Cationic core-shell nanoparticles with carmustine contained within O6-benzylguanine shell for glioma therapy.

The application of carmustine (BCNU) for glioma treatment is limited due to its poor selectivity for tumor and tumor resistance caused by O6-methylguanine-DNA-methyl transferase (MGMT). To improve the efficacy of BCNU, we constructed chitosan surface-modified poly (lactide-co-glycolides) nanoparticles (PLGA/CS NPs) for targeting glioma, loading BCNU along with O6-benzylguanine (BG), which could directly deplete MGMT. With core-shell structure, PLGA/CS NPs in the diameter around 177 nm showed positive zeta potential. In vitro plasma stability of BCNU in NPs was improved compared with free BCNU. The cellular uptake of NPs increased with surface modification of CS and decreasing particle size. The cytotoxicity of BCNU against glioblastoma cells was enhanced after being encapsulated into NPs; furthermore, with the co-encapsulation of BCNU and BG into NPs, BCNU + BG PLGA/CS NPs showed the strongest inhibiting ability. Compared to free drugs, PLGA/CS NPs could prolong circulation time and enhance accumulation in tumor and brain. Among all treatment groups, F98 glioma-bearing rats treated with BCNU + BG PLGA/CS NPs showed the longest survival time and the smallest tumor size. The studies suggested that the co-encapsulation of BCNU and BG into PLGA/CS NPs could remarkably enhance the efficacy of BCNU, accompanied with greater convenience for therapy.

RGD-modified PEG-PAMAM-DOX conjugates: in vitro and in vivo studies for glioma.

This work was based on our recent studies that a promising conjugate, RGD-modified PEGylated polyamidoamine (PAMAM) dendrimer with doxorubicin (DOX) conjugated by acid-sensitive cis-aconityl linkage (RGD-PPCD), could increase tumor targeting by binding with the integrin receptors overexpressed on tumor cells and control release of free DOX in weakly acidic lysosomes. To explore the application of RGD-PPCD to glioma therapy, the effects of the conjugate were further evaluated in glioma model. For comparative studies, DOX was also conjugated to PEG-PAMAM by acid-insensitive succinic linkage to produce the PPSD conjugates, which was further modified by RGD to form RGD-PPSD. In vitro cytotoxicity of the acid-sensitive conjugates against C6 cells was higher than that of the acid-insensitive ones, and further the modification of RGD enhanced the cytotoxicity of the DOX-polymer conjugates as a result of the increased cellular uptake of the RGD-modified conjugates by C6 cells. In vivo pharmacokinetics, biodistribution and antitumor activity were investigated in an orthotopic murine model of C6 glioma by i.v. administration of DOX-polymer conjugates. In comparison with DOX solution, all the conjugates showed significantly prolonged half-life and increased AUC and exhibited higher accumulation in brain tumor than normal brain tissue. Although RGD-PPCD was more than 2-fold lower tumor accumulation than RGD-PPSD, it exhibited the longest survival times among all treatment groups, and therefore, RGD-PPCD conjugate provide a desirable candidate for targeted therapy of glioma.

PEGylated PAMAM dendrimers as a potential drug delivery carrier: in vitro and in vivo comparative evaluation of covalently conjugated drug and noncovalent drug inclusion complex.

To understand more about the influence of the types of interaction between drug and PEGylated PAMAM dendrimers on the in vitro and in vivo behavior of drug, methotrexate (MTX) was coupled to PEGylated or non-PEGylated generation 4 PAMAM (G4) through complexing drug within the dendritic architecture and covalently conjugated onto the surface of the dendrimer, respectively. PAMAM was first modified with PEG(5000) chains at three different degrees of substitution. The ability of PEGylated G4 complexing MTX was higher than that of non-PEGylated one. MTX-G4 and MTX-G4-PEG conjugates were synthesized via amide linkages. MTX was readily released from all complexes in isotonic solution, while the conjugates hardly released MTX in the same medium and keep stable in human plasma and the lysosomal medium. There were no obvious differences between complexes and free MTX in cytotoxicity against KB cell line, whereas the conjugates showed the relatively low activity. In vivo study in rodents found that the MTX-G4-PEG conjugate exhibited significantly prolonged blood residence time and the strongest antitumor effects, as compared with MTX-G4, the complexes and MTX. The results indicated that the covalent attachment of drug to PEGylated PAMAM could be more effective for targeted drug delivery.

PEGylated PAMAM dendrimer-doxorubicin conjugates: in vitro evaluation and in vivo tumor accumulation.

