Protein liposomes-mediated targeted acetylcholinesterase gene delivery for effective liver cancer therapy.

BACKGROUND: Effective methods to deliver therapeutic genes to solid tumors and improve their bioavailability are the main challenges of current medical research on gene therapy. The development of efficient non-viral gene vector with tumor-targeting has very important application value in the field of cancer therapy. Proteolipid integrated with tumor-targeting potential of functional protein and excellent gene delivery performance has shown potential for targeted gene therapy. RESULTS: Herein, we prepared transferrin-modified liposomes (Tf-PL) for the targeted delivery of acetylcholinesterase (AChE) therapeutic gene to liver cancer. We found that the derived Tf-PL/AChE liposomes exhibited much higher transfection efficiency than the commercial product Lipo 2000 and shown premium targeting efficacy to liver cancer SMMC-7721 cells in vitro. In vivo, the Tf-PL/AChE could effectively target liver cancer, and significantly inhibit the growth of liver cancer xenografts grafted in nude mice by subcutaneous administration. CONCLUSIONS: This study proposed a transferrin-modified proteolipid-mediated gene delivery strategy for targeted liver cancer treatment, which has a promising potential for precise personalized cancer therapy.

Properties and evaluation of quaternized chitosan/lipid cation polymeric liposomes for cancer-targeted gene delivery.

Development of high-stability and efficient nonviral vectors with low cytoxicity is important for targeted tumor gene therapy. In this study, cationic polymeric liposomes (CPLs), with similar lipid bilayer structure and high thermal stability, were prepared from polymeric surfactants of quaternized (carboxymethyl)chitosan with different carbon chains (dodecyl, tetradecyl, hexadecyl, and octadecyl). By comparing different factors that influence gene delivery, tetradecyl-quaternized (carboxymethy)chitosan (TQCMC) CPLs, with suitable size (184.4 ± 17.1 nm), ζ potentials (27.5 ± 4.9 mV), and productivity for synthesis TQCMC (weight yield 13.1%), were selected for gene transfection evaluation in various cancer cell lines. Although TQCMC CPLs have lower gene transfection efficiency compared with cationic liposomes (Lipofectamine 2000) in vitro, they displayed higher reporter gene delivery ability for cancer tissues (bearing U87 and SMMC-7721 tumors) in vivo after intravenous injection. TQCMC CPLs also have lower cell cytotoxicity and lower cytokine production or liver injury for BALB/c mice. We conclude that the CPLs are promising gene delivery systems that may be used to target various cancers.

Calcium phosphate/octadecyl-quatemized carboxymethyl chitosan nanoparticles: an efficient and promising carrier for gene transfection.

Calcium phosphate (CaP) has been widely used as the vector for gene transfection in the past three decades. However, clinical application is still not popular due to the poor-controlling of DNA/CaP complexes preparation, cytotoxicity and its low transfection efficiency. In this study, a novel amphipathic octadecyl-quatemized carboxymethyl chitosan (OQCMC) derivative from chitosan was combined with calcium phosphate to synthesize CaP/OQCMC nanoparticles (CaP/OQCMC NPs). The nanoparticles were 122-177 nm in diameter exhibited neutral zeta potential (from -0.115 mV to 0.216 mV), and they were applied as DNA vectors for DNA loading and in vitro transfection. The results showed that CaP/OQCMC NPs displayed high DNA loading capacity and enhanced transfection efficiency with extremely low cytotoxicity. In addition, both CaP and OQCMC are biocompatible and biodegradable, thus the as-prepared CaP/OQCMC NPs are promising in gene delivery.

[Study of TAT-PEG loaded magnetic liposomes crossing blood spinal cord barrier after spinal cord injury in rats].

OBJECTIVE: To evaluate the ability of a kind of novel magnetic liposomes modified with polyethylene glycol (PEG) and transactivating-transduction protein (TAT) to cross the blood spinal cord barrier (BSCB) so as to demonstrate whether or not they can accumulate at the lesions of injured spinal cord. METHODS: The novel liposomes were made through reverse-phase evaporation method modified with polyethylene glycol (PEG) and transactivating-transduction protein (TAT) with an iron core. Thirty-six Wistar rats subject to spinal cord injury (SCI) at T10 were randomly divided into three groups (Groups I, II and III). The rats of Group III were injected with TAT-PEG loaded magnetic liposomes (4.55 mg/kg). The rats of GroupII received an injection of the equivalent PEG loaded magnetic liposomes while those of control group (GroupI) the equivalent normal saline. The accumulation of liposomes was observed by MRI (magnetic resonance imaging), Prussian blue staining, electron microscope and flame atomic absorption spectrophotometer. RESULTS: This kind of TAT-PEG loaded magnetic liposomes could cross the BSCB and enter into the cells around the injured tissue. A low signal of T2WI on MRI could also be found in Group III. The results of flame atomic absorption spectrophotometer showed that the iron content accumulated around the lesion site in Group III was obviously higher than the other two groups (P < 0.05). CONCLUSION: The TAT-PEG loaded magnetic liposomes may be employed as one kind of novel drug carrier to cross the BSCB and accumulate at tissue cells of spinal cord. It is likely to become a new therapy for SCI.

