Inhibition of Breast Cancer Metastasis by Pluronic Copolymers with Moderate Hydrophilic-Lipophilic Balance.

Metastasis is the primary cause resulting in the high mortality of breast cancer. The inherent antimetastasis bioactivity of Pluronic copolymers with a wide range of hydrophilic-lipophilic balance (HLB) including Pluronic L61, P85, P123, F127, F68, and F108 was first explored on metastatic 4T1 breast cancer cells. The results indicated that P85 and P123 could strongly inhibit the migration and invasion of 4T1 cells. The effects of the polymers on cell healing, migration, and invasion exhibited bell-shaped dependencies on HLB of Pluronic copolymers, and the better antimetastasis effects of Pluronic copolymers could be achieved with the HLB between 8 and 16. P85 and P123 themselves could significantly inhibit pulmonary metastasis in 4T1 mammary tumor metastasis model in situ. In addition, a synergetic antimetastasis effect could be achieved during drug combination of doxorubicin hydrochloride (DOX) and P85 or P123 intravenously. The metastasis effects of P85 and P123 both in vitro and in vivo were partially attributed to the downregulation of matrix metalloproteinase-9 (MMP-9). Therefore, Pluronic copolymers with moderate HLB 8-16 such as P85 and P123 could be promising excipients with therapeutics in drug delivery systems to inhibit breast cancer metastasis.

Directed self-assembled nanoparticles of probucol improve oral delivery: fabrication, performance and correlation.

PURPOSE: We are reporting on the development of a unique drug delivery platform by directed self-assembly technique to improve the oral delivery of hydrophobic drugs. METHODS: Herein, a series of probucol directed self-assembled nanoparticles (PDN) were developed with two components of probucol and surfactant such as Tween 20, Tween 80, D-alpha-tocopheryl polyethylene glycol 1,000 succinate (TPGS) and HS-15, which was respectively named as T20-PDN, T80-PDN, TP-PDN and HS-PDN. The formation of various PDNs was determined by in vitro characterization and the physicochemical properties of these PDNs were determined. Moreover, the performance of PDN in enhancing the oral delivery and possible correlation between the in vitro properties and in vivo performances were investigated. RESULTS: PDN was homogenous nanometer-sized particles with negative surface charge. The cellular uptake of probucol in Caco-2 cell monolayer was respectively increased 1.15, 1.82, 1.59 and 5.31-fold by these PDN. In particular, the oral bioavailability of these PDN was significantly improved 3.0, 4.1, 5.4 and 10.4 folds compared with the free drug suspension. The enhanced cellular uptake and oral bioavailability were correlated with the characters of involved surfactants and the particle size of PDN. CONCLUSIONS: Thereby, the directed self-assembled nanoparticles could provide a new strategy for enhancing the oral delivery of hydrophobic drugs.

Multifunctional nanoparticles improve therapeutic effect for breast cancer by simultaneously antagonizing multiple mechanisms of multidrug resistance.

For efficient reversal of multidrug resistance (MDR) in chemotherapy for breast cancer, multifunctional self-assembled nanoparticles (MSN) based on a new amphiphilic copolymer consisting of bioreducible poly[bis(2-hydroxylethyl)-disulfide-diacrylate-ß-tetraethylenepentamine] and polycaprolactone (PBD-PCL) were constructed and characterized. shRNA targeting the apoptosis-inhibiting gene, Survivin, was incorporated into the nanoparticles with high RNA interference efficiency. PBD-PCL significantly inhibited the activity of P-glycoprotein, one of the most well-described drug-efflux pumps, and glutathione S-transferase, an important detoxification enzyme. MSN achieved colocalization of RNA and doxorubicin in tumors after intravenous administration and showed remarkable antitumor efficacy in MDR tumor-bearing mice with less side-effect than drug combination therapy. This was a new attempt to overcome MDR against three different mechanisms of MDR simutaneously: overexpression of drug efflux protein, activation of detoxification system, and blockage of apoptosis. These results indicated that the PBD-PCL-based MSN had obvious potential for therapy of breast cancer.

Urocanic acid improves transfection efficiency of polyphosphazene with primary amino groups for gene delivery.

