Construction And Gene Transfection Evaluation Of Two Types Of Cyclodextrin-based Gene Delivery Carriers

BackgroundGene therapy is one of the promising biological paradigms for many diseases, which has broad application in the treatment of cardiovascular, severe combined immunodeficiency(SCID-X1), Parkinson’s disease, Wiskott-Aldrich syndrome and neurodegenerative diseases as well as an alternative to traditional chemotherapy for the treatment of cancer. Non-viral vectors have attracted more and more attention for gene delivery. Non-viral vectors have the advantages of low immunogenicity, low cytotoxicity, low production cost, unlimited gene size for packaging and can be administered repeatedly without causing immune response. However, there are still some serious problems in the gene delivery system, such as endosome escape, the difficulty of unpacking pDNA into the cytoplasm, and relatively low transfection efficiency and so on. So it is still of great importance for the research and development of new gene delivery carriers with low toxicity and high gene transfection.Cyclodextrins(CDs), the commonly used medicinal biomaterials, which have low toxicity and can be modified functionally, show potential ability for gene delivery. Cyclodextrins derivatives can be used as gene delivery carriers, and it can also be applied to construct other gene carriers as linker or modifier. What’s more, cyclodextrins can be used to construct gene delivery carriers in the form of polypseudorotaxane or polyrotaxanes. Unfortunately, these cyclodextrin based gene carriers still show some drawback, such as poor endosome escape ability, poor stability of polyplexes suffering dilution in vivo and relatively low transfection efficiency and so on. ObjectivesConsidering the above background, we selected β-cyclodextrin and α-cyclodextrin as starting materials to design and synthesize two categories of novel gene vectors. The first category was based on β-cyclodextrin, and a series of amphipathic polycations β-cyclodextrin derivatives were formed by introducing hydrophilic cationic amino groups and hydrophobic tails. Different types and numbers of cationic amino groups were introduced to the β-cyclodextrin derivatives to obtain gene vectors with good biocompatibility and high transfection efficiency. The second category based on α-cyclodextrin, to form pseudopolyrotaxane with COOH-PEG-COOH, then capped with acid-sensitive hydrazone bond, follwed by introducing different types and different numbers of amino groups to form α-cyclodextrin based acid-sensitive polyrotaxane supermolecules as gene carrier. Our goal was to construct biocompatible, highly effective and endosome stimuli-responsive gene vectors with high dilution stability for gene delivery. Methods1. The first category gene delivery vectors were prepared starting from β-CD, by reacting with 4-toluene sulfochloride to give mono-substituted β-CD-OTs, followed by substitution reaction with ethylenediamine to form β-CD-EDA, then by amidation with stearic acid to give β-CD-EDA-C18 with an hydrophobic tail. By controlling the reaction equivalent, different number of DMEDA and branched PEI600 was introduced as the hydrophilic amino groups respectively. Thus we synthesized two series(DME series and PEI series), six kinds of polycationic amphiphilic β-cyclodextrin derivatives.2. The second category gene delivery vectors were a series of α-cyclodextrin-based acid-sensitive polyrotaxane supermolecules. First, the polypseudorotaxanes were formed with α-CDs threaded onto COOH-PEG-COOH chain. Then the obtained polypseudorotaxane was capped with bulky benzophenone hydrazone. The number of amine groups(DMEDA or PEI600) linked to per α-CD ring was adjusted by controlling the feed ratio of CDI, DMEDA or PEI600 to hydroxyl groups of α-CD.3. The toxicity of two categories including 12 kinds of gene carriers toward 293 T cells was evaluated using the MTT reduction assay; The size and zeta potential of polyplexes was determined by Delsa? Nano C Particle Analyzer; The ability of gene carries to compact DNA was evaluated by agarose gel electrophoresis; The transfection efficiency of 12 kinds of gene carries was evaluated in HEK293 T cell lines by flow cytometry and fluorescence microscopy with pEGFP as a reporter gene, with " gene transfection gold standard" PEI25000 as the positive control; The transfection activity of optimal gene carries among the two categories was further investigated in the the presence of serum.4. Acidic stimuli-responsive properties of PRPEI-2/DNA polyplex were observed by Delsa? Nano C Particle Analyzer(Beckman Coulter) and transmission electron microscopy; Hemolysis experiments were performed to imitate endosome escape under acidic stimulus; The dilution stability of PRPEI-2/DNA polyplex was also investigated through dilution stability assay. To elucidate