Nanocrystal-loaded liposome for targeted delivery of poorly water-soluble antitumor drugs with high drug loading and stability towards efficient cancer therapy.

Nanocrystals (NCs) enable the delivery of poorly water-soluble drugs with improved dissolution and bioavailability. However, their uncontrolled release and instability make targeted delivery challenging. Herein, a nano-in-nano delivery system composed of a drug nanocrystal core and liposome shell (NC@Lipo) is presented, which merges the advantages of drug nanocrystals (high drug loading) and liposomes (easy surface functionalization and high stability) for targeted delivery of hydrophobic drugs to tumors. CHMFL-ABL-053 (053), a hydrophobic drug candidate discovered by our group, was employed as a model drug to demonstrate the performance of NC@Lipo delivery system. Surface PEGylated (053-NC@PEG-Lipo) and folic acid-functionalized (053-NC@FA-Lipo) formulations were fabricated by wet ball milling combined with probe sonication. 053-NC@Lipo enabled high drug loading (up to 19.51%), considerably better colloidal stability, and longer circulation in vivo than 053-NC. Compared with free 053, 053-NC@PEG-Lipo and 053-NC@FA-Lipo exhibited higher tumor accumulation and considerably better in vivo antitumor efficacy in K562 xenograft mice with tumor growth inhibition rate (TGI) of up to 98%. Additionally, more effective tumor cell targeting in vitro and higher TGI in vivo were achieved with 053-NC@FA-Lipo. The NC@Lipo strategy may contribute to the targeted delivery of poorly water-soluble drugs with high drug loading, high stability, and tailorable surface, and has potential for the development of more efficient nanocrystal- and liposome-based formulations for commercial and clinical applications. It may also provide new opportunities for potential clinical application of candidate 053.

Construction of a novel cationic polymeric liposomes formed from PEGlated octadecyl-quaternized lysine modified chitosan/cholesterol for enhancing storage stability and cellular uptake efficiency.

The design and construction of delivery vectors with high stability and effective cellular uptake efficiency is very important. In this study, a novel polymeric liposomes (PLs) formed from PEGlated octadecyl-quaternized lysine modified chitosan (OQLCS) and cholesterol with higher size stability and cellular uptake efficiency has been synthesized successfully. Compared to conventional liposomes (CLs; phosphatidyl choline/cholesterol), the calcein-loaded PLs exhibited a multi-lamellar structure with homogenous size diameter (200 nm) and high calcein encapsulation efficiency (about 92%). PLs could be stored at different temperature (25, 4, and -20 degrees C) and different medium (deionized water, phosphate-buffered saline, and human plasma solution) for up to 4 weeks without significant size change. The spectrophotometer fluorometry analysis and the flow cytometry analysis indicated that in comparison with CL, PLs with positive zeta potential facilitates the uptake of calcein by MCF-7 tumor cells. The data suggests that PLs may provide a new method to overcome the stability and enhance the uptake efficiency of CLs.

Automatic Time-Resolved Fluorescence Immunoassay of Serum Alpha Fetoprotein-L3 Variant via LCA Magnetic Cationic Polymeric Liposomes Improves the Diagnostic Accuracy of Liver Cancer.

PURPOSE: The aim of this study was to develop an avidin-modified macromolecular lipid magnetic sphere and its application in differential diagnosis of liver disease and liver cancer. MATERIALS AND METHODS: Lectin-modified macromolecular lipid magnetic spheres were prepared by thin-film hydration method using lentil lectin derivatives (LCA-HQ) and cholesterol as raw materials. Alpha-fetoprotein variants (AFP-L3) in serum from healthy people, liver disease and liver cancer patients were isolated using the prepared lectin-modified macromolecular lipid magnetic spheres, and alpha-fetoprotein (AFP) and AFP-L3 were detected by fully automatic time-resolved fluorescence immunoassay. RESULTS: The lectin polymer lipid magnetic sphere prepared in this study was superparamagnetic and encapsulated by a lectin derivative. There was no significant difference in the recovery rate of AFP-L3 between avidin magnetic ball-automatic time-resolved fluorescence immunoassay and manual micro-affinity column method (p>0.05). We found that AFP-L3 can be used as a differential indicator between liver cancer and liver disease. The positive rate of AFP and AFP-L3 in liver cancer patients was higher than that in healthy people and liver disease patients (p<0.001). The AUC (95% CI) of AFP and AFP-L3 were 0.743 ± 0.031 and 0.850 ± 0.024, respectively. AFP-L3 AUC value is greater than AFP; therefore, AFP-L3 distinguishes liver cancer more accurately, and the difference is statistically different, p<0.05. CONCLUSION: We proposed a novel method for integration of the lectin polymer lipid magnetic spheres and time-resolved fluorescence immunoassay that enables simple, accurate and rapid determination of AFP-L3 in clinical samples. To be noted, fully automatic time-resolved fluorescence immunoassay compared with the commonly used techniques in clinical practice, the measurement procedure is simple and is expected to be used for the detection and accurate diagnosis of liver cancer.

