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.

Codelivery of sorafenib and curcumin by directed self-assembled nanoparticles enhances therapeutic effect on hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most common causes of cancer-related mortality worldwide. Herein, we first reported the codelivery of sorafenib and curcumin by directed self-assembled nanoparticles (SCN) to enhance the therapeutic effect on HCC. SCN was formed by employing the hydrophobic interactions among the lipophilic structure in sorafenib, curcumin, and similar hydrophobic segments of polyethylene glycol derivative of vitamin E succinate (PEG-VES), which comprised uniform spherical particles with particle size of 84.97 ± 6.03 nm. SCN presented superior effects over sorafenib, curcumin, and their physical mixture (Sora + Cur) on enhancing in vitro cytotoxicity and cell apoptosis in BEL-7402 cells and Hep G2 cells, and antiangiogenesis activities in tube formation and microvessel formation from aortic rings. Moreover, the tissue concentration of sorafenib and curcumin in gastrointestinal tract and major organs were significantly improved after their coassembly into SCN. In particular, in BEL-7402 cells induced tumor xenograft, SCN treatment displayed the obviously enhanced inhibitory effect on tumor progression over free drug monotherapy or their physical mixture, with significantly increased antiproliferation and antiangiogenesis capability. Thereby, the codelivered nanoassemblies of sorafenib and curcumin provided a promising strategy to enhance the combinational therapy of HCC.

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.

Galactosylated poly(2-(2-aminoethyoxy)ethoxy)phosphazene/DNA complex nanoparticles: in vitro and in vivo evaluation for gene delivery.

To achieve efficient gene delivery to the tumor after intravenous administration, biodegradable poly(2-(2-aminoethyoxy)ethoxy)phosphazene (PAEP) was modified by lactobionic acid, bearing a galactose group as a targeting ligand. Galactosylated poly(2-(2-aminoethyoxy)ethoxy)phosphazene (Gal-PAEP) with 4.9% substitution degree of galactose could condense pDNA into nanoparticles with a size around 130 nm at the polymer/DNA ratio (N/P) of 2-40. For BEL-7402 cells, the in vitro transfection efficiency of gal-PAEP/DNA complex nanoparticles (gal-PACNs) was much higher than that of the PAEP/DNA complex nanoparticles (PACNs). MTT assay indicated that the cytotoxicity of PACNs significantly decreased after conjugating with the galactose moiety. Gal-PACNs displayed the selective gene expression in the tumor and liver with relatively low gene expression in the lung or other organs compared with PACNs. These results suggested that gal-PACNs could be a promising targeting gene carrier to deliver a therapeutic gene in future.

Synthesis of 6-N,N,N-trimethyltriazole chitosan via "click chemistry" and evaluation for gene delivery.

The selective introduction of a trimethylammonium cationic group into the C-6 position of chitosan (Cs) was successfully performed by "click chemistry" for the first time, and the 6-N,N,N-trimethyltriazole-Cs (TCs) showed good solubility in water. TCs showed strong DNA binding ability and high protection of DNA against nuclease degradation assessed by gel electrophoresis assay. TCNs showed lower degree of flocculation than Cs/DNA self-assembled nanoparticles (CsNs) in the presence of medium containing serum within 60 min. The introduction of trimethyltriazole group led to significantly increased cellular uptake compared with unmodified Cs, which resulted in higher transfection efficiency in HEK 293 and MDA-MB-468 cells. TCs were noncytotoxic, and viability of cells exposure to TCNs for 24 h was over 80% even at 50 microg/mL of polymer. These results suggested that TCs could be an efficient and safe material for gene delivery.

The role of daidzein-loaded sterically stabilized solid lipid nanoparticles in therapy for cardio-cerebrovascular diseases.