cellular internalization of PRPEI-2/DNA polyplex and the effects of stimuli-responsive profiles on gene transfection efficiency, specific inhibitors were applied to investigate the endocytic uptake mechanisms. Results Part one: Polycationic amphiphilic β-cyclodextrin derivatives1. The structure of six amphiphilic polycations β-cyclodextrin derivatives was identified by 1H-NMR and IR spectra. The final number of DMEDA amine groups in each α-CD ring were 3.0, 7.1 and 10.3 for DME-1, DME-2 and DME-3 respectively, and that of PEI600 amine groups in each α-CD ring were 1.9, 3.2 and 4.6 for PEI-1, PEI-2 and PEI-3, respectively.2. All amphipathic polycations β-cyclodextrin derivatives exhibited good biocompatibility. Among them, the DME series showed lower cytotoxicity. With the increasing number of PEI600, these derivatives showed toxic effect at high concentration, but still lower than PEI25000.3. Among the DME series, the size of DME-2 or DME-3/DNA complexes was about 1171~2828 nm, while DME-1 formed relatively stable complexes with the size of 140~369 nm. As for the PEI series, these derivatives exhibited rather small and regular size about 97~181 nm. The zeta potential of all complexes increased with the increasing of N/P ratio.4. All these β-cyclodextrin derivatives completely retarded DNA over the N/P weight ratio of 5, except for DME-3 and PEI-3, for which, free DNA disappeared at the N/P weight ratio of 1.5. Among the amphiphilic polycations β-cyclodextrin derivatives containing DMEDA amino groups, DME-2 and DME-3 showed comparable transfection efficiency with naked DNA. DME-1 showed high transfection activity at the N/P ratio of 30 and 50, although the number of positive transfected cells of DME-1 was lower than PEI25000, the mean fluorescence intensity of it was higher than PEI25000. PEI-1/2/3 with PEI600 amino groups showed higher transfection efficiency both in the number of positive transfected cells and the mean fluorescence intensity than naked DNA. Although the number of positive transfected cells of PEI-2 and PEI-3 was less than PEI25000, the mean fluorescence intensity was better than PEI25000 at all N/P ratio. PEI-1 showed superior transfection activity among all the derivatives, it exhibited comparable positive transfected cells with PEI25000 at the N/P ratio of 30, and higher mean fluorescence intensity than PEI25000. The number of positive transfected cells and MFI of PEI-1 were not affected by the presence of serum, while the positive transfected cells and the mean fluorescence intensity of PEI25000 reduced in the presence of serum. PEI-1 delivered pDNA into cells mainly through caveolae-mediated pathway. Part two: α-cyclodextrin-based acid-sensitive polyrotaxane supermolecules1.The structure of six α-cyclodextrin-based acid-sensitive polyrotaxane supermolecules was identified by 1H-NMR and IR spectra. The final number of DMEDA amine groups in each α-CD ring were 1.3, 6.3 and 10 for PRDME-1, PRDME-2 and PRDME-3 respectively, and that of PEI600 amine groups in each α-CD ring were 1.7, 2.9 and 4.1 for PRPEI-1, PRPEI-2 and PRPEI-3, respectively.2. All of the six polyrotaxanes showed far lower toxicity than PEI25000, exhibiting better biocompatibility and safety for further biomedical applications.3. Among the PRDME series, the size of PRDME-1/DNA polyplex was about 1973~3468 nm,while PRDME-2 or PRDME-3 formed relatively stable polyplexes with pDNA(the size was about 92~232 nm). As for the PRPEI series, these derivatives exhibited rather small and regular size about 81~123 nm. The zeta potential of all polyplexes increased with the increasing of N/P ratio.4. When polyrotaxanes/DNA polyplexes were formed over the N/P weight ratio of 1, all these polyrotaxanes completely retarded DNA except for PRDME-1, for which, free DNA disappeared at the N/P weight ratio of 10.5. PRDME-1/2/3 with DMEDA groups showed very poor transfection activity comparable to that of naked DNA. Fortunately, as for PRPEI series, all of the PRPEI-1/2/3 achieved favorable transfection efficiency, higher than that of naked DNA both in transfected cells% and MFI. Among the PRPEI series, PRPEI-2 with moderate density of PEI amine group showed highest transfection efficiency both in the percent of positive transfected cells and MFI than the other two at the same weight ratio. Moreover, the percent of positive transfected cells of PRPEI-2 was comparable to that of PEI25000, and PRPEI-2 even showed higher MFI at weight ratio of 30 and 50, almost 1.4 and 1.3-fold compared with that of PEI25000 respectively. The positive transfected cells and MFI of PRPEI-2 were not affected by the presence of serum, while the positive transfected cells and the mean fluorescence intensity of PEI25000 reduced in the presence of serum.6. When incubated at pH5.0, typical size changing and morphological transformation of PRPEI-2/DNA polyplex