The Design And Preparation Of Delivery System Of Dendritic Peptides-small Nucleic Acid Molecules And Its Delivery Efficiency And Characteristics In Bacteria

AIMNowadays, the infections of drug-resistant bacteria, especially the multi-drug resistant and pan-drug resistant bugs, have been becoming more and more urgent, which is the primary cause of death from severe infectious in clinic. What’s worse, the development of antibiotics couldn’t keep pace with the emergence of drug-resistant bacteria. Searching for and developing new strategies and new drugs to fight against drug resistance has become an imperative task for medical scientists in the world.Antisense antibacterial strategy targets to the key genes of bacteria, such as pathogenic genes, drug resistant genes or live essential genes, and adapts antisense nucleic acid molecules to block the expression of these genes to inhibit the reproduction of bacteria or kill them. Compared to classical antibiotics, antisense antibacterial nucleic acid molecule has the advantages of clear and highly specific target gene, easy design of drug molecules, difficult to induce drug resistance, rapid and large-scale production, and shorter research and development circle. It breaks out the stereotyped development of antibiotics that target to the protein of bacteria, and it is the new hope for the therapy of drug-resistant bacteria. Several targeted genes of antisense nucleic acid in bacteria have been found in our previous study and we have successfully screened several antisense antibacterial molecules that could inhibit the expression of target genes or reverse the drug resistance of bacteria or kill them in vitro and in vivo. However, antisense nucleic acid molecule always has a high molecular weight, negative charges and super hydrophilicity, the naked antisense nucleic acid molecules is difficult to penetrate the plasma membrane to enter into bacteria, which has become the bottleneck of the further development of antisense antibacterial strategy. So, to develop a delivery system that could effectively transport the small nucleic acid into bacteria is a significant project for the antisense antibacterial drug research, and it is also the key point for the clinical application of antisense antibacterial strategy.An ideal vector of small nucleic acid should have the ability to deliver the nucleic acid into bacteria effectively and block the expression of targeted genes to exert the antibacterial activity. It has been reported that cell penetrating peptides could deliver the antisense nucleic acid molecules into bacteria by covalent method. However, this strategy is difficult to be popularized due to the limitation of the low loading capacity and the difficulties of the design and synthesis of the connecting arm. Dendritic poly-peptide is favorable for the attraction of small nucleic acid molecules and form nanoparticles as it has the cavity structure. And it is also reported that dendritic poly-peptides is more efficient than the linear counterpart to deliver small nucleic acid into cells. However, it is not reported that dendritic poly-peptides could form nanoparticles with small nucleic acid in a non-covalent manner and penetrating the cytomembrane to enter into bacteria. In order to design an effective dendritic poly-peptide, following questions must come first:(1) which factors affect the formation of dendritic/small nucleic acid nanoparticles?(2) which factors directly impact the penetrating capacity of dendritic poly-peptide?(3) Is there any difference on the penetrating mechanism of nanoparticles on bacterial and mammalian cells?(4) Is there any difference on the penetrating efficiency between the Gram-negative and Gram-positive bacteria? How about the sensitive and drug-resistant bacteria? To resolve above questions, we designed seven dendritic poly-peptide(DPP) based on the structure of peptides with four branches with the repeat sequence of leucine-tryptophan-arginine. Several factors were manily considered in the DPP design, including the number and distribution of positive charge, hydrophilicity and amphiphilicity, the amino acid with hydroxyl group, histidine and lipid additive of DPP. And the first generation of RNA mimics, Phosphorothioate oligodeoxyribonucleotides(PS-ODN), was used as model drug to systemically research the preparation of dendritic poly-peptide/PS-ODN(ODN/DPP) nanoparticles and the factors that influence the formation and the penetrating capacity of DPP nanoparticles, as well as the probably mechanism of the delivery process.METHODS1. The design and synthesis of DPPWe designed 7 dendritic poly-peptide, named DPP1-7. Several factors were mainly considered:(1) hydrophilicity and amphiphilicity: DPP1, DPP4, DPP5 and DPP6 were hydrophilic peptides, but DPP2, DPP3 and DPP7 were amphipathic peptides.