PURPOSE: To investigate the effects of PEGylation degree and drug conjugation style on the in vitro and in vivo behavior of PEGylated polyamidoamine (PAMAM) dendrimers-based drug delivery system. METHODS: Doxorubicin (DOX) was conjugated to differently PEGylated PAMAM dendrimers by acid-sensitive cis-aconityl linkage and acid-insensitive succinic linkage to produce the products of PPCD and PPSD conjugates, respectively. In vitro evaluations including pH-dependent DOX release, cytotoxicity, cellular uptake, cell internalization mechanism, and intracellular localization were performed. Tumor accumulation was also visualized by in vivo fluorescence imaging. RESULTS: DOX release from PPCD conjugates followed an acid-triggered manner and increased with increasing PEGylation degree. In vitro cytotoxicity of PPCD conjugates against ovarian cancer (SKOV-3) cells increased, while cellular uptake decreased with increasing PEGylation degree. PPSD conjugates released negligible drug at any tested pH condition and were less cytotoxic. The conjugates were internalized by SKOV-3 cells via clathrin-mediated and adsorptive endocytosis, and were delivered to acidic lysosomes where DOX was released from PPCD conjugates and diffused into the nuclei. PPCD conjugates with highest PEGylation degree showed the highest tumor accumulation in mice inoculated with SKOV-3 cells. CONCLUSION: The obtained results suggested that PPCD conjugates with highest PEGylation degree would be a potential candidate for solid tumor treatment.

Gene therapy using lactoferrin-modified nanoparticles in a rotenone-induced chronic Parkinson model.

BACKGROUND: Gene therapy is considered one of the most promising approaches to develop an effective treatment for Parkinson's disease (PD). The existence of blood-brain barrier (BBB) significantly limits its development. In this study, lactoferrin (Lf)-modified nanoparticles (NPs) were used as a potential non-viral gene vector due to its brain-targeting and BBB-crossing ability. METHODS AND RESULTS: The neuroprotective effects were examined in a rotenone-induced chronic rat model of PD after treatment with NPs encapsulating human glial cell line-derived neurotrophic factor gene (hGDNF) via a regimen of multiple dosing intravenous administration. The results showed that multiple injections of Lf-modified NPs obtained higher GDNF expression and this gene expression was maintained for a longer time than the one with a single injection. Multiple dosing intravenous administration of Lf-modified NPs could significantly improve locomotor activity, reduce dopaminergic neuronal loss, and enhance monoamine neurotransmitter levels on rotenone-induced PD rats, which indicates its powerful neuroprotective effects. CONCLUSION: The findings may have implications for long-term non-invasive gene therapy for neurodegenerative diseases in general.

Novel anti-tumor strategy: PEG-hydroxycamptothecin conjugate loaded transferrin-PEG-nanoparticles.

The aim of the study was to prepare transferrin modified stealth nanoparticles (Tf-PEG-NP) encapsulating poly(ethylene) glycol-hydroxycamptothecin conjugate (PEG-HCPT) and exploit the possiblility of combination of the functions of passive and active targeting by Tf-PEG-NP, as well as sustained drug release in tumor by PEGylated drug for most efficient tumor targeting and anti-tumor effects enhancement. PEG was covalently linked to the 10-hydroxyl group of HCPT to produce PEG-HCPT conjugate. The conjugate was stable, highly water soluable with the cytotoxicity similar to the parent drug. By encapsulation of the drug conjugate in active targeting, long circulating nanoparticles, we further improved its therapeutic efficacy. The prepared Tf-PEG-NP with average diameters of 110nm showed more sustained in vitro release profile. The pharmacokinetic and biodistribution studies found that Tf-PEG-NP demostrated the longest retention time in blood (8.94-fold that of PEG-HCPT), the highest tumor accumulation (9.03-fold, 3.11-fold that of PEG-HCPT and HCPT-loaded counterpart, respectively), as well as the most powerful anti-tumor activity with the inhibition rate up to 93% against S180 tumor in mice (1.85-fold, 1.23-fold that of PEG-HCPT and HCPT-loaded counterpart, respectively). Such Tf-PEG-NP loaded with PEGylated drug conjugates could be one of the promising strategies to deliver anti-tumor drugs to tumor.

Lactoferrin-modified nanoparticles could mediate efficient gene delivery to the brain in vivo.