took place compared to pH7.4. The multiple peaks appeared when incubated at pH5.0 and the diameters of polyplex changed from 158 nm(pH7.4) to 555 nm(pH5.0). The polyplexes were regular at pH7.4, but irregular and loose polyplexes appeared when incubated at pH5.0. These results suggested that the PRPEI-2/DNA polyplex could keep stable in blood circulation and extracellular environment(pH7.4) and turn into relatively loose and swelling particles in acidic endosomal compartment(pH5.0), which facilitated the intracellular release of DNA from polyplex.7. Polyrotaxane PRPEI-2 exhibited higher membrane disruptive capability at pH5.0(endosome) than that at pH7.4(extracellular) either incubated for 1 h or 4 h. In addition, when incubated at pH5.0, the hemolytic capability increased with the prolonging of incubation time. The membrane disruptive property of PRPEI-2/DNA polyplex was further confirmed by scanning electron microscopy. When incubated with PRPEI-2/DNA polyplex at pH7.4, the membrane of red blood cell remained intact. While incubated with PRPEI-2/DNA polyplex at pH5.0, the cell membrane appeared some pores, and the cells swelled or even bursted. On the whole, these results indicated that the stimuli-responsive supermolecule PRPEI-2 was a promising nanomaterial applied to construct intracellular release vector for gene delivery, which was helpful for endosome escape during intracellular trafficking.8. PRPEI-2 could form stable polyplexes with DNA at a mean diameter of 160 nm and keep their particle size and distribution relatively stable even undergoing strong dilution up to 128-fold. In contrast, the diameter of PEI25000/DNA polyplex at 16-fold/32-fold dilution was about 1.5 times/2 times larger than its original diameter, and broad peaks appeared in their size distribution, which was in accord with the increase of PDI to 0.37. These results revealed that the well-formed PRPEI-2/DNA polyplex was able to withstand a high blood dilution in vivo, indicating a promising prospect as the hopeful nonviral vector for gene therapy.9. When treated with chlorpromazine at the concentration of 15, 20 and 30 μM, the transfection efficiency was reduced by 45.7%, 70.8% and 99.4%, respectively. When treated with genistein at the concentration of 75 μM, the transfection efficiency was comparable with the control group; with the concentration increasing to 150 and 200 μM, the transfection efficiency was reduced by 46.9% and 49.7% respectively. When treated with amiloride, the transfection efficiency of PRPEI-2/DNA polyplex was unaffected even up to 200 μM. These results indicated that the endocytic uptake involved in the gene transfection of PRPEI-2/DNA polyplexes were through the clathrin- and caveolae-mediated endocytosis, among which the clathrin-mediated endocytosis had greater effects on transfection efficiency. Clathrin-mediated pathway is associated with clathrin-based internalization and subsequent intracellular endolysosomal trafficking of nucleic acid molecules by gene release vectors. Thus it is very important to improve the endosome escape activities for gene vectors through the clathrin-mediated endocytic pathway. Conclusions1. We designed and synthesized two types of novel gene vectors, including six polycationic amphiphilic β-cyclodextrin derivatives and six α-cyclodextrin-based acid-sensitive polyrotaxane supermolecules. The structure of each derivative was identified by 1H-NMR and IR spectra.2. All of these designed novel gene vectors exhibited low cytotoxicity. Among two types of gene delivery carriers, α-cyclodextrin based acid-sensitive polyrotaxane supermolecules showed better biocompatibility.3. Among the two types of gene vectors, those with PEI600 amine groups exhibited better transfection efficiency than those with DMEDA amine groups. Appropriate density of amino groups had important effects on the transfection efficiency. PEI-1 and PRPEI-2 with suitable amine group achieved the best transfection efficiecy among the same type of gene vectors, which showed comparable transfected cells% with PEI25000, and the MFI was higher than PEI25000. PEI-1 and PRPEI-2 remained relatively high transfection activity in serum-containing conditions, and were expected to be promising and competitive gene vectors used for in vivo transfection.4. The PEI-1/DNA complex was uptake mainly through caveolae-mediated endocytosis, which can avoid lysosomal degradation and was beneficial for gene transfection. Polyrotaxane supermolecule PRPEI-2 had acid stimuli-responsive profiles, endosome escape capacity, and high dilution stability, which makes it hopeful and competitive stimuli-responsive nonviral vector for further application. Moreover, the modular endosome stimuli-responsive polyrotaxane supermolecules offer new opportunities to certain fields attempting to deliver other therapeutic and diagnostic genes.