(2) Charge distribution: The positive charge was located at the two extremities of each branch in hydrophilic DPP1, but averagely located at every branch in DPP6; the positive charge of DPP2 showed a sparse distribution at the hydrophobic branches and a dense distribution at the hydrophilic branches, but just located at hydrophilic branches of DPP3.(3) Hydroxyl group amino acid: the leucine and tryptophan in hydrophilic DPP1 and DPP6 was substituted by serine and threonine which contained hydroxyl group in DPP4 and DPP5.(4) Histidine: the lysine located at the hydrophilic side on amphipathic DPP3 was substituted with histidine in DPP7.2. The preparation and characterization of ODN/DPP nanoparticlesDPP and PS-ODN was mixed completely at different N: P ratio, followed by co-incubating at 37°C to form ODN/DPP nanoparticles. Then agarose gel electrophoresis was adapted to measure the encapsulation of PS-ODN, and screened the best N: P ratio, followed by preparing ODN/DPP nanoparticles under this condition. The characterization of ODN/DPP nanoparticles contained three parts:(1) TEM was used to observe themorphology of ODN/DPP nanoparticles.(2) DLS was adapted to measure the size and zeta potential of ODN/DPP nanoparticles.(3) Fluorescence spectrophotometry was used to measure the encapsulation of PS-ODN by DPP.3. The factors that affect the formation of ODN/DPP nanoparticles and the study of thestability of ODN/DPP nanoparticlesThe solvents with different concentration of phosphate buffer or phosphate buffer with different p H value were used to prepare ODN/DPP nanoparticles, followed by comparing the size by DLS. Circular dichroism was adapted to observe the secondary structures of DPP in different solvents. ODN/DPP/DOTAP nanoparticles were prepared by two steps, followed by comparing to the size of ODN/DPP by DLS. ODN/DPP nanoparticles were store at 4°C and measured the size by DLS every week for 4 weeks to evaluate the stability.4. The delivery efficiency and characteristics of ODN/DPP nanoparticles into bacteriaThe tested strains contained four Gram-negative strains: E. coli, ESBLs-E. coli, ESBLs-K. pneumonia and S. typhimurium and four Gram-positive strains: S. aureus, MRSA, MRSE and E. faecalis. FITC-labeled PS-ODN was used to prepare the FITC-labeled ODN/DPP nanoparticles, followed by co-incubating with bacteria at 37°C for a certain period time in dark. Then, flow cytometry was adapted to measure the positive ratio of bacteria, which represented the delivery capacity of ODN/DPP nanoparticles.5. The inhibition of expression of targeted gene and the retardation of growth ofbacteriaThe tested strain was ESBLs-E. coli. RT-PCR was adapted to measure the inhibition effect of the expression of rpo D in ESBLs-E. coli after being treated with ODN/DPP and ODN/DPP/DOTAP nanoparticles, while growth curve of ESBLs-E. coli was used to measure the inhibition of bacterial growth.6. The analysis of the roles of ODN/DPP/DOTAP nanoparticles components and theprobable mechanism of penetrating cell membrane of ODN/DPP nanoparticlesThe tested strains were ESBLs-E. coli and MRSA. We prepared ODN/DPP2, ODN/DPP2/DOTAP and ODN/DOTAP nanoparticles and compared the delivery efficiencies of these nanaoparticles on bacteria, to confirm the roles of each component in nanoparticles. The mechanism of delivery of ODN/DPP was divided into two parts: FITC-labeled ODN/DPP nanoparticles were co-incubated with bacteria at 4°C and 37°C, respectively, to test if the delivery process was energy dependent; Bacterial solutions were treated with inhibitors of endocytosis pathway in mammalian cells, followed by adding FITC-labeled ODN/DPP nanoparticles and co-incubating in dark before measuring on flow cytometry.RESULTS1. The morphology and characterization of ODN/DPP nanoparticlesFrom the results of the agarose gel electrophoresis, we found that the best N: P ratio of