyhydroxyalkanoate (PHA) family synthesized by many bacteria, has been studied as a feed additive for large yellow croaker fish and weaned piglets. The fish grow faster and gain more weight when 1% and 2% PHB is added as a feed additive, accompanied by increased survival rates. Weaned piglets are found to grow normally and showed no significant change in average daily weight gains, average daily feed intakes, feed efficiency, and organ developments when 0.5% PHB is added to the feed. It can therefore be concluded that biodegradable and biocompatible PHB is not harmful as a feed additive for marine large yellow croakers and sensitive weaned piglets. PHB therefore holds great promise as a plastic that combines biodegradability and biocompatibility with good tolerability as a feed supplement for animals.

A novel stealth liposomal topotecan with amlodipine: apoptotic effect is associated with deletion of intracellular Ca2+ by amlodipine thus leading to an enhanced antitumor activity in leukemia.

The objectives of the present study were to define whether amlodipine induces apoptosis and what mechanism is involved in the process in human resistant and non-resistant leukemia cells following co-administration of stealth liposomal topotecan with amlodipine, a novel antiresistant liposomes developed by our institution. In three leukemias, K562, HL-60, and multidrug resistant (MDR) HL-60, cytotoxicity of topotecan was potentiated by amlodipine, while topotecan alone was resistant to MDR HL-60 cells. In two selected K562 or MDR HL-60 cells, the apoptotic effects were increased by addition of amlodipine, showing a dose-dependent manner. The activities of caspase 3 and 7 (marked as caspase 3/7), and caspase 8 were significantly activated by topotecan with amlodipine co-treated as the stealth liposomes. The deletions of intracellular Ca2+ stores induced by amlodipine correlated with the activated activities of caspase 3/7, or 8, respectively. In xenograft model with MDR HL-60 in nude mice, antitumor activity of stealth liposomal topotecan with amlodipine was significantly enhanced as compared to that of stealth liposomal topotecan or topotecan alone. In conclusion, apoptotic effect is associated with deletion of intracellular Ca2+ by amlodipine through activation of caspase 8 and then 3/7 activities. The enhanced antitumor activities by stealth liposomal topotecan with amlodipine are mainly due to the potentiating apoptotic effect and reversing the resistance by amlodipine. Stealth liposomal encapsulation of anticancer agent with a modulator may provide a novel strategy for improving the chemotherapeutic effects.