Lactoferrin (Lf)-modified nanoparticles (NPs) have been demonstrated to mediate efficient expression of exogenous genes in the brain via intravenous administration. The brain-targeting properties of Lf-modified NPs were investigated in this study. In vivo imaging results showed that the accumulation of Lf-modified NPs was higher in the brain but lower in the other organs than that of unmodified counterparts. The results of analytical transmission electron microscopy showed that some Lf-modified NPs crossed the blood-brain barrier (BBB) and reached the neural tissues, while some remained within the BBB. Similar results were observed in the distribution of exogenous gene products. All the results demonstrated the successful delivery of Lf-modified NPs into the brain. Lf-modified NPs could be exploited as potential brain-targeting delivery systems for exogenous genes, especially for those encoding secretive proteins.

Partly PEGylated polyamidoamine dendrimer for tumor-selective targeting of doxorubicin: the effects of PEGylation degree and drug conjugation style.

Partly PEGylated polyamidoamine (PAMAM) dendrimers were used as the carrier for tumor-selective targeting of the anticancer drug doxorubicin (DOX). Acid-sensitive cis-aconityl linkage or acid-insensitive succinic linkage was introduced between DOX and polymeric carriers to produce PPCD or PPSD conjugates, respectively. DOX release from PPCD conjugates followed an acid-triggered manner and increased with increasing PEGylation degree. In vitro cytotoxicity of PPCD conjugates against murine B16 melanoma cells increased with, while cellular uptake decreased with increasing PEGylation degree. PPSD conjugates released negligible drug at any tested pH condition and were less cytotoxic. Confocal laser scanning microscopy confirmed the acid-sensitive release of DOX from PPCD conjugates in the lysosomes and the entrance into nuclei. Pharmacokinetic and biodistribution studies demonstrated that increasing PEGylation degree resulted in reduced liver and splenic accumulation, longer circulation time and more tumor accumulation of the conjugates. Although PPSD conjugates showed more tumor accumulation than PPCD conjugates at the same PEGylation degree, the acid-sensitive DOX release from PPCD conjugates ensured higher concentration of free DOX in tumor and more pronounced antitumor activity. Besides, the antitumor activity of PPCD conjugates increased with increasing PEGylation degree. Overall, PPCD conjugate with the highest PEGylation would be a promising candidate for solid tumor therapy.

Application of Box-Behnken design in understanding the quality of genistein self-nanoemulsified drug delivery systems and optimizing its formulation.

The purpose of the present study was to understand the effect of formulation variables of self- nanoemulsified drug delivery systems (SNEDDS) on the rapid dissolution of a model drug, genistein (GN). A three-factor, three-level Box-Behnken design was used to explore the main and interaction effect of several independent formulation variables including the amount of Maisine 35-1 and Labrafac Lipophile WL 1349 (1:1, w/w) (X1), Cremophor EL and Labrasol (3:1, w/w) (X2), and Transcutol P (X3). Droplet size (Y1), turbidity (Y2), and dissolution percentage of GN after 5 (Y3) and 30 (Y4) min were the dependent variables. A mathematical relationship, Y3 = - 89.3447 + 5.9524X1 + 1.0683X2 + 0.462X3 - 0.0825X(1)(2) - 0.0075X(2)(2) - 0.0009X(3)(2) + 0.0104X1X2 - 0.0113X1X3 + 0.0009X2X3 (r2 = 0.9604), was obtained to explain the effect of all factors and their co-linearities on the dissolution of GN at 5 min. Formulation optimization was then performed to maximize dissolution percentage of GN at 5 min (Y3). The optimized formulation was predicted to dissolution 93.34% of GN at 5 min, when X1, X2 and X3 values were 37.1, 101.7 and 77.3 mg, respectively. A new batch was prepared according to the optimized formulation, and the observed and predicted values of Y3 were in close agreement. In conclusion, the Box-Behnken experimental design allowed us to understand the effect of formulation variables on the rapid dissolution of GN from SNEDDS, and optimize the formulation to obtain a rapid drug dissolution at 5 min.

Brain-targeting mechanisms of lactoferrin-modified DNA-loaded nanoparticles.

Ligand-mediated brain-targeting drug delivery is one of the focuses at present. Elucidation of exact targeting mechanisms serves to efficiently design these drug delivery systems. In our previous studies, lactoferrin (Lf) was successfully exploited as a brain-targeting ligand to modify cationic dendrimer-based nanoparticles (NPs). The mechanisms of Lf-modified NPs to the brain were systematically investigated in this study for the first time. The uptake of Lf-modified vectors and NPs by brain capillary endothelial cells (BCECs) was related to clathrin-dependent endocytosis, caveolae-mediated endocytosis, and macropinocytosis. The intracellular trafficking results showed that Lf-modified NPs could rapidly enter the acidic endolysosomal compartments within 5 mins and then partly escape within 30 mins. Both Lf-modified vectors and NPs showed higher blood-brain barrier-crossing efficiency than unmodified counterparts. All the results suggest that both receptor- and adsorptive-mediated mechanisms contribute to the cellular uptake of Lf-modified vectors and NPs. Enhanced brain-targeting delivery could be achieved through the synergistic effect of the macromolecular polymers and the ligand.

