The purpose of the study is to construct R8 peptide (RRRRRRRR) and pH sensitive polyethylene glycols (PEG) co-modified liposomes (Cl-Lip) and utilize them in breast cancer treatment. The co-modified liposomes were prepared with soybean phospholipid, cholesterol, DSPE-PEG2K-R8 and PEG5K-Hz-PE (pH sensitive PEG). The size and zeta potential of Cl-Lip were also characterized. The in vitro experiment demonstrated that the Cl-Lip had high serum stability in 50% fetal bovine serum. The cellular uptake of Cl-Lip under different pre-incubated conditions was evaluated on 4T1 cells. And the endocytosis pathway, lysosome escape ability and tumor spheroid penetration ability were also evaluated. The results showed the particle size of the Cl-Lip was (110.4 ± 5.2) nm, PDI of the Cl-Lip was 0.207 ± 0.039 and zeta potential of the Cl-Lip was (-3.46 ± 0.05) mV. The cellular uptake of Cl-Lip on 4T1 cells was pH sensitive, as the cellular uptake of Cl-Lip pre-incubated in pH 6.0 was higher than that of pH 7.4 under each time point. The main endocytosis pathways of Cl-Lip under pH 6.0 were micropinocytosis and energy-dependent pathway. At the same time, the Cl-Lip with pre-incubation in pH 6.0 had high lysosome escape ability and high tumor spheroid penetration ability. All the above results demonstrated that the Cl-Lip we constructed had high pH sensitivity and is a promising drug delivery system.
Animals ; Cell Line, Tumor ; Cell-Penetrating Peptides ; chemical synthesis ; chemistry ; Cholesterol ; chemistry ; Drug Delivery Systems ; Liposomes ; Mice ; Oligopeptides ; chemical synthesis ; chemistry ; Particle Size ; Phospholipids ; chemistry ; Polyethylene Glycols

OBJECTIVETo synthesize a biodegradable non-viral gene carrier with a high transfection efficiency and a low cytotoxicity.

METHODSPoly(ethylene glycol)-block-(poly(L-glutamic acid)-graft-polyethylenimine) was prepared via ammonolysis of poly(ethylene glycol)-block-poly (γ-benzyl L-glutamate) with the low-molecular-mass polyethylenimine (600 Da). The synthesized copolymer was characterized by 1H nuclear magnetic resonance spectroscopy and gel permeation chromatography. The polyplex micelle from PEG-b-(PG-g-PEI) and plasmid DNA (pDNA) was studied using dynamic light scattering, zeta-potential measurements, and gel retardation assay. The in vitro cytotoxicity and transfection efficiency of PEG-b-(PG-g-PEI) were tested by MTT assay and luciferase assay in HEK 293T cells using PEI (25 kDa) as the control.

RESULTSPEG-b-(PG-g-PEI) could efficiently condense DNA into nanosized particles with positive surface charges when the N/P ratio of polymer and DNA was above 5:1. The zeta potential of the polyplexes was about 25 mV, and the particle size was 120 nm at a N/P ratio of 10. The cell toxicity and gene transfection evaluations showed a lower cytotoxicity and a higher gene transfection efficiency of the copolymer than PEI 25000 in HEK 293T cells.

CONCLUSIONSThe polymer can be used as a potential non-viral gene carrier for gene therapy.

Cell Survival ; Gene Transfer Techniques ; Genetic Vectors ; Glutamic Acid ; chemistry ; HEK293 Cells ; Humans ; Particle Size ; Plasmids ; Polyethylene Glycols ; chemical synthesis ; chemistry ; Polyethyleneimine ; analogs & derivatives ; chemical synthesis ; chemistry ; Polyglutamic Acid ; analogs & derivatives ; chemical synthesis ; chemistry ; Polymers ; Transfection