The biodegradable cationic poly(2-(2-aminoethoxy)ethoxy)phosphazene (PAEP) bearing primary amino groups and a new PAEP derivative, urocanic acid (UA) modified PAEP (UA-PAEP), were synthesized and investigated for gene delivery. The results indicated that PAEP was able to condense DNA into complex nanoparticles with the size around 120 nm at the polymer/DNA ratio (N/P) of 35, at which PAEP/DNA complex nanoparticles (PACNs) showed efficient transfection activity in complete medium. After conjugating with UA at the substitution degree of 7% (UA-PAEP7), UA-PAEP7/DNA complex nanoparticles (UP7CNs) exhibited higher transfection efficiency than PACNs and UA-PAEP25/DNA complex nanoparticles (UP25CNs) and much lower cytotoxicity compared with PEI/DNA complex nanoparticles (PEICNs). The transfection experiment using a proton pump inhibitor suggested that the gene expression of PACNs and UP-PAEP/DNA complex nanoparticles (UPCNs) was dependent on the endosomal acidification process. The acetate solution (20 mM, pH5.7) improved the transfection activity of UP7CNs in HeLa and COS 7 cell lines, which was almost comparable to PEICNs at the N/P ratio of 35. Therefore, the results suggested that UP7CNs could be a promising carrier for gene delivery.

Linear cationic click polymer for gene delivery: synthesis, biocompatibility, and in vitro transfection.

Sixteen novel cationic click polymers (CPs) were parallelly synthesized via the conjugation of four alkyne-functionalized monomers to four azide-functionalized monomers by "click chemistry". The biocompatibility of CPs was evaluated by in vitro cytotoxicity (MTT assay, Hoechst/PI apoptosis/necrosis assay, and cell cycle analysis) and blood compatibility tests (hemolysis and erythrocyte aggregation). The experimental results showed that the kind of amine groups, charge density, and number of methylene or ethylene glycol groups brought about the effect on toxicity of CPs. Among all polymers, two polymers (B1 and B2) showed good biocompatibility, inducing neither apoptosis nor necrosis at the test concentration and low hemolysis ratio and erythrocyte aggregation. In particular, B1 and B2 exhibited the comparable transfection efficiency compared with PEI (25 kDa) but much lower cytotoxicity. These results suggested that the novel cationic CPs could be promising carriers for gene delivery.

A smart nanoassembly consisting of acid-labile vinyl ether PEG-DOPE and protamine for gene delivery: preparation and in vitro transfection.

The conception of a modular designed and viruslike nonviral vector has been presented for gene delivery. Recently, we constructed a new smart nanoassembly (SNA) with multifunctional components that was composed of a condensed core of pDNA with protamine sulfate (PS) and a dioleoyl phosphatidylethanolamine (DOPE)-based lipid envelope containing poly(ethylene glycol)--vinyl ether--DOPE (PVD). SNAs with mPEG 2000 (SNAs1) or mPEG 5000 (SNAs2) loading PS/DNA were prepared by the lipid film hydration technique. The particle size was about 160 nm for SNAs1 and 240 nm for SNAs2 loading PS/DNA (10:1 w/w), and the zeta potential was about 4 mV for two SNAs. The in vitro release experiment indicated that PVD possessed a good ability for self-dePEGylation, which could result in the recovery of an excellent fusogenic capacity of DOPE at low pH. SNAs showed a higher transfection efficiency and much lower cytotoxicity than did Lipofectamine 2000 on HEK 293, HeLa, and COS-7 cells. The cellular uptake and subcellular localization demonstrated that the superior transfection efficiency of SNAs could result from the fact that the DOPE-based lipid envelope containing PVD increased PS/DNA in the cytoplasm, and protamine enhanced the nuclear delivery or overcame the nuclear membrane barrier. These results implied that the PVD-based nanoassembly loading PS/DNA could be a promising gene delivery system.

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.

Inhibition of metastasis and growth of breast cancer by pH-sensitive poly (ß-amino ester) nanoparticles co-delivering two siRNA and paclitaxel.

Breast cancer is the most vicious killer for women's health, while metastasis is the main culprit, which leads to failure of treatment by increasing relapse rate. In this work, a new complexes nanoparticles loading two siRNA (Snail siRNA (siSna) and Twist siRNA (siTwi)) and paclitaxel (PTX) were designed and constructed using two new amphiphilic polymer, polyethyleneimine-block-poly[(1,4-butanediol)-diacrylate-ß-5-hydroxyamylamine] (PEI-PDHA) and polyethylene glycol-block-poly[(1,4-butanediol)-diacrylate-ß-5-hydroxyamylamine] (PEG-PDHA) by self-assembly. The experimental results showed that in the 4T1 tumor-bearing mice models, PEI-PDHA/PEG-PDHA/PTX/siSna/siTwi) complex nanoparticles (PPSTs) raised the accumulation and retention of both PTX and siRNA in tumor after administrated intravenously, resulted in the strong inhibition of the tumor growth and metastasis simultaneously. It was found that co-delivery of siSna and siTwi had more significant anti-metastasis effect than delivering a single siRNA, as a result of simultaneously inhibiting the motility of cancer cells and degradation of ECM. Therefore, PPSTs could be a promising co-delivery vector for effective therapy of metastatic breast cancer.

