Comparison of in vivo pharmacokinetic behaviors of R- and S-flurbiprofen after intravenous injection of flurbiprofen axetil.

Flurbiprofen axetil (FA) is a prodrug of flurbiprofen (FP), and it is hydrolyzed to the active FP by carboxylesterase in plasma after intravenous injection. The pharmacological action of FP is closely related to its chirality, and S-FP shows better analgesic effects than R-FP. Therefore, it is of great significance to compare the in vivo pharmacokinetic behaviors of R-FP and S-FP. In this study, we designed a sensitive high performance liquid chromatography-tandem mass spectrometry method and used CHIRALPAK-IG3 column for chiral separation to quantify the concentrations of R-FP and S-FP in rat plasma. The results show that this method can accurately and effectively analyze the contents of R-FP and S-FP in plasma. In addition, the systemic exposure was approximately 3.09-folds for the S-FP compared with the R-FP following intravenous administration of the FA to rats at a single dose of 4.5 mg/kg. More importantly, the clearance rate of S-FP is significantly smaller than that of R-FP. Therefore, the development of S-FA injectable emulsion for clinical treatment of postoperative pain is very necessary.

Interaction kinetics of peptide lipids-mediated gene delivery.

BACKGROUND: During the course of gene transfection, the interaction kinetics between liposomes and DNA is speculated to play very important role for blood stability, cellular uptake, DNA release and finally transfection efficiency. RESULTS: As cationic peptide liposomes exhibited great gene transfer activities both in vitro and in vivo, two peptide lipids, containing a tri-ornithine head (LOrn3) and a mono-ornithine head (LOrn1), were chosen to further clarify the process of liposome-mediated gene delivery in this study. The results show that the electrostatically-driven binding between DNA and liposomes reached nearly 100% at equilibrium, and high affinity of LOrn3 to DNA led to fast binding rate between them. The binding process between LOrn3 and DNA conformed to the kinetics equation: y = 1.663631 × exp (- 0.003427x) + 6.278163. Compared to liposome LOrn1, the liposome LOrn3/DNA lipoplex exhibited a faster and more uniform uptake in HeLa cells, as LOrn3 with a tri-ornithine peptide headgroup had a stronger interaction with the negatively charged cell membrane than LOrn1. The efficient endosomal escape of DNA from LOrn3 lipoplex was facilitated by the acidity in late endosomes, resulting in broken carbamate bonds, as well as the "proton sponge effect" of the lipid. CONCLUSIONS: The interaction kinetics is a key factor for DNA transfection efficiency. This work provided insights into peptide lipid-mediated DNA delivery that could guide the development of the next generation of delivery systems for gene therapeutics.

Novel carbamate-linked quaternary ammonium lipids containing unsaturated hydrophobic chains for gene delivery.

In this paper, two novel carbamate-linked quaternary ammonium lipids (MU18: a lipid with a mono-ammonium head; GU18: a lipid with a Gemini-ammonium head) containing unsaturated hydrophobic chains were designed and synthesized. The chemical structures of the synthetic lipids were characterized by infrared spectrum, ESI-MS, 1H NMR, 13C NMR, and HPLC. For investigating the effect of unsaturation on gene delivery, the previous reported saturated cationic liposomes (MS18 and GS18) were used as comparison. Cationic liposomes were prepared by using these cationic lipids and neutral lipid DOPE at the molar ratio of 1:1. Particle sizes and zeta potentials of the cationic liposomes were studied to show that they were suitable for gene transfection. The binding abilities of the cationic liposomes were investigated by gel electrophoresis at various N/P ratios from 0.5/1 to 8/1. The results indicated that the binding ability of GU18 was much better than MU18 and the saturated cationic liposomes (MS18 and GS18). DNA transfection of these liposomes comparable to commercially available reagent (DOTAP) was achieved in vitro against Hela, HepG-2 and NCI-H460 cell lines. GU18 showed higher transfection at the N/P ratio of 3/1 than other cationic liposomes and the positive control, DOTAP. All of the liposomes presented a relatively low cytotoxicity, which was measured by MTT. Therefore, the synthetic lipids bearing unsaturated hydrophobic chains and Gemini-head could be promising candidates for gene delivery.