A novel amphiphilic copolymer, folate-poly (PEG-cyanoacrylate-co-cholesteryl cyanoacrylate) (FA-PEG-PCHL) was synthesized as liposomal modifying material with folate receptor targeting and long circulating property. FA-PEG-PCHL-modified docetaxel-loaded liposomes (FA-PDCT-L) were prepared by organic solvent injection method, and the system was optimized using central composite design-response surface methodology. The structure of the FA-PEG-PCHL copolymer was confirmed by FT-IR and 1H NMR. Ultrafiltration technique, transmission electron microscope, dynamic light scattering and electrophoretic light scattering, and fluorescence polarization method were used to study the physicochemical parameters of FA-PDCT-L. FA-PDCT-L showed spherical or ellipsoid shape. The mean particle sizes were in the range of 111.6-126.9 nm, zeta potentials were from -6.54 mV to -14.13 mV and the drug encapsulation efficiency achieved 97.8%. The observed values agreed well with model predicted values. The membrane fluidity increased with the increment of the molecular weight of PEG and the decrement of the amount of FA-PEG-PCHL. The in vitro release test showed that the drug could be sustained-released from liposomes without a burst release and with stability for 6 months. After 24 h only 31.1%, 27.2% and 19.5% of encapsulated docetaxel were released for FA-PDCT10000-L, FA-PDCT4000-L and FA-PDCT2000-L, respectively. This work is useful for further research on the application of the synthesized copolymer-modified long circulating liposomes for cancer therapy.
Antineoplastic Agents ; administration & dosage ; Cholesterol Esters ; chemistry ; Cyanoacrylates ; chemistry ; Delayed-Action Preparations ; Drug Carriers ; chemical synthesis ; chemistry ; Drug Delivery Systems ; Folate Receptors, GPI-Anchored ; chemistry ; Liposomes ; administration & dosage ; chemistry ; Molecular Weight ; Particle Size ; Polyethylene Glycols ; chemistry ; Polymers ; chemical synthesis ; chemistry ; Taxoids ; administration & dosage

This study is to prepare the in situ forming sustained-release injection which can perform sustained release behavior at the periodontal site for 7 days and to evaluate its in vitro and in vivo properties. After preparation of in situ forming sustained-release injection the in situ time was studied. And the surface of the solid injection was characterized by SEM. The rheological curve at 0 degrees C, 25 degrees C, 37 degrees C was determined and the impact of the temperature on the viscosity was examined. The in vitro release behavior was investigated. At last, rabbit periodontitis model was established to study its pharmacokinetics. The injection was stable, hard to stratify and decompose. The in situ forming time was about 6 seconds. It can easily adhere into periodontal pockets. There were lots of holes on the surface of the solid injection for the drug to diffuse. The drug releasing curves could be fit by Korsmeyer-Peppas equation. The drug smoothly released for 7 days at pH 7.4 PBS buffer with a very slight burst release and maintained a certain concentration. In vivo pharmacokinetics results indicated that after administration with the in situ forming injection, achievement of tinidazole (TNZ) concentration in gingival crevicular fluid (GCF) was more comparable and long-lasting than usual solution of TNZ management and relatively constant TNZ levels were attained until 168 h. All these results supported the prospect of tinidazole in situ forming sustained-release injection in clinical applications.
Animals ; Antitrichomonal Agents ; administration & dosage ; pharmacokinetics ; Delayed-Action Preparations ; Drug Carriers ; Drug Compounding ; methods ; Endotoxins ; Gingival Crevicular Fluid ; metabolism ; Injections ; Periodontal Pocket ; metabolism ; Periodontitis ; chemically induced ; metabolism ; Polyesters ; chemical synthesis ; pharmacokinetics ; Polyethylene Glycols ; chemical synthesis ; pharmacokinetics ; Rabbits ; Random Allocation ; Rheology ; Tinidazole ; administration & dosage ; pharmacokinetics

Polyamidoamine-polyethylene glycol (PAMAM-PEG) copolymers were synthesized using IPDI as coupling reagent by two-step method. The copolymers were characterized by IR spectrum and 1H NMR spectrum, and the PEG conjugating ratios of the copolymers were calculated equal to 10% and 30% separately. MTT assay indicated that after PEGylation a lower cytotoxicity of the copolymers could be found, and with increasing PEG conjugating ratio the cytotoxicity decreased obviously. Agarose gel retardation assay demonstrated that PAMAM-PEG copolymers could be combined with DNA and PAMAM-PEG/DNA complexes were prepared by self-assembly. DLS measurement showed that when N/P > or = 50, the particle size of copolymer/ gene complexes was in a range of 150-200 nm, and the zeta potential was in a range of 10-25 mV. In vitro gene transfection illustrated that when N/P < or = 50, the gene transfection efficiency of PAMAM-PEG copolymers was a little less than that of PAMAM-G5, but the transfection efficiency can be raised by increasing N/P ratio or transfection time. Considering both cytotoxicity and transfection efficiency aspects PAMAM-PEG-13 was more effect than PAMAM-PEG-39 in PEGylation.
Carcinoma, Hepatocellular ; pathology ; Cell Line, Tumor ; Cell Survival ; drug effects ; DNA ; chemistry ; pharmacology ; Dendrimers ; chemical synthesis ; pharmacology ; Gene Transfer Techniques ; Genetic Vectors ; Humans ; Isocyanates ; chemistry ; Liver Neoplasms ; pathology ; Particle Size ; Polyamines ; chemistry ; Polyethylene Glycols ; chemical synthesis ; chemistry ; pharmacology ; Transfection