ODN/DPP nanoparticles was 8. TEM showed that the morphology of these nanoparticles was spherical with the size ranging from 45 to 80 nm, and the results were identified by DLS. The zeta potential of ODN/DPP nanoparticles was less than 5 m V. And the DPP with hydroxyl group couldn’t form stable nanoparticles with PS-ODN. The hydrophilicity and amphiphilicity, charge distribution and histidine of DPP didn’t influence the size of ODN/DPP nanoparticles. These nanoparticles had a high encapsulation, more than 80%.2. The factors that impact the size of ODN/DPP nanoparticles and the study of stability.With the increase of concentration of phosphate buffer in solvents, the sizes of ODN/DPP nanoparticles increased sharply. The p H value of solvents didn’t significantly influence the sizes of these nanoparticles except for DPP2. The circular dichroism spectra showed that the secondary structures of DPPs in salt solutions and in water were obviously different, which could lead to assembles of DPPs and increase the size of ODN/DPP nanoparticles. The secondary structure of DPP2 in acidic solvent was different from that in water, which led to the larger size of ODN/DPP2 nanoparticle. When stored at 4°C, the size of the nanoparticles kept stable within 2 weeks.3. The delivery efficiency and characterization of ODN/DPP nanoparticles on bacteriaThe hydrophilic DPP1, DPP4, DPP5 and DPP6 had low delivery efficiency in our experiments, the positive ratios of all the tested strains were less than 50%, however, the amphipathic DPP2, DPP3 and DPP7 higher delivery efficiency on all these strains, most of the positive ratios of all the tested strains were more than 90%. The delivery process of these amphipathic ODN/DPP nanoparticles on ESBLs-E. coli was very fast, reaching the highest efficiency within 5 to 10 min; but the process was slightly slower on MRSA, which reached the highest positive ratio after co-incubating for 30 min.4. The inhibition of the expression of targeted gene and the retardation of growth ofbacteria by ODN/DPP nanoparticlesAmphipathic ODN/DPP and ODN/DPP/DOTAP nanoparticles could significantly inhibited the expression of targeted gene in ESBLs-E. coli, as well as retarded the growth of ESBLs-E. coli, and the latter had higher efficiency.5. The analysis of the roles of the components of ODN/DPP nanoparticles and themechanism of penetrating cell membrane of nanoparticlesThere was no significant difference between the delivery efficiency of ODN/DPP2 and ODN/DPP/DOTAP nanoparticles, but ODN/DOTAP could not delivery PS-ODN into bacteria. The results indicated that DPP was account for the penetrating cell membrane, while DOTAP at the tested dose didn’t affect the delivery efficiency. When incubated with ODN/DPP nanoparticles at different temperature, the positive ratios of bacteria were the same, indicateing that the delivery process of DPP was energy independent. On ESBLs-E. coli, all the three inhibitors of endocytosis didn’t influence the delivery efficiency of ODN/DPP nanoparticles; on MRSA, however, chlorpromazine, the inhibitor of clathrin-mediated endocytosis, could significantly decrease the delivery efficiency of ODN/DPP nanoparticles, but amiloride, the inhibitor of micropinocytosis, and genistein, the inhibitors of caveolin-mediated endocytosis, didn’t influence the delivery efficiency of these nanoparticles.CONCLUSIONS 1. ODN/DPP nanoparticles was spherical-like with size scale ranging from 45 nm to 80nm when N: P was 8, and had high encapsulation and stability. The concentration ofion could significantly influence the size of nanoparticles and was the key factors inthe preparation of ODN/DPP nanoparticles. 2. The nanoparticles prepared by amphipathic DPP2, DPP3 and DPP7 had much higherdelivery efficiency than hydrophilic DPP1, DPP4, DPP5 and DPP6 and couldsignificantly inhibit the expression of targeted gene and retard the growth of bacteriain the presence of DOTAP. 3. The delivery mechanisms of amphipathic ODN/DPP nanoparticles on Gram-negativeand Gram-positive bacteria were different. 4. It was confirmed that DOTAP could enhanced the inhibitory effects of amphipathicODN/DPP nanoparticles on the expression of targeted gene and the retardation ofbacterial growth. 5. The amphipathic ODN/DPP/DOTAP nanoparticles has potential application value asan ideal delivery system for small nucleic acids.