Hydrophobic interaction mediating self-assembled nanoparticles of succinobucol suppress lung metastasis of breast cancer by inhibition of VCAM-1 expression.

The prevention and treatment of lung metastasis of breast cancer remain a major challenge. The vascular cell adhesion molecule-1 (VCAM-1) could provide a potential therapeutic target in lung metastasis. Herein, succinobucol (SCB), a water-insoluble potent and selective VCAM-1 inhibitor, was assembled with triblock polymer poloxamer P188 into nanoparticles due to the intermolecular hydrophobic interactions. The experimental results showed that the SCB loaded nanoparticles (SN) could greatly improve the oral delivery and suppress the lung metastasis of breast cancer. The cell migration and invasion abilities of metastatic 4T1 breast cancer cells were obviously inhibited by SN. Moreover, the VCAM-1 expression on 4T1 cells was significantly reduced by SN, and the cell-cell binding ratio of RAW 264.7 cells to 4T1 cells greatly decreased from 47.4% to 3.2%. Furthermore, the oral bioavailability of SCB was greatly improved about 13-fold by SN, and the biodistribution in major organs was evidently enhanced. In particular, in the metastatic breast cancer model, the lung metastasis was notably reduced by SN treatment, and the VCAM-1 expression in lung tissues was significantly inhibited. Thereby, SN could evoke a new effective therapeutic efficacy of SCB on lung metastasis of breast cancer by inhibition of VCAM-1 expression.

Effective delivery of p65 shRNA by optimized Tween 85-polyethyleneimine conjugate for inhibition of tumor growth and lymphatic metastasis.

To maximize the interference efficacy of pGPU6/Neo-p65 shRNA-expressing pDNA (p65 shRNA) and subsequently more effectively inhibit tumor growth and lymphatic metastasis through blocking the nuclear factor-kappa B (NF-κB) signaling pathway, seven Tween 85-polyethyleneimine (PEI) conjugates (TnPs, n=2, 3, 4, 5, 6, 7 and 8), which differed in the length of the polymethylene [-(CH2)n-] spacer between Tween 85 and PEI, were synthesized and investigated. The results showed that the transfection efficiency and cytotoxicity both increased with the spacer chain length. Then, TnPs with a [-(CH2)6-] spacer (T6P) were chosen to deliver p65 shRNA to a tumor and subsequently inhibit tumor growth and lymphatic metastasis. The T6P/p65 shRNA complex nanoparticles (T6Ns) could significantly down-regulate p65 expression in breast cancer cells, and consequently inhibit cell invasion and disrupt the tube formation. Most importantly, T6Ns accumulated greatly in tumor tissue, and as a result, significantly inhibited the growth and lymphatic metastasis of breast cancer xenograft. All these results indicated that the transfection efficacies of cationic amphiphiles could be significantly modulated by minor structural variations, and that T6P was promising for the effective delivery of p65 shRNA to knock down the expression of the key metastasis-driving genes and inhibit tumor growth and metastasis.

Induction of apoptosis in non-small cell lung cancer by downregulation of MDM2 using pH-responsive PMPC-b-PDPA/siRNA complex nanoparticles.

Non-small cell lung cancer (NSCLC) accounts for the majority of lung cancer caused human death. In this work, we selected oncogene mouse double minute 2 (MDM2) as a therapeutic target for NSCLC treatment and proposed that sufficient MDM2 knockdown could inhibit tumor growth via induction of cell cycle arrest and cancer cell apoptosis. On this regard, a new pH-responsive diblock copolymer of poly(methacryloyloxy ethyl phosphorylcholine)-block-poly(diisopropanolamine ethyl methacrylate) (PMPC-b-PDPA)/siRNA-MDM2 complex nanoparticle with minimized surface charge and suitable particle size was designed and developed for siRNA-MDM2 delivery in vitro and in vivo. The experimental results showed that the nanoparticles were spherical with particle size around 50 nm. MDM2 knockdown in p53 mutant NSCLC H2009 cells induced significant cell cycle arrest, apoptosis and growth inhibition through upregulation of p21 and activation of caspase-3. Furthermore, the growth of H2009 xenograft tumor in nude mice was inhibited via repeated injection of PMPC-b-PDPA/siRNA-MDM2 complex nanoparticles. These results suggested that PMPC-b-PDPA/siRNA complex nanoparticles targeting a unique set of oncogenes could be developed into a new therapeutic approach for NSCLC treatment.

Overcoming multidrug resistance by co-delivery of Mdr-1 and survivin-targeting RNA with reduction-responsible cationic poly(ß-amino esters).

Multidrug resistance (MDR) remains one of the main challenges in the successful chemotherapy of human cancer. RNA interference (RNAi) strategy aiming at only one cause of MDR was widely applied, nevertheless hardly obtained satisfactory tumor-suppressing effect. In this work, a new attempt to package two kinds of RNA with different functions into one vector and reverse MDR against two different mechanisms via RNAi was carried out. A new bioreducible poly (ß-amino esters) (PAEs), poly[bis(2-hydroxylethyl)-disulfide-diacrylate-ß-tetraethylenepentamine] (PAP) was synthesized by Michael addition reaction. The PAEs/RNA complex nanoparticles (PAEN) were prepared. The experimental results demonstrated that co-delivery of iMdr-1-shRNA and iSurvivin-shRNA could be achieved by a single vector, and interfering two genes simultaneously had a synergistic effect on overcoming MDR. PAEN lowered the IC(50) value of doxorubicin (DOX) in MDR tumor cells to a comparable level to that in the sensitive cell line through down-regulating the expression of P-gp and Survivin, and decreased the tumor volumes in mice xenograft model bearing DOX-resistant human breast cancer when combined with DOX. These results illustrated that PAEN could be applied as potential efficient non-viral RNA carriers for reversing MDR.

Poly(imidazole/DMAEA)phosphazene/DNA self-assembled nanoparticles for gene delivery: synthesis and in vitro transfection.

A new cationic derivate of polyphosphazene with imidazole and 2-dimethylaminoethylamino (DMAEA) as side groups, poly(imidazole/DMAEA)phosphazene (PIDP), was synthesized and investigated for gene delivery. The half-lives of PIDP degradation under neutral (pH 7.4) and acidic conditions (pH 5.0) were 22 and 3 days at 37 degrees C, respectively. The cytotoxicity of PIDP assayed by MTT was much lower than that of poly(2-dimethylaminoethylamino)phosphazene (PDAP) and PEI 25K. PIDP could condense DNA into nanoparticles with a size around 100 nm and zeta potential (+25 mV) at the ratio of 10:1 (PIDP/DNA, w/w). The transfection efficiency of PIDP/DNA complex nanoparticles (PICNs) against 293T, COS-7 and Hela cells was much higher than that of PDAP/DNA complexes nanoparticles (PDCNs) and PEI/DNA complexes nanoparticles (PECNs) at 10:1 (polymer/DNA, w/w). Therefore, PIDP could be a safe, efficient and promising cationic polymer for gene therapy.

Targeted nanoassembly loaded with docetaxel improves intracellular drug delivery and efficacy in murine breast cancer model.

Docetaxel is one of the most promising chemotherapeutic agents for the treatment of metastatic breast cancer, but it shows fearful side effects. We hypothesized that a novel targeted nanoassembly (TNA) could provide efficient intracellular drug delivery in breast tumor cells overexpressing epidermal growth factor (EGF) receptor and thus improve the efficacy and reduce the side effects of docetaxel. We prepared the novel docetaxel loaded TNAs formed by polyethylene glycol-distearoylphosphatidylethanolamine (PEG-DSPE) and modified with EGF. Compared with nontargeted nanoassemblies (NNAs), TNAs showed obvious improvement of cell-specific uptake and internalization, and revealed more cytotoxicity against MDA-MB-468 cells by inducing more late apoptosis and subG1 cells at low drug concentration, or more G2/M arrest at high drug concentration than NNAs or Taxotere. In BALB/c mice bearing breast tumor xenografts, TNAs showed stronger inhibition of tumor growth compared with NNAs (relative tumor volume in mice treated with 5 mg/kg TNAs = 0.99 and 10 mg/kg NNAs = 1.71, p < 0.05) or Taxotere (relative tumor volume in mice treated with 5 mg/kg TNAs = 0.99 and 10 mg/kg Taxotere = 4.20, p < 0.01). In particular, tumor disappeared completely in the TNA group at a dose of 10 mg/kg. The maximum tolerated dose (MTD) of TNAs was about four times higher than that of Taxotere. TNAs also demonstrated a much longer circulation time in vivo and more drug accumulation in tumor in a murine breast cancer model than Taxotere. TNA treatment also prolonged survival of mice. These results suggested that TNAs could have more potential as a delivery system for breast cancer chemotherapy.