A new class of dendrimer polylysine poly(ethylene glycol)-lipid was designed and synthesized. The cationic polymer liposomes were prepared by the lipid film-extrusion and post-insertion two methods with these dendrimer polylysine poly(ethylene glycol)-lipid and other lipids. The structural properties of obtained cationic polymer liposomes were studied by laser light scattering and fluorescence spectrometer. It was demonstrated that the nano sized liposomes with different density of surface cationic charges can be prepared by either lipid film-extrusion or post-insertion methods, but post-insertion process did not affect drug loading, did not influence drug loading capacity and did not induce liposomal morphology and particle size. The density of positive charge does not affect the size and distribution of different liposomes size and distribution. It was the better choice for manufacture because post-insertion method did not cause early release of drug and size changes. Cell binding experiments show that cationic polymer liposomes, especially dendrimer polymer liposomes had higher local charge density, and therefore have dramatic non specific cell targeting ability.
Animals ; Biological Transport ; Cations ; Cell Line ; Cricetinae ; Drug Delivery Systems ; Kinetics ; Lipids ; chemistry ; pharmacokinetics ; Liposomes ; chemical synthesis ; chemistry ; pharmacokinetics ; Molecular Structure ; Nanoparticles ; Particle Size ; Polyethylene Glycols ; chemistry ; pharmacokinetics ; Polymers ; chemistry ; pharmacokinetics

The purpose of this study was to develop a novel polymer cuff for the local delivery of alpha-lipoic acid (ALA) to inhibit neointimal formation in vivo. The polymer cuff was fabricated by incorporating the ALA into poly-(D,L-lactide-co-caprolactone) 40:60 (PLC), with or without methoxy polyethylene glycol (MethoxyPEG). The release kinetics of ALA and in vitro degradation by hydrolysis were analyzed by HPLC and field emission scanning electron microscopy (FE-SEM), respectively. In vivo evaluation of the effect of the ALA-containing polymer cuff was carried out using a rat femoral artery cuff injury model. At 24 h, 48% or 87% of the ALA was released from PCL cuffs with or without MethoxyPEG. FE-SEM results indicated that ALA was blended homogenously in the PLC with MethoxyPEG, whereas ALA was distributed on the surface of the PLC cuff without MethoxyPEG. The PLC cuff with MethoxyPEG showed prolonged and controlled release of ALA in PBS, in contrast to the PLC cuff without MethoxyPEG. Both ALA-containing polymer cuffs had a significant effect on the inhibition of neointimal formation in rat femoral artery. Novel ALA-containing polymer cuffs made of PLC were found to be biocompatible and effective in inhibiting neointimal formation in vivo. Polymer cuffs containing MethoxyPEG allowed the release of ALA for one additional week, and the rate of drug release from the PLC could be controlled by changing the composition of the polymer. These findings demonstrate that polymer cuffs may be an easy tool for the evaluation of anti-restenotic agents in animal models.
Animals ; Coronary Restenosis/*therapy ; Delayed-Action Preparations ; Male ; Materials Testing ; Polyesters/*administration & dosage/*chemical synthesis ; Polyethylene Glycols/chemical synthesis ; Rats ; Rats, Sprague-Dawley ; Surface Properties