Cationic lioposomes with folic acid as targeting ligand for gene delivery.

In our previous Letter, we have carried out the synthesis of a novel DDCTMA cationic lipid which was formulated with DOPE for gene delivery. Herein, we used folic acid (FA) as targeting ligand and cholesterol (Chol) as helper lipid instead of DOPE for enhancing the stability of the liposomes. These liposomes were characterized by dynamic laser scattering (DLS), transmission electron microscopy (TEM) and agarose gel electrophoresis assays of pDNA binding affinity. The lipoplexes were prepared by using different weight ratios of DDCTMA/Chol (1:1, 2:1, 3:1, 4:1) liposomes and different concentrations of FA (50-200µg/mL) combining with pDNA. The transfection efficiencies of the lipoplexes were evaluated using pGFP-N2 and pGL3 plasmid DNA against NCI-H460 cells in vitro. Among them, the optimum gene transfection efficiency with DDCTMA/Chol (3:1)/FA (100µg/mL) was obtained. The results showed that FA could improve the gene transfection efficiencies of DDCTMA/Chol cationic liposome. Our results also convincingly demonstrated FA (100µg/mL)-coated DDCTMA/Chol (3:1) cationic liposome could serve as a promising candidate for the gene delivery.

Transmembrane routes of cationic liposome-mediated gene delivery using human throat epidermis cancer cells.

For studying the mechanism of cationic liposome-mediated transmembrane routes for gene delivery, various inhibitors of endocytosis were used to treat human throat epidermis cancer cells, Hep-2, before transfection with Lipofectamine 2000/pGFP-N2 or Lipofectamine 2000/pGL3. To eliminate the effect of inhibitor toxicity on transfection, the RLU/survival rate was used to represent the transfection efficiency. Chlorpromazine and wortmannin, clathrin inhibitors, decreased transfection efficiency by 44 % (100 µM) and 31 % (100 nM), respectively. At the same time, genistein, a caveolin inhibitor, decreased it by 30 % (200 µM). Thus combined transmembrane routes through the clathrin and caveolae-mediated pathways were major mechanisms of cell uptake for the cationic liposome-mediated gene delivery. After entering the cells, microtubules played an important role on gene delivery as vinblastine, a microtubulin inhibitor, could reduce transfection efficiency by 41 % (200 nM).

The headgroup evolution of cationic lipids for gene delivery.

Cationic lipids are one of the most widely used nonviral vectors for gene delivery and are especially attractive because they can be easily synthesized and extensively characterized. Additionally, they can best facilitate the elucidation of structure-activity relationships by modifying each of their constituent domains. The polar hydrophilic headgroups enable the condensation of nucleic acids by electrostatic interactions with the negatively charged phosphate groups of the genes, and further govern transfection efficiency. The headgroups of cationic lipids play a crucial role for gene delivery; they can be quaternary ammoniums, amines, aminoacids or peptides, guanidiniums, heterocyclic headgroups, and some unusual headgroups. This review summarizes recent research results concerning the nature (such as the structure and shape of cationic headgroup) and density (such as the number and the spacing of cationic headgroup) of head functional groups for improving the design of efficient cationic lipids to overcome the critical barriers of in vitro and in vivo transfection.

Dual functionalized hyaluronic acid micelles loading paclitaxel for the therapy of breast cancer.

Although many carriers for the delivery of chemotherapeutic drugs have been investigated, the disadvantages of passive targeting and uncontrolled drug release limit their utility. Herein, hyaluronic acid (HA) was hydrophobically modified to serve as a carrier for binding to cluster determinant 44 (CD44) overexpressed on tumor cell surfaces. Specifically, after deacetylation, HA was grafted to dodecylamine or tetradecylamine to afford amphiphilic zwitterionic polymer micelles, designated dHAD and dHAT, respectively, for the delivery of paclitaxel (PTX). The micelles were negatively charged at pH 7.4 and positively charged at pH 5.6, and this pH sensitivity facilitated PTX release under acidic conditions. The cell uptake efficiencies of the dHAD-PTX and dHAT-PTX micelles by MCF-7 cells after 4 h of incubation were 96.9% and 95.4%, respectively, and their affinities for CD44 were twice that of HA. Furthermore, the micelles markedly inhibited tumor growth both in vitro and in vivo, with IC50 values of 1.943 µg/mL for dHAD-PTX and 1.874 µg/mL for dHAT-PTX for MCF-7 cells; the tumor inhibition rate of dHAD-PTX (92.96%) was higher than that of dHAT-PTX (78.65%). Importantly, dHAD and dHAT micelles showed negligible systemic toxicity. Our findings suggest that these micelles are promising delivery vehicles for antitumor drugs.