Neuroprotection in a 6-hydroxydopamine-lesioned Parkinson model using lactoferrin-modified nanoparticles.

BACKGROUND: Nonviral gene therapy of chronic degenerative diseases such as Parkinson's disease (PD) is a great challenge as a result of the low tranfection efficiency of nonviral gene vectors. We previously constructed a lactoferrin (Lf)-modified vector, which was demonstrated to be potential for brain gene delivery both in vitro and in vivo. In the present study, this type of vector was applied to load human glial cell line-derived neurotrophic factor gene (hGDNF). METHODS: A rat PD model was constructed by the unilateral lesion of striatum using 6-hydroxydopamine (6-OHDA). Lf-modified nanoparticles (NPs) were prepared and characterized. Neuroprotective effects of Lf-modified NPs were examined in the 6-OHDA-lesioned PD model via a regimen of multiple dosing intravenous administrations. RESULTS: The size of Lf-modified NPs was 196 +/- 10.1 nm, whereas the zeta potential value was 29.35 +/- 3.27 mV. Lf-modified NPs could protect themselves from heparin displacement and DNase digestion. The results of the neuroprotective evaluation show that increasing the number of injections of Lf-modified NPs loading hGDNF improved locomotor activity, reduced dopaminergic neuronal loss and enhanced monoamine neurotransmitter levels in PD rats. Five injections of Lf-modified NPs loading hGDNF exhibited much more powerful neuroprotection than a single injection, indicating the effectiveness and feasibility of multiple dosing administrations. The results of toxicity tests demonstrated that the dosage of NPs used in the present study was safe enough for brain gene delivery. CONCLUSIONS: The findings obtained in the present study suggest that Lf-modified NPs could be developed for potential nonviral gene therapy of chronic brain disorders.

Effects of polyoxyethylene (40) stearate on the activity of P-glycoprotein and cytochrome P450.

The present study was aimed to investigate the effects of polyoxyethylene (40) stearate (PS), a non-ionic surfactant, on the activity of P-glycoprotein (P-gp) and six major cytochrome P450 (CYP) isoforms. An in vitro diffusion chamber system was utilized to estimate the effects of PS concentration on the transport characteristics of Rhodamine 123 (R123) and Rhodamine 110 (R110), a standard P-gp substrate and nonsubstrate, respectively, across the excised intestinal segments of rat. Caco-2 cells were cultured to investigate the mechanisms by estimating the effects of PS on intracellular ATP levels, P-gp ATPase activity and membrane fluidity. The obtained results showed that PS inhibited P-gp mediated efflux in a concentration-dependent manner mainly by modulating substrate-stimulated P-gp ATPase activity. On the other hand, human liver microsomes were utilized to examine the inhibitive potential of PS on six major CYP isoforms. Inhibitive potential on two of these CYP2C9 and CYP2C19 was found to be clinically significant. In conclusion, PS is potentially useful as a pharmaceutical ingredient to improve the oral bioavailability of coadministered P-gp substrates and substrates for certain CYP isoforms.

[PEGylated polyamidoamine dendrimer/methotrexate complex: pharmacokinetics and anti-tumor activity in normal and tumor-bearing rodents].

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.

Efficient tumor targeting of hydroxycamptothecin loaded PEGylated niosomes modified with transferrin.

The aim of the present report was to exploit the possibility of combination of the stealth action by polyethylene glycol cyanoacrylate-co-hexadecyl cyanoacrylate (PEG-PHDCA) modified niosomes and active targeting function of transferrin (Tf) by transferrin receptor-mediated endocytosis to promote drug delivery to solid tumor following intravenous administration with hydroxycamptothecin (HCPT) as model drug. HCPT-loaded PEG-niosomes (PEG-NS) were prepared by thin-film hydration and ultrasound method; the periodate-oxidated Tf was coupled to terminal amino group of PEG to produce the active targeting vesicles with average diameters of 116 nm. The uptake of Tf-PEG-NS into KB cells was concentration and time dependent, which could be inhibited by low temperature and free Tf, indicating that the endocytosis process was energy-driven and receptor specific. Compared with HCPT injection, non-stealth niosomes and PEG-NS, Tf-PEG-NS demonstrated the strongest cytotoxicity to three carcinomatous cell lines (KB, K562 and S180 cells), the greatest intracellular uptake especially in nuclei, the highest tumor concentration and largest area under the intratumoral hydroxycamptothecin concentration curve, as well as the most powerful anti-tumor activity with the inhibition rate of 71% against S180 tumor in mice. The results showed that the transferrin modified PEGylated niosomes could be one of the promising solutions to the delivery of anti-tumor drugs to tumor.