Luteolin-loading of Her-2-poly (lactic-co-glycolic acid) nanoparticles and proliferative inhibition of gastric cancer cells via targeted regulation of forkhead box protein O1.

BACKGROUND: Developing the natural medicine that allow for the specific targeting cytotoxicity is a very important research area in the development of anti-tumor drugs. AIMS AND OBJECTIVES: This study was conducted to determine the targeted inhibitory effects of luteolin-loaded Her-2-poly (lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) on gastric cancer cells and to delineate the mechanism underlying the inhibition of tumors by luteolin. MATERIALS AND METHODS: Luteolin-loaded Her-2-PLGA NPs (Her-2-NPs) were prepared, physically and chemically characterized, and their effects on gastric cancer cells were investigated. The rate of NP uptake by cells and the cell morphology were observed using confocal microscopy; the rates of cell proliferation and apoptosis were identified using the 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide assay and flow cytometry, respectively; and the mRNA and protein expression levels of forkhead box protein O1 (FOXO1) were determined using quantitative polymerase chain reaction and Western blotting, respectively. RESULTS: Compared with nontargeted microspheres, Her-2-NPs led to significantly enhanced uptake of luteolin by SGC-7901 cells. Luteolin-loaded Her-2-NPs also significantly inhibited the proliferation of gastric cancer cells, weakened their migratory ability, and increased both the mRNA and protein expression levels of FOXO1. CONCLUSION: Luteolin-loading of Her-2-NPs could potentially be used as a novel anti-cancer drugs for targeted cancer therapy.

Application of Paclitaxel-loaded EGFR Peptide-conjugated Magnetic Polymeric Liposomes for Liver Cancer Therapy.

Developing the methodologies that allow for safe and effective delivery of therapeutic drugs to target sites is a very important research area in cancer therapy. In this study, polyethylene glycol (PEG)-coated magnetic polymeric liposome (MPL) nanoparticles (NPs) assembled from octadecyl quaternized carboxymethyl chitosan (OQC), PEGylated OQC, cholesterol, and magnetic NPs, and functionalized with epithelial growth factor receptor (EGFR) peptide, were successfully prepared for in-vivo liver targeting. The two-step liver targeting strategy, based on both magnetic force and EGFR peptide conjugation, was evaluated in a subcutaneous hepatocellular carcinoma model of nude mouse. The results showed that EGFR-conjugated MPLs not only accumulated in the liver by magnetic force, but could also diffuse into tumor cells as a result of EGFR targeting. In addition, paclitaxel (PTX) was incorporated into small EGFR-conjugated MPLs (102.0±0.7 nm), resulting in spherical particles with high drug encapsulation efficiency (>90%). The use of the magnetic targeting for enhancing the transport of PTX-loaded EGFR-conjugated MPLs to the tumor site was further confirmed by detecting PTX levels. In conclusion, PTX-loaded EGFR-conjugated MPLs could potentially be used as an effective drug delivery system for targeted liver cancer therapy.

Epidermal growth factor receptor-targeted immunomagnetic liposomes for circulating tumor cell enumeration in non-small cell lung cancer treated with epidermal growth factor receptor-tyrosine kinase inhibitors.