Synthesis and biological activity of carbamate-linked cationic lipids for gene delivery in vitro.

We have introduced a convenient synthesis method for carbamate-linked cationic lipids. Two cationic lipids N-[1-(2,3-didodecylcarbamoyloxy)propyl]-N,N,N-trimethylammonium iodide (DDCTMA) and N-[1-(2,3-didodecyl carbamoyloxy)propyl]-N-ethyl-N,N-dimethylammonium iodide (DDCEDMA), with identical length of hydrocarbon chains, alternative quaternary ammonium heads, carbamate linkages between hydrocarbon chains and quaternary ammonium heads, were synthesized for liposome-mediated gene delivery. Liposomes composed of DDCEDMA and DOPE in 1:1 ratio exhibited a lower zeta potential as compared to those made of pure DDCEDMA alone, which influences their DNA-binding ability. pGFP-N2 plasmid was transferred by cationic liposomes formed from the above cationic lipids into Hela and Hep-2 cells, and the transfection efficiency of some of cationic liposomes was superior or parallel to that of two commercial transfection agents, Lipofectamine2000 and DOTAP. Combined with the results of the agarose gel electrophoresis and transfection experiment, the DNA-binding ability of cationic lipids was too strong to release DNA from complex in the transfection, which could lead to relative low transfection efficiency and high cytotoxicity.

Non-viral vectors for the mediation of RNAi.

Though the delivery of siRNA into cells, tissues or organs remains to be a big obstacle for its applications, recently siRNA therapeutics has developed rapidly and already there are clinical trials ongoing or planned. Some non-viral vectors have attracted much more attention and shown the great potential for combating the delivery obstacle. As a novel class of lipid like materials lipidoids have the advantages of easy synthesis and large library of compounds. Cell penetrating peptides and chitosans have been used for the delivery of bioactive molecules for many years, but they are showing great promise for the delivery of siRNA. The hybrids of inorganic particles and the conjugates of siRNA have indicated the complex utilization different materials may provide another solution to the delivery problem. The most exciting thing is some clinical trials are undergoing, which provokes the hope of real curing method by using RNAi mediated by some non-viral vectors.

Structure-activity relationships of pH-responsive and ionizable lipids for gene delivery.

Ionizable lipids are the leading vectors for gene therapy. Understanding the effects of molecular structure on efficient gene delivery is one of the most important challenges for maximizing the utility of such lipid vectors. We synthesized an array of pH-responsive and ionizable lipids to investigate the relationship between lipid structure and activity. The optimized lipid (EDM) has double tertiary amines in the headgroup and an ester linker. EDM exhibited efficient DNA and siRNA delivery to, and gene silencing of, A549 cells. EDM has a pKa value of 6.67, which enabled it to quickly escape from the endosome after entering the cell; the ester linkages rapidly degraded and enabled gene release into the cytoplasm. EDM also delivered IGF-1R siRNA to inhibit tumor growth and induce cancer cell apoptosis by efficient inhibition of IGF-1R expression in mice. Our study on the structure-activity relationships of ionizable lipids will facilitate clinical applications.

pH-sensitive, tail-modified, ester-linked ionizable cationic lipids for gene delivery.