In situ intestinal absorption of cyclosporine A solid dispersion in rats.

Effects of concentration of Polyoxyethylene (40) stearate, Na(+) and P-gp inhibitor on cyclosporin A (CyA-SD) absorption were investigated by in situ circulation method. The results showed that the absorption of CyA increased linearly with its concentration, indicating a passive diffusion process was dominated. CyA absorption decreased with the carrier concentration. The concentration of Na(+) didn't influence the drug absorbed (P > 0.05). The P-gp inhibitor enhanced the CyA absorption significantly (P < 0.05). The passive diffusion process during the intestinal absorption indicated that the solubility enhancement of CyA is one of the mechanisms for the absorption of this water insoluble drug.

Histidylated cationic polyorganophosphazene/DNA self-assembled nanoparticles for gene delivery.

Cationic polyorganophosphazene has shown the ability to deliver gene. To obtain more efficient transfection, His(Boc)-OMe bearing histidine moiety was introduced to synthesize a new derivative of cationic polyphosphazenes with another side group of 2-dimethylaminoethylamine (DMAEA). The poly(DMAEA/His(Boc)-OMe)phosphazene (PDHP) and DNA could self-assemble into nanoparticles with a size around 110 nm and zeta potential of +15 mV at the PDHP/DNA ratio of 10:1 (w/w). The maximum transfection efficiency of PDHP/DNA self-assembled nanoparticles (PHSNs) against 293 T cells was much higher than that of poly(di-2-dimethylaminoethylamine) phosphazenes (PDAP)/DNA self-assembled nanoparticles (PASNs) and PEI 25/DNA self-assembled nanoparticles (PESNs) at the polymer/DNA ratio of 10:1, but the cytotoxicity of PDHP assayed by MTT was much lower than that of PDAP and PEI 25. These results suggested that PDHP could be a good candidate with high transfection efficiency and low cytotoxicity for gene delivery.

The use of lactoferrin as a ligand for targeting the polyamidoamine-based gene delivery system to the brain.

Development of an efficient gene vector is a key-limiting factor of brain gene therapy. In this study, lactoferrin (Lf), for the first time, was investigated as a brain-targeting ligand in the design of polyamidoamine (PAMAM)-based non-viral gene vector to the brain. Using polyethyleneglycol (PEG) as a spacer, PAMAM-PEG-Lf was successfully synthesized. This vector showed a concentration-dependent manner in the uptake in brain capillary endothelial cells (BCECs). The brain uptake of PAMAM-PEG-Lf was 2.2-fold compared to that of PAMAM-PEG-transferrin (Tf) in vivo. The transfection efficiency of PAMAM-PEG-Lf/DNA complex was higher than that of PAMAM-PEG-Tf/DNA complex in vitro and in vivo. The results of frozen sections showed the widespread expression of an exogenous gene in mouse brain via intravenous administration. With a PAMAM/DNA weight ratio of 10:1, the brain gene expression of the PAMAM-PEG-Lf/DNA complex was about 2.3-fold when compared to that of the PAMAM-PEG-Tf/DNA complex. These results provide evidence that PAMAM-PEG-Lf can be exploited as a potential non-viral gene vector targeting to the brain via noninvasive administration. Lf is a promising ligand for the design of gene delivery systems targeting to the brain.

Enhanced oral bioavailability of doxorubicin in a dendrimer drug delivery system.

PAMAM dendrimers can permeate across intestinal epithelial barriers suggesting their potential as oral drug carriers. In the present study, we have developed a drug-PAMAM complex for oral administration. The loading of a model drug, doxorubicin into PAMAM, the cellular uptake and pharmacokinetics of the doxorubicin-PAMAM complex were studied. As the results, the cellular uptake of doxorubicin in Caco-2 cells treated with the doxorubicin-PAMAM complex was increased significantly with an increase in concentration and time, as compared to that treated with free doxorubicin. And the transport efficiency of the doxorubicin-PAMAM complex from the mucosal side to the serosal side was 4-7 times higher than that of free doxorubicin in different segments of small intestines of rat. The doxorubicin-PAMAM complex led to the bioavailability that was more than 200-fold higher than that of free doxorubicin after oral administration. These results indicate that PAMAM dendrimer is a promising novel carrier to enhance the oral bioavailability of drug, especially for the P-glycoprotein (P-gp) substrates.