Generation 4 polyamidoamine (PAMAM) dendrimer was PEGylated with polyethylene glycol (PEG) at an average molecular weight 5 000 via amide bond. PAMAM and PEGylated PAMAM (PAMAM-PEG) dendrimer were used as drug nanocarriers. Methotrexate (MTX), an antineoplastic agent, was selected as a model drug. PAMAM/MTX and PAMAM-PEG/MTX complexes were prepared. The pharmacokinetic characters and anti-tumor activity of the PAMAM-PEG/MTX complex were studied as compared with MTX injection and PAMAM/MTX complex by intravenous injection in rats and S180 tumor bearing mice, separately. The plasma samples from normal rats were analyzed by HPLC method, and concentration-time data were analyzed using a non-compartmental analysis. Their anti-tumor effects in vivo were evaluated against S180 solid tumors in mice by measuring average tumor weight and calculating the inhibitory rate of tumor on day 17 after successive injections. The results showed that both plasma half-life and mean retention time (MRT) of the complexes were longer than that of MTX injection (P<0.01), while the area under the plasma concentration vs time curve (AUC) of PAMAM-PEG/MTX was the largest as compared with that of free drug and PAMAM/MTX complex (P<0.01). The inhibitory rate of tumor of PAMAM-PEG/MTX complex enhanced 2.1 and 1.8 times over that of free drug and PAMAM/MTX complex, respectively, indicating that PAMAM-PEG/MTX exhibited the highest antitumor activity. In summary, PEGylated PAMAM could be useful as a potential drug delivery carrier.
Animals ; Antimetabolites, Antineoplastic ; blood ; pharmacokinetics ; pharmacology ; Area Under Curve ; Cell Line, Tumor ; Dendrimers ; chemical synthesis ; pharmacokinetics ; Drug Carriers ; Female ; Male ; Methotrexate ; blood ; pharmacokinetics ; pharmacology ; Mice ; Neoplasm Transplantation ; Nylons ; chemical synthesis ; pharmacokinetics ; Polyethylene Glycols ; chemistry ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Sarcoma 180 ; pathology ; Tumor Burden ; drug effects

The poly(ethylene glycol)-lipid derivatives were synthesized for constructing pH-sensitive liposomes. The polyethylene glycol polymer MePEG2000-NH2 and phospholipids POPA were connected by phosphorus-amide linkage. The poly(ethylene glycol)-lipid derivatives acidic sensitive liposomes were prepared. Factor effects on polymer insertion into liposomes were evaluated and the pH-sensitivity of the polymer associated liposomes were studied by calcein release assay. The poly(ethylene glycol)-lipid derivatives acidic sensitive liposomes were prepared successfully by the extruding linkage device. The liposomes constructing by poly(ethylene glycol)-lipid derivatives was stable at pH 6.5-7.5, the stability was closely related to phospholipid types and cholesterol content of the preparation of liposomes. At pH 5.0 occurred when divulging fluorescence occurred obviously, the leakage rate and the strength was with a positive correlation between time of in the acidic environment and intensity of acid. The acidic sensitive liposomes prepared by poly(ethylene glycol)-lipid derivatives were developed as a potential pH sensitive delivery system.
Cholesterol ; chemistry ; Drug Delivery Systems ; methods ; Drug Stability ; Hydrogen-Ion Concentration ; Liposomes ; chemical synthesis ; chemistry ; Particle Size ; Phospholipids ; chemistry ; Polyethylene Glycols ; chemistry ; Polymers

V(E) acetate-loaded methoxy poly(ethylene glycol)-b-poly(lactic acid) amphiphilic diblock copolymer nano-dispersion (PMV) was prepared by self-emulsification/solvent evaporation method. The drug-loaded amount, size distribution of PMV nanoparticles, and entrapment efficiency of V(E) acetate (V(E)A) were determined by UV and laser particle analyzer. Drug release in vitro was primarily investigated by UV. The results indicate that the size of PMV nanoparticles is less than 300 nm and PMV is largely influenced by preparation methods, property of solvents, V(E)A-fed amount, and the concentration of dispersion. The initial burst release is not observed and the accumulated release is more than 79% after 14 h. This study develops a new formulation for V(E)A and provides an experimental basis for the novel drug delivery systems of V(E)A.
Delayed-Action Preparations ; chemical synthesis ; Drug Carriers ; administration & dosage ; chemistry ; Hydrophobic and Hydrophilic Interactions ; Nanoparticles ; Polyesters ; administration & dosage ; Polyethylene Glycols ; administration & dosage ; Vitamin E ; administration & dosage