OBJECTIVES: To establish a circulating tumor cell (CTC) enrichment system for non-small cell lung cancer (NSCLC) patients who received first-line treatment with epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (EGFR-TKI), using EGFR magnetic liposomes (EGFR-ML). MATERIALS AND METHODS: An inverted evaporation method was used to develop antibody modified EGFR-ML. Peripheral blood was collected from NSCLC patients who underwent first-line EGFR-TKI treatment for CTC enumeration. RESULTS: Protein electrophoresis, magnetic saturation curve, and ultraviolet absorption spectrum showed successful incorporation of the EGFR antibody on the surface of the magnetic microspheres, and the development of EGFR-ML was ascertained based on cell morphology and particle size. Using EGFR-ML, CTC were successfully enriched from blood samples and were identified in 77.3% (99/128) of the cohort. When compared to the 21L858R variant, EGFR-19del showed lower CTC counts by EGFR-ML (CTCEGFR). At one month after EGFR-TKI, a lower CTCEGFR was associated with partial response (PR) during treatment (CTCEGFR < 6 vs. ≥ 6/7.5 mL, 75% vs. 49%, P = 0.027). In addition, patients with a lower CTCEGFR at 3 months after EGFR-TKI achieved a longer progression-free survival (PFS) [CTCEGFR < 6 vs. ≥ 6/7.5 mL, 13 months vs. 10.4 months, HR = 2.4, P = 0.042]. CTCEGFR significantly increased at the time of RECIST-progressive disease (RECIST-PD). Representative cases showed that CTCEGFR might increase before and beyond RECIST-PD until no clinical benefit could be acquired from EGFR-TKI. CONCLUSION: We showed that establishing a CTC enrichment system by antibody modified EGFR-ML in NSCLC is feasible. CTC enumeration by EGFR-ML may have the potential to supplement RECIST in dynamically monitoring the response of NSCLC patients' to first-line EGFR-TKI.

Repair effect of diabetic ulcers with recombinant human epidermal growth factor loaded by sustained-release microspheres.

In this study the w/o/w extraction-evaporation technique was adopted to prepare poly(lactic-co-glycolic acid) (PLGA) microspheres loading recombinant human epidermal growth factor (rhEGF). The microspheres were characterized for morphology by transmission electron microscopy (TEM) and particle size distribution. The release performances, the proliferation effects and therapeutic effects of rhEGF-loaded PLGA microspheres were all studied. The results showed that these spherical microspheres had a narrow size distribution and a high drug encapsulation efficiency (85.6%). RhEGF-loaded microspheres enhanced the growth rate of fibroblasts and wound healing more efficiently than pure rhEGF. The number of the proliferating cell nuclear antigen (PCNA) in the epidermis layer with the microsphere treatment was significantly larger than those of the control groups. Overall locally sustained delivery of rhEGF from biodegradable PLGA microspheres may serve as a novel therapeutic strategy for diabetic ulcer repair.

Folate-PEG coated cationic modified chitosan--cholesterol liposomes for tumor-targeted drug delivery.

In this paper, a folate-PEG coated polymeric liposome (FPL) formed from octadecyl-quaternized lysine modified chitosan (OQLCS) and cholesterol has been prepared successfully. The OQLCS and its derivatives were characterized using (1)H NMR and infrared spectrum analysis. The FPLs properties were extensively studied by dynamic light scattering (DLS), fluorescence spectroscopy, and transmission electron microscopy (TEM). Due to the amphiphilic property and positive zeta potential of OQLCS, the OQLCS and cholesterol can form stable core-shell FPLs with small size (effective diameter: 163.5 nm) and narrow distribution (polydispersity: 0.108) in aqueous solutions. The PLs could form multi-lamellar structure similar to that of traditional liposomes prepared from phosphatidylcholine/cholesterol (PC/Chol). Compared with traditional liposome, calcein-loaded Polymeric Liposome exhibited high encapsulation efficiency in aqueous solution and slow, controlled release under different pH conditions. Most important, in cellular uptake experiment, folate coated FPLs showed significant higher uptake by MCF-7 cells as compared to FPLs without folate and traditional liposomes, because of the folate-receptor mediated endocytosis. The data suggest that the folate-PEG coated polymeric liposomes (FPLs) may be a useful drug delivery system.

Novel quaternized chitosan and polymeric micelles with cross-linked ionic cores for prolonged release of minocycline.

Novel multifunctional octadecyl quaternized carboxymethyl chitosans (OQCMCs) with varying degree of quaternary substitution (DS) and molecular mass were prepared and compared with quaternized chitosan. OQCMCs exhibited excellent solubility both in water and organic solvents. Nanoparticles of OQCMCs offered many advantages, such as easier fabrication and modulation of their size and degree of positive charge, and a lower cytotoxic effect compared with PEI (25 kDa). DNA can be successfully adsorbed on its surface. Electrostatic attraction of carboxymethyl and quaternary groups in OQCMCs was utilized as micellar template for the synthesis of cross-linked micelles. Formation and characteristics of OQCMC polymeric micelles were studied by fluorescence spectroscopy, tensiometry, SEM, TEM and particle size analysis. Self-assembled OQCMC micelles were evaluated as carrier of the lipophilic drug, minocycline hydrochloride (MH). MH was incorporated into cross-linked ionic cores of micelles with remarkably high efficiency (22.7%, w/w). MH-loaded OQCMC polymeric micelles exhibited a slow steady release profile over a 1-week period at 37 degrees C. The OQCMC micelles are potentially useful for gene and lipophilic drug delivery applications.