Ionizable cationic lipids have great potential for gene delivery, yet the effect of the molecular structure of such lipids on gene delivery efficiency is an ongoing research challenge. To better understand corresponding structure-function activity relationships, we synthesized four ester-linked, pH-responsive, ionizable cationic lipids. The screened DEDM4 lipid, containing 2-ethylenedimethylamine in the headgroup and a branched-chain tail, exhibited a high delivery efficacy of plasmid DNA and siRNA in A549 cells, which was comparable with that of the commercial reagent lipofectamine 3000 (lipo3000). Moreover, because of its pKa value of 6.35 and pH-sensitivity under acidic conditions, DEDM4 could carry sufficient positive charge in the acidic environment of endosomes and interact with the endosome lumen, leading to destruction of the endomembrane and subsequent release of siRNA into the cytoplasm with endosomal escape. Furthermore, we used DEDM4 to deliver IGF-1R siRNA to induce cancer cell apoptosis, thereby leading to great tumor inhibition. More importantly, it also showed very low toxicity in vivo. These structure-activity data for DEDM4 demonstrate potential clinical applications of DEDM4-mediated gene delivery for cancer.

pH-sensitive hyaluronic acid-targeted prodrug micelles constructed via a one-step reaction for enhanced chemotherapy.

Although many chemotherapy prodrugs have been developed for tumor therapy, non-targeted delivery, uncontrolled release and tedious construction procedure of prodrugs still limit their clinical application in tumor treatment. In this work, hyaluronic acid (HA) which has tumor-targeting ability was used to conjugate to antitumor drug podophyllotoxin (PPT) to construct a pH-sensitive prodrug named HA-CO-O-PPT just via a one-step esterification reaction. The HA-CO-O-PPT spontaneously assembled into nano spherical micelles in aqueous medium, which had outstanding serum stability and blood compatibility. The obtained prodrug micelles (named HP micelles) exhibited a pH-responsive drug release mode with cumulative release reaching 81.2% due to their dissociation in response to acid stimulus, and had a high cellular uptake efficiency beyond 97% owing to HA receptor-mediated targeting. Furthermore, it was found that the prodrug micelles showed excellent antitumor activities in vivo with the tumor inhibition ratio up to 85% and negligible systemic toxicity. Accordingly, the pH-responsive HP micelles constructed by a simple one-step reaction, could be a promising candidate as a chemotherapeutic agent for cancer therapy.

Microneedles for gene and drug delivery in skin cancer therapy.

The success of skin cancer treatment is severely limited in the route of administration because most genes and drugs cannot break through the excellent stratum corneum (SC) barrier of the skin, leading to their inability to reach the tumor tissues at therapeutic doses. As an excellent minimally invasive delivery method, microneedles (MNs) can bypass the SC and enter the skin microcirculation to achieve drug and gene delivery when used to puncture the skin. Compared with traditional administration approaches, MN-assisted gene and drug delivery have obvious advantages, in that they are simple, safe, painless, easily transport genes (such as DNA and siRNA (small interfering RNA)) and macromolecule drugs (including proteins and antibodies), and have good reproducibility. Besides, other treatment strategies including photothermal therapy (PTT) have been combined with MN arrays containing genes or drugs, which is expected to improve the therapeutic effect of skin cancer. Therefore, this review summarizes the latest developments in MNs for gene and/or drug delivery, with a focus on their performances as effective MNs for skin cancer treatment.

Integrin αvß3-targeted liposomal drug delivery system for enhanced lung cancer therapy.

Conventional chemotherapy for tumor treatment remains flawed because it fails to limit cytotoxicity to a small set of selectable tissues. Active targeting techniques for the delivery of drugs to specific sites are increasingly used to enhance drug accumulation at tumor sites with the aim of reducing side effects in vivo. Liposomes, modified with different targeting ligands, are considered to be one of the most promising targeted drug carriers. Herein, novel linear and cyclic arginine-glycine-aspartate (RGD) peptide-based lipids were synthesized to develop modified liposomal drug delivery systems with active targeting and pH-sensitivity. The RGD-modified liposomes showed excellent active targeting ability for integrin αvß3 receptors, resulting in improved cellular uptake. The modified liposomes also enhanced intracellular doxorubicin (DOX) release because of their degradation in an acidic environment. Consequently, the RGD-modified, DOX-loaded liposomes exhibited significant antitumor efficacy and low toxicity in vitro and in vivo. In particular, 5% cRGD-lipid modified DOX-loaded liposome showed the greatest inhibition of tumor growth in mice among the tested formulations, and much less toxicity than free DOX. In conclusion, the DOX-loaded pH-sensitive liposome modified with 5% cRGD-lipid developed in the current study provides a potential approach for improved tumor therapy.