Daidzein is a very good candidate for treating cardio-cerebrovascular diseases, but its poor oral absorption and bioavailability limit its curative efficacy. In this work, daidzein-loaded solid lipid nanoparticles (SLNs) with PEGylated phospholipid as stabilizer were successfully prepared by hot homogenization method. SLNs showed the mean particle size 126+/-14 nm with entrapment efficiency 82.5+/-3.7%. In vitro release of SLNs demonstrated a sustained release manner with cumulative release over 90% within 120 h in bovine serum albumin solution (4%, w/v). The pharmacokinetic behavior showed that SLNs loading daidzein could significantly increase circulation time compared with orally administrated daidzein suspension or intravenously delivered daidzein solution. SLNs showed the better effect on cardiovascular system of the anesthetic dogs by reducing the myocardial oxygen consumption (MOC) and the coronary resistance (CR) in heart compared with oral suspension or intravenous solution. The SLNs demonstrated the best effect on cerebrovascular system by increasing cerebral blood flow (CeBF) and reducing cerebrovascular resistance (CeR) in anesthetized dogs, and the protective effect on rats with ischemia-reperfusion injury model among three formulations. These results suggested that SLNs could be a potential candidate for the treatment of cardio-cerebrovascular diseases.

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.

Arginine-chitosan/DNA self-assemble nanoparticles for gene delivery: In vitro characteristics and transfection efficiency.

Chitosan (Cs) is a natural cationic polysaccharide that has shown potential as non-viral vector for gene delivery because of its biocompatibility and low toxicity. However, chitosan used for gene delivery is limited due to its poor water solubility and low transfection efficiency. The purpose of this work was to prepare Arginine-chitosan (Arg-Cs)/DNA self-assemble nanoparticles (ACSNs), and determine their in vitro characteristics and transfection efficiency against HEK 293 and COS-7 cells. Our experimental results showed that the particle size and zeta potential of ACSNs prepared with different N/P ratios were 200-400nm and 0.23-12.25mV, respectively. The in vitro transfection efficiency of ACSNs showed dependence on pH of transfection medium, and the highest expression efficiency was obtained at pH 7.2. The transfection efficiency increased with the ratio of chitosan-amine/DNA phosphate (N/P ratio) from 1 to 5, and reached the highest level with the N/P ratio 5. Effect of plasmid dosage on the transfection efficiency showed the highest transfection efficiency was obtained at 4microg/well for HEK 293 cells and 6microg/well for COS-7 cells. The transfection efficiency of ACSNs was much higher than that of Cs/DNA self-assemble nanoparticles (CSNs). The average cell viability of ACSNs was over 90%. These results suggested that ACSNs could be a safe and effective non-viral vector for gene delivery.

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.

In vitro and in vivo evaluation of donepezil-sustained release microparticles for the treatment of Alzheimer's disease.

The purpose of this work is to prepare donepezil microparticles (DM) and evaluate its advantage as a sustained release delivery system with subcutaneous injection once a month. DM was prepared using poly (d,l-lactide-co-glycolide) (PLGA) by an oil-water emulsion solvent evaporation technique. DM showed the loading ratio 13.2+/-2.1% (w/w) and yield 54.8+/-0.8% with mean particle size about 75mum. In vitro release of DM showed that donepezil completely released within 28 days in water, but the cumulative release percentages up to day 30 were 98.4% and 49.1% for phosphate buffer saline (PBS, pH 5.8) and PBS (pH 7.4), respectively. The in vivo experiment demonstrated that DM (90mg/kg) produced a sustained release process in rats, and reached steady-state concentration at day 8 and maintained until day 27 with steady-state levels of donepezil between 130.3+/-7.8 and 121+/-9.8ng/ml, which was accordance with that of free donepezil by oral application route (3mg/kgday). DM (90mg/kg) by subcutaneous infusion in rats produced the same pharmacological role as free donepezil (3mg/kgday) by oral application route. These results implicated that DM as a sustained release delivery strategy could substitute for its oral formulation for therapy of AD and come true its administration once a month.

Dual pH-sensitive micelles with charge-switch for controlling cellular uptake and drug release to treat metastatic breast cancer.

For successful chemotherapy against metastatic breast cancer, the great efforts are still required for designing drug delivery systems that can be selectively internalized by tumor cells and release the cargo in a controlled manner. In this work, the chemotherapeutic agent paclitaxel (PTX) was loaded with the dual-pH sensitive micelle (DPM), which consisted of a pH-sensitive core, an acid-cleavable anionic shell, and a polyethylene glycol (PEG) corona. In the slightly acidic environment of tumor tissues, the anionic shell was taken off, inducing the conversion of the surface charge of DPM from negative to positive, which resulted in more efficient cellular uptake, stronger cytotoxicity and higher intra-tumor accumulation of PTX in the murine breast cancer 4T1 tumor-bearing mice models compared to the micelles with irremovable anionic or non-ionic shell. Meanwhile, the pH-sensitive core endowed DPM with rapid drug release in endo/lysosomes. The inhibitory rates of DPM against tumor growth and lung metastasis achieved 77.7% and 88.3%, respectively, without significant toxicity. Therefore, DPM is a promising nanocarrier for effective therapy of metastatic breast cancer due to satisfying the requirements of both selective uptake by tumor cells and sufficient and fast intracellular drug release.

A delivery strategy for rotenone microspheres in an animal model of Parkinson's disease.

In order to study the pathogenesis of Parkinson's disease (PD), and explore therapeutic drug or approaches, the accurate animal model of PD with inexpensive, biocompatible and convenient administration was necessary. The aim of the present work was to investigate a delivery strategy for rotenone microspheres in an animal model of PD. The rotenone microspheres were prepared by solvent evaporation technique. The rotenone microspheres showed high entrapment efficiency (97.4+/-2.2%) with particle size about 100 microm. In vitro release of rotenone microspheres demonstrated different profiles from medium with different pH or concentration of isopropyl alcohol. The most consistent medium with in vivo rotenone levels in rat plasma was PBS (pH 5.8) with 20% isopropyl alcohol, and the cumulated release amount of rotenone over 30 days was 95.4% in it. The rotenone microspheres (9 mg/kg) produced typical PD symptoms in rats, for example, the cataleptic behavior test demonstrated a obviously prolonged descent latency compared with control animals after administration, and the tyrosine hydroxylase (TH) immunohistochemistry tests showed typical histological evidence of selective degeneration of the nigrostriatal dopaminergic system (striatum and substantia nigra) in rotenone microspheres-treated rats. In addition, this delivery system for rotenone model showed many noticeable advantages such as inexpensive, biocompatible and expedient administration by direct subcutaneous injection. This information suggested that rotenone microspheres as a delivery strategy for setting up an ideal animal model of PD was feasible.

Cancer-Cell-Biomimetic Nanoparticles for Targeted Therapy of Homotypic Tumors.

A unique biomimetic drug-delivery system composed of 4T1-breast-cancer-cell membranes and paclitaxel-loaded polymeric nanoparticles (PPNs) (cell-membrane-coated PPNs), demonstrates superior interactions to its source tumor cells and elongated blood circulation, and displays highly cell-specific targeting of the homotypic primary tumor and metastases, with successful inhibition of the growth and lung metastasis of the breast cancer cells.

In vitro and in vivo evaluation of actively targetable nanoparticles for paclitaxel delivery.

The aim of the present work was to assess the merits of an actively targetable nanoparticles (ATN), PEG-coated biodegradable polycyanoacrylate nanoparticles (PEG-nanoparticles) conjugated to transferrin, for paclitaxel delivery. PEG-nanoparticles loading paclitaxel were prepared by solvent evaporation technique in advance. ATN were prepared by coupling of transferrin to PEG-nanoparticles. The results showed that the average encapsulation efficiency of ATN was 93.4+/-3.6% with particle size (101.4+/-7.2 nm) and zeta-potential (-13.6+/-1.1 mV). The paclitaxel loaded ATN exhibited a low burst effect with about only 16.2% drug release within the first phase. Subsequently, paclitaxel release profiles displayed a sustained release phase. The amount of cumulated paclitaxel release over 30 days was 81.6%. ATN exhibited a markedly delayed blood clearance in mice, and the paclitaxel level from ATN remained much higher at 24 h compared with that of free drug from paclitaxel injection. The distribution profiles of ATN in S-180 solid tumor-bearing mice after intravenous administration showed the tumor accumulation of paclitaxel increase with time, and the paclitaxel concentration in tumor was about 4.8 and 2.1 times higher than those from paclitaxel injection and PEG-nanoparticles at 6 h after intravenous injection. For mice treated with 20 mg/kg x 5 of ATN, the decrease in body weight was limited within 4% of the initial weight at 5 days after the final administration, and tumor regression was significantly observed with complete tumor regression for five out of nine mice. The tumor burden with ATN-treated mice was much smaller compared with free paclitaxel or NTN-treated mice. In addition, the life span of tumor-bearing mice was significantly increased when they were treated with ATN, in particular, three mice survived over 60 days. Thus, PEG-coated biodegradable polycyanoacrylate nanoparticles conjugated to transferrin could be an effective carrier for paclitaxel 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.