Transfection efficiency of cationic lipids with different hydrophobic domains in gene delivery.

The structure of cationic lipids is a major factor for their transfection activity. A cationic lipid generally contains four functional domains: a hydrophilic headgroup, a linker, a backbone domain, and a hydrophobic domain. The structure of the hydrophobic domain determines the phase transition temperature and the fluidity of the bilayer and influences the stability of liposomes, the DNA protection from nucleases, the endosomal escape, the DNA release from complex, and the nuclear penetration. Also, toxicity of the lipids is influenced by the hydrophobic domain. The compounds used for gene delivery are classified according to the structure of the hydrophobic domain as follows: aliphatic chains, steroid domain, and fluorinated domain. In this review, we summarized recent research results concerning the structures of the hydrophobic domain, in order to find the effect of the hydrophobic domain on transfection efficiency. Understanding these would be very important for scientists to prepare novel cationic lipids and design novel formulations with high transfection efficiency.

Targeting strategies for superparamagnetic iron oxide nanoparticles in cancer therapy.

Among various nanoparticles, superparamagnetic iron oxide nanoparticles (SPIONs) have been increasingly studied for their excellent superparamagnetism, magnetic heating properties, and enhanced magnetic resonance imaging (MRI). The conjugation of SPIONs with drugs to obtain delivery nanosystems has several advantages including magnetic targeted functionalization, in vivo imaging, magnetic thermotherapy, and combined delivery of anticancer agents. To further increase the targeting efficiency of drugs through a delivery nanosystem based on SPIONs, additional targeting moieties including transferrin, antibodies, aptamers, hyaluronic acid, folate, and targeting peptides are coated onto the surface of SPIONs. Therefore, this review summarizes the latest progresses in the conjugation of targeting molecules and drug delivery nanosystems based on SPIONs, especially focusing on their performances to develop efficient targeted drug delivery systems for tumor therapy. STATEMENT OF SIGNIFICANCE: Some magnetic nanoparticle-based nanocarriers loaded with drugs were evaluated in patients and did not produce convincing results, leading to termination of clinical development in phase II/III. An alternative strategy for drug delivery systems based on SPIONs is the conjugation of these systems with targeting segments such as transferrin, antibodies, aptamers, hyaluronic acid, folate, and targeting peptides. These targeting moieties can be recognized by specific integrin/receptors that are overexpressed specifically on the tumor cell surface, resulting in minimizing dosage and reducing off-target effects. This review focuses on magnetic nanoparticle-based nonviral drug delivery systems with targeting moieties to deliver anticancer drugs, with an aim to provide suggestions on the development of SPIONs through discussion.

Photothermal therapy.

Microneedles (MNs), as an effective minimally invasive delivery route, when used to puncture the skin, can bypass the skin's stratum corneum (SC) to enter the skin microcirculation and achieve systemic administration. Additionally, the MN route has obvious advantages over other routes of administration, including simplicity, non-pain, readily-permitted transport of drugs (including DNA and metformin) and macromolecules (such as antibodies and proteins), good repeatability, and wide range of clinical applications and safety. MNs have been combined with various therapy strategies including photodynamic therapy (PDT) and photothermal therapy (PTT) to treat many diseases, and hold great promise for improving the diagnosis and treatment of diseases. Both MN-assisted PDT and PTT are light-mediated phototherapy methods and have unique advantages, including improved selectivity, and minimal invasiveness and side effects. MN-assisted PDT or PTT has been studied for various applications by many research groups and pharmaceutical companies worldwide. Therefore, this review summarizes recent advances in MNs for PDT or PTT.

Co-delivery of paclitaxel and anti-VEGF siRNA by tripeptide lipid nanoparticle to enhance the anti-tumor activity for lung cancer therapy.

The combination of chemotherapeutic drug paclitaxel (PTX) and VEGF siRNA could inhibit cancer development with synergistic efficacy. However, efficient and safe delivery systems with high encapsulation efficiency of PTX and a long-time release of drugs are urgently needed. In this study, novel nanoparticles (PTX/siRNA/FALS) were constructed by using tripeptide lipid (L), sucrose laurate (S), and folate-PEG2000-DSPE (FA) to co-deliver PTX and siRNA. The cancer cell targeting nanoparticle carrier (PTX/siRNA/FALS) showed anticipated PTX encapsulation efficiency, siRNA retardation ability, improved cell uptake and sustained and controlled drug release. It led to significant anti-tumor activity in vitro and in vivo by efficient inhibition of VEGF expression and induction of cancer cell apoptosis. Importantly, the biocompatibility of the carriers and low dosage of PTX required for effective therapy greatly reduced the toxicity to mice. The targeting nanoparticles show potential as an effective co-delivery platform for RNAi and chemotherapy drugs, aiming to improve the efficacy of cancer therapy.

Stimuli-Responsive Polysaccharide Enveloped Liposome for Targeting and Penetrating Delivery of survivin-shRNA into Breast Tumor.

Silencing the inhibitor of apoptosis (IAP) by RNAi is a promising method for tumor therapy. One of the major challenges lies in how to sequentially overcome the system barriers in the course of the tumor targeting delivery, especially in the tumor accumulation and penetration. Herein we developed a novel stimuli-responsive polysaccharide enveloped liposome carrier, which was constructed by layer-by-layer depositing redox-sensitive amphiphilic chitosan (CS) and hyaluronic acid (HA) onto the liposome and then loading IAP inhibitor survivin-shRNA gene and permeation promoter hyaluronidase (HAase) sequentially. The as-prepared HA/HAase/CS/liposome/shRNA (HCLR) nanocarrier was verified to be stable in blood circulation due to the negative charged HA shield. The tumor targeting recognition and the enhanced tumor accumulation of HCLR were visualized by fluorescence resonance energy transfer (FRET) and in vivo fluorescence biodistribution. The deshielding of HA and the protonizing of CS in slightly acidic tumor extracellular pH environment (pHe, 6.8-6.5) were demonstrated by ζ potential change from -23.1 to 29.9 mV. The pHe-responsive HAase release was confirmed in the tumor extracellular mimicking environments, and the intratumoral biodistribution showed that the tumor penetration of HCLR was improved. The cell uptake of HCLR in pHe environment was significantly enhanced compared with that in physiological pH environment. The increased shRNA release of HCLR was approved in 10 mM glutathione (GSH) and tumor cells. Surprisingly, HCLR suppressed the tumor growth markedly through survivin silencing and meanwhile maintained low toxicity to mice. This study indicates that the novel polysaccharide enveloped HCLR is promising in clinical translation, thanks to the stimuli-triggered tumor accumulation, tumor penetration, cell uptake, and intracellular gene release.

[Comparison of two kinds of cationic vectors-mediated gene delivery].

In order to study the important factors involved in cationic liposome-mediated gene transfer, Lipofectamine 2000 or DOTAP was evaluated using three types of cells (Hep-2, MCF-7 and SW-480) in vitro transfection efficiencies. Different properties of the two reagents were analyzed and compared by DNA arrearage assay and MTT assay. Both Lipofectamine 2000 and DOTAP had strong capability to combine with DNA; Lipofectamine 2000 can get higher transfection efficiency of the three cells by using GFP as report gene, meanwhile, DOTAP can also get higher transfection efficiency against Hep-2 cell. However, DOTAP showed lower transfection efficiency against MCF-7 and SW-480 cell. On the other hand, the cytotoxicity assay showed that over 85% cell viability of MCF-7 cell could be achieved both by Lipofectamine 2000 and DOTAP under the optimal transfection condition. Relatively speaking, Lipofectamine 2000 has very high transfection efficiency in a broad range of cell lines, but because of the special selectivity of cell type on liposome, DOTAP